![Chapter 1
A Historical Perspective on Bacterial Persistence
Natalie Verstraeten, Wouter Knapen, Maarten Fauvart,
and Jan Michiels
Abstract
Bactericidal antibiotics quickly kill the majority of a bacterial population. However, a small fraction of cells
typically survive through entering the so-called persister state. Persister cells are increasingly being viewed as
a major cause of the recurrence of chronic infectious disease and could be an important factor in the
emergence of antibiotic resistance. The phenomenon of persistence was first described in the 1940s, but
remained poorly understood for decades afterwards. Only recently, a series of breakthrough discoveries has
started to shed light on persister physiology and the molecular and genetic underpinnings of persister
formation. We here provide an overview of the key studies that have paved the way for the current boom in
persistence research, with a special focus on the technological and methodological advances that have
enabled this progress.
Keywords: Persisters, Persistence, Antibiotic tolerance, Dormancy, Antibiotics, Review
1 The Early Days
The first report on the survival of a small fraction of streptococci
cells following treatment with penicillin dates from 1942 [1].
Two years later, Joseph Bigger established that addition of penicil-
lin to staphylococci does not result in complete sterilization of all
cells in a clonal population. One out of a million cells survived even
prolonged treatment with antibiotics. He appropriately named the
surviving cells persisters [2]. More recently, it was shown that in
most bacterial species, the majority of cells are efficiently killed by
relatively low concentrations of bactericidal antibiotics. However,
killing shows a biphasic pattern and beyond a certain threshold,
further increasing the concentration of the antibacterial does not
result in complete clearing of the culture (Fig. 1) [3].
For 40 years following its discovery, the persistence phenome-
non was largely neglected, at least by molecular geneticists.
This was partly due to the fact that the clinical relevance of persister
Jan Michiels and Maarten Fauvart (eds.), Bacterial Persistence: Methods and Protocols,
Methods in Molecular Biology, vol. 1333, DOI 10.1007/978-1-4939-2854-5_1,
© Springer Science+Business Media New York 2016
3](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-20-320.jpg)
![cells was not clear. In contrast, the threat posed by inherited
antibiotic resistance was generally recognized, adding incentives
to resistance research. The problem was compounded by technical
challenges that inevitably accompany the study of a transient
phenotype that is associated with only a very small fraction of cells.
A breakthrough discovery came in the early 1980s, from
research carried out by Harris Moyed during a sabbatical leave in
the lab of Alexander Tomasz [4]. Mutagenesis of Escherichia coli
populations with ethyl methanesulfonate (EMS) led to the
identification of three highly persistent (hip) mutants exhibiting
10–10,000-fold increased persister fractions upon incubation
with penicillin [4, 5]. Moyed’s pioneering work led to the identifi-
cation of two mutants hit in the hipA locus that up until now
remains the best studied persister gene [6–10]. Furthermore,
because of their increased persister fraction, hipA mutants have
frequently been used as a tool in persistence research. Crucially
and for the first time, hipA mutants enabled the direct observation
of persister cells. Using a combination of microfluidics and live cell
microscopy, Nathalie Balaban recorded how persisters survived
killing by antibiotics through dormancy and subsequent resuscita-
tion [11]. In addition, the hipBA locus is a representative for other
toxin– antitoxin (TA) loci that are now intensively studied in rela-
tion to persistence. TA modules consist of a stable toxin, typically
targeting essential cellular functions, and an unstable antitoxin,
Fig. 1 Illustration of persistence: The majority of cells in a bacterial culture are efficiently killed by relatively
low concentrations of antibiotics. However, beyond a certain threshold, a killing plateau is observed as only
persister cells remain viable. When regrown in fresh medium, the surviving cells generate a population as
sensitive to the antibiotic as the original population
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![which counteracts the activity of its cognate toxin [12, 13]. TA
systems were originally identified on plasmids, where they play a
role in plasmid maintenance; yet a significant number of TA loci are
chromosomally encoded and these have been implicated in persis-
tence [14]. Examples include RelE [6], MqsR [15–17], TisB [18,
19], MazF [20], and YafQ [21]. Interestingly, with the notable
exception of Salmonella persisters residing within macrophage
vacuoles [22], deletion of a single toxin generally does not affect
persistence. This can partly be explained by redundancy of TA
systems in most bacteria. Deletion of multiple TA systems, on the
other hand, causes a decrease in E. coli persistence [23].
2 The Rise of Persistence Research
Following the discovery of hipA, persistence as a field of study
steadily gained attention. This was partly due to the acknowledge-
ment of its clinical significance (summarized by [24]). In 1944,
Bigger already alluded to the role of persisters in the resuscitation of
chronic infections [2]. Decades later, Kim Lewis postulated that
persisters might contribute to the recalcitrance of biofilm infections
[25, 26]. This is of particular interest as biofilms are known to
withstand antibiotic treatment, thereby causing chronic infections
[27]. Subsequently, mathematical modeling demonstrated that
persistence could extend the duration of antibiotic treatment,
thereby causing treatment failure and promoting the emergence
of resistance [28]. Finally, two studies have unambiguously demon-
strated that prolonged antimicrobial therapy selects for high-
persistence strains of Candida albicans during candidiasis and
of Pseudomonas aeruginosa during cystic fibrosis lung infections
[29, 30]. In addition, the role of persister cells in the development
of resistance is becoming increasingly clear [31]. Apart from
providing incentives to further intensify persistence research,
these findings also promoted the search for anti-persister therapies.
At present, several strategies have been described, but their in vivo
effectiveness remains to be investigated. Examples include the
use of resonant activation [32], electrochemical currents [33],
cadaverine [34], metabolites [35, 36], antimicrobial peptides
[37], brominated furanones [38–41], and activated ClpP [42]
(summarized by [43]).
Apart from increased interest due to the clinical importance of
persistence, the development of novel techniques also caused per-
sistence research to boom. An overview of these novel techniques is
provided below.
2.1 Screening
Approaches
Over the years, several screening procedures have been developed
that led to the identification of persister genes. In a first approach, a
non-redundant E. coli knockout library was screened for mutants
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![with altered persistence [44]. Persister cells of individual mutants
were quantified by treating a stationary-phase culture with oflox-
acin and plating the surviving cells on agar medium containing
amdinocillin. As the number of spontaneous amdinocillin-resistant
mutants is a fraction of the original number of cells, this obviates
the need for dilution steps and greatly reduces the laborious task of
screening several thousands of strains.
A second screening approach employed a P. aeruginosa plaspo-
son knockout library. Individual mutants were grown until station-
ary phase and treated with either ofloxacin to kill non-persister cells
or water, the latter serving as a control. Subsequently, samples were
diluted and incubated in an automated plate reader (Bioscreen C,
Oy Growth Curves Ab Ltd), allowing the optical density of 200
samples to be measured simultaneously as a function of time.
Given the linear relationship between the number of cells in an
inoculum and the lag phase, this allowed for the selection of
mutants displaying altered persister levels [45].
Both screenings led to the identification of a number of
interesting persister genes including some global regulators. In
addition, not a single mutant lacking persisters was identified. As a
general conclusion, these screenings therefore provided evidence
pointing to the multiplicity of persister formation mechanisms.
In a final approach, a random overexpression library was
generated in E. coli. Cells from the recombinant library were pooled
and logarithmically growing cultures of library clones were exposed
to multiple rounds of exposure to ampicillin. This led to the enrich-
ment of mutants with increased probability of persister formation
and ultimately to the identification of glpD as a genuine persister
gene [46].
2.2 Single-Cell
Studies
As persistence is a phenotypic trait expressed in only a subfraction of
a population, advances in single-cell research signified an era of vast
new possibilities. First used by the Balaban group [11], transparent
microfluidic devices proved instrumental for microscopic examina-
tion of persister cells [47–49]. The strength of this technique lies in
the possibility to monitor individual cells for prolonged periods of
time while adapting growth conditions. For example, normal
growth conditions can be alternated with antibiotic treatments in
order to kill non-persister cells. This allows to pinpoint persister
cells surviving treatment. Subsequently, the history of persister cells
can be traced back through the recorded images. Several studies
have used this technique to demonstrate preexisting heterogeneity
in bacterial populations [11], to characterize the dormant state of
single persister cells [47], to monitor persister formation following
administration of indole [48], and to correlate high TA expression
to cessation of growth [49].
Also developed by the Balaban group, a colony-appearance assay
was elaborated to quantify single-cell persister lag phases [50].
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![Experiments demonstrated that a threshold concentration of toxin
molecules is required for induction of persistence.
A major drawback of microfluidic devices, or more precisely of
microscopy, is the limited number of cells that can be studied
simultaneously. This can be circumvented by using flow cytometry,
allowing thousands or even millions of cells to be evaluated in a
high-throughput manner. A shortcoming of this technique is the
inability to continuously monitor individual cells over time. None-
theless, flow cytometry has been successfully used to study the
kinetics of persister awakening [51]. In addition, while Bigger
postulated that persisters are in a dormant, nondividing state [2],
flow cytometry has been used to demonstrate that dormancy is not
a requirement for entry into the persister state [52]. Finally, a recent
study performed by the Holden group showed how to characterize
the dynamics of intracellular bacterial replication at the single-cell
level. They used a fluorescence dilution technique to quantify the
number of replication cycles of internalized Salmonella [53]. This
showed the existence of different Salmonella subpopulations in
bone marrow-derived macrophages including a non-replicating
but metabolically active subpopulation, comprising the persister
cells, possibly capable of resuming growth and causing relapsing
infections [22]. Similarly, the Bumann group exploited a DsRed
variant called TIMERbac
, which spontaneously changes color from
green to green/orange over time, as a dynamic growth rate
reporter to identify persister cells in vivo [54].
2.3 Transcriptomics Insight into global transcriptional changes in persister cells came
from several elegant studies by the Lewis group. To enrich for
persisters, all three approaches conveniently employed the meta-
bolic inactivity of these cells. In the first report, logarithmically
growing populations of the high-persistence E. coli mutant hipA7
[4] were treated with ampicillin, thereby lysing non-persister cells.
Isolated RNA was enriched for mRNA, labeled, and hybridized
to E. coli GeneChips [6]. Similarly, gene expression profiling of
persisters was performed after treating an exponentially growing
population of Mycobacterium tuberculosis with D-cycloserine and
collecting surviving persister cells by centrifugation. Transcriptome
analysis was performed by microarray hybridization [55]. The third
study followed a slightly different approach. It was based on the
assumption that persisters are dormant cells with low levels
of protein synthesis and corresponding low levels of rRNA tran-
scription. E. coli persister cells were isolated by linking the rrnB
promoter to a gene encoding an unstable fluorescent protein. In so
doing, persister cells are dim as compared to normal cells in the
population, which allows for the isolation of persisters using
fluorescence-activated cell sorting (FACS). cDNA was prepared
from purified RNA and hybridized to spotted E. coli DNA micro-
arrays [56].
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![Based on the studies cited above, stress response pathways as
well as TA loci were shown to be highly expressed in isolated
persister cells. On the other hand, biosynthetic functions including
energy production were downregulated [6, 55, 56].
2.4 Experimental
Evolution
The use of experimental evolution for elucidating antibacterial
resistance mechanisms is a widely used method. A recent study by
the Balaban group used this technique for enriching a population
with persisters by repeated exposure of a bacterial population to
high concentrations of antibiotics. This resulted in evolved strains
showing very high persister fractions caused by fixed specific
genetic mutations. The increased survival appeared to be the result
of an adjustment in the single-cell lag-time distribution, which was
correlated with the extent of the antibiotic exposure interval [57].
They implemented the ScanLag method, which allows the simulta-
neous measurement of lag times of hundreds of cells [58]. These
findings resulted in a new theory regarding persister cells and their
ability to adapt to high doses of drugs called tolerance by lag.
2.5 Modeling Apart from these wet lab techniques, mathematical modeling has
provided interesting insights [28, 59–62] (summarized by [63]).
Briefly, two main strategies can be discerned: the first one relies on
estimating the switching rates between persister and non-persister
growth states and assumes this process to take place continuously
and stochastically (e.g., [11, 28, 59, 61, 64]). The balance between
both switching rates provides a straightforward way to model a
given persister level, although ignoring exactly what determines
the switching rates. The second strategy focuses on the molecular
mechanisms of persister formation by TA systems, with the ratio of
(free) toxin over antitoxin ultimately determining, at the single-cell
level, the decision to switch to the persister state (e.g., [50, 60,
65, 66]). A crucial factor in this type of models is the generation of
phenotypic bistability at the population level, typically requiring
noisy gene expression and noise amplification through positive
feedback mechanisms [67]. Both modeling strategies have their
merits, and until a more integrated approach is presented, the
choice between both will depend on the goal and specific focus of
the study at hand.
Mathematical modeling of persistence poses several advantages.
Experiments that are not feasible in the lab can be simulated to
predict the outcome. It also allows to explain empirically observed
persister levels in terms of the parameters encompassed by the
model, and why varying some parameters has more impact on
persistence than others. Consequently, evolutionary forces that
shape persister levels can be identified, which should help to devise
strategies to avoid high persister levels emerging in the clinic.
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![3 State of the Art and Future Perspectives
Recently, the field of microbial persistence research has exploded, as
evidenced by a host of publications in top-tier journals [10, 22, 23,
35, 48–50, 54, 68–70]. Currently, it is generally accepted that
persister cells are present in a bacterial population preceding antibi-
otic treatment [71]. It is postulated that their formation results
from noisy gene expression [72] as was first suggested by Kim
Lewis [6]. However, over the years, several stimuli have been
shown to induce persistence. For example, sub-inhibitory concen-
trations of fluoroquinolones are known to induce persistence via
activation of the tisAB/istR TA locus [18, 19]. Other examples
include quorum sensing molecules [73, 74], carbon source transi-
tions [70], and nutrient deprivation leading to activation of the
stringent response [75]. As was earlier described for HipA [76],
a recent model ascribes TA-regulated persistence to stochastic
fluctuations in cellular concentrations of the alarmone (p)ppGpp.
High (p)ppGpp levels activate TA loci through a regulatory cascade
requiring inorganic polyphosphate and Lon protease targeting
protein toxins [49]. For an elaborate discussion on the role of
these mechanisms in persistence, the reader is referred to some
excellent reviews on the topic [3, 77–81].
Adding to the significance of these studies is the recent obser-
vation of a phenotypically distinct subpopulation of transiently
drug-tolerant persisters in cancer cell populations. These cells are
held responsible for “fractional killing” upon chemotherapy [82].
Cell-to-cell variations in protein levels were suggested to contribute
to this phenomenon in which each round of therapy kills some but
not all of the cells in a tumor [83]. There is a striking analogy
between bacterial and cancer cell-derived persistence as both phe-
nomena reflect a transiently phenotypic heterogeneity causing mul-
tidrug tolerance and recurrence of disease symptoms upon removal
of treatment [84]. Added insight into bacterial persistence may
therefore impact research areas far beyond infectious disease.
Acknowledgements
Research in the lab of JM is supported by grants from KU Leuven
Research Council (PF/10/010; IDO/09/010; IDO/13/008),
IAP-BELSPO, FWO, and IWT.
References
1. Hobby GL, Meyer K, Chaffee E (1942) Obser-
vations on the mechanism of action of penicil-
lin. Exp Biol Med 50(2):281–285
2. Bigger JW (1944) Treatment of staphylococcal
infections with penicillin. Lancet 244:497–500
3. Lewis K (2010) Persister cells. Annu Rev
Microbiol 64:357–372
4. Moyed HS, Bertrand KP (1983) hipA, a newly
recognized gene of Escherichia coli K-12
that affects frequency of persistence after
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![Chapter 2
Persisters: Methods for Isolation and Identifying
Contributing Factors—A Review
Sarah E. Rowe, Brian P. Conlon, Iris Keren, and Kim Lewis
Abstract
Persister cells are phenotypic variants surviving a lethal dose of antibiotic, sufficient to kill the bulk of an
exponential phase population. In this chapter we summarize current techniques to isolate persisters and
discuss limitations associated with identifying mechanisms of persister formation.
Keywords: Hip mutant, Toxin-antitoxin (TA) modules, Antibiotic, Biofilm
1 Introduction
Persisters were first described in 1944 by Joseph Bigger, a medical
doctor from Trinity College Dublin [1]. Bigger reported that
addition of a lethal dose of penicillin to a population of Staphylococ-
cus aureus yielded a small subpopulation of surviving cells. Upon
reinoculation, these colonies grew into a culture that once again
lysed in the presence of penicillin and again yielded a subpopulation
of survivors. Bigger referred to these surviving cells as persisters [1].
These cells are contrary to resistant cells as they cannot grow in
the presence of the antibiotic. The proportion of persisters in a
population varies dramatically depending on growth phase and
environmental conditions [2, 3]. Specifically, persister numbers
are greatly increased in stationary phase and biofilms [4, 5].
Persisters can be enumerated by adding a lethal concentration
of a bactericidal antibiotic to a population of cells. The bulk of the
population dies rapidly and the surviving persister cells either die
much more slowly or do not die during the experiment (Fig. 1).
The persister fraction can be enumerated at various time intervals
by removing an aliquot, washing with 1 % NaCl, and plating serial
dilutions for colony counting. The surviving persister fraction
emerges in a clear biphasic curve in response to the antibiotic.
Jan Michiels and Maarten Fauvart (eds.), Bacterial Persistence: Methods and Protocols,
Methods in Molecular Biology, vol. 1333, DOI 10.1007/978-1-4939-2854-5_2,
© Springer Science+Business Media New York 2016
17](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-34-320.jpg)
![The time period where the persister fraction remains constant is
referred to as the “persister plateau” (Fig. 1).
The majority of research has concentrated on persisters in
Escherichia coli but persisters have been shown to be produced
by many other bacterial species including Pseudomonas aeruginosa,
Mycobacterium tuberculosis, Salmonella enterica serovar typhimur-
ium, Staphylococcus aureus, and Streptococcus mutans Burkholderia
cepacia and even in the fungal pathogen Candida albicans [6–13].
2 High-Persister (hip) Mutants
Although persisters were discovered in the 1940s, they were largely
ignored until the early 1980s when Harris Moyed and co-workers
isolated high-persistence (hip) mutants [14, 15]. They identified
the gene hipA, which was the first gene known to contribute to
persister formation; this gene was later identified as the toxin of an
antitoxin/toxin (TA) module. The hipA7 allele of the gene pro-
duced 1000 times more persisters to two classes of antibiotics: beta-
lactams and fluoroquinolones [16]. This was the first indication
that some persisters are multidrug tolerant. Importantly the hipA7
mutant has the same minimal inhibitory concentration (MIC) as
the parent strain [16], indicating that the increased level of persis-
ters is not due to acquisition of resistance.
Time [h]
Log
CFU/ml
3 6
Antibiotic added here
108
Persister plateau
Drug
susceptible
cells
Resistant mutant
Fig. 1 Typical biphasic kill curve of a bacterial population in exponential phase of growth: A culture of E. coli
grown to mid-exponential phase is challenged with 10 MIC of a bactericidal antibiotic (such as ciprofloxa-
cin). The majority of cells die rapidly. The persister fraction is enumerated by removing an aliquot of cells after
3 and 6 h of exposure to the antibiotic. Cells are washed with 1 % NaCl and serial dilutions are plated for
counting. Persister cells differ from resistant cells as they can tolerate the antibiotic but cannot grow in its
presence
18 Sarah E. Rowe et al.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-35-320.jpg)
![Hip mutants can be identified in vitro by generating a mutant
library and exposing it to several rounds of a lethal dose of an
antibiotic [17]. The bacterial mutant library can be obtained either
by chemical mutagenesis (ethyl methanesulfonate) [18] or by trans-
poson mutagenesis [19]. Following several rounds of antibiotic
challenge, the genetic basis for hip mutant phenotypes is identified
by subjecting surviving cells to whole-genome sequencing or
micro-array-based genetic footprinting [17].
Persisters represent transient phenotypic variants that are
genetically identical to their drug-susceptible kin. Such traits have
proved taxing for in vivo investigations as these persisters tend to
resuscitate. Mulcahy et al. took a different approach [20]. They
hypothesized that if hip mutants had an advantage then they will be
selected for in vivo during infection or in response to antibiotic
treatment. Longitudinal isolates of Pseudomonas aeruginosa were
obtained from patients throughout the course of a cystic fibrosis
infection. The study demonstrated that strains developed a hip
phenotype over the course of the infection (Fig. 2). A similar
finding was observed in Candida albicans with patients suffering
from chronic oral thrush [21]. These studies indicate that persisters
are clinically relevant and may contribute to treatment failures.
0.8
0.00001
0.0001
0.001
0.01
0.1
1
AMT47 AMT60 AMT66 AMT71 AMT74 AMT76 AMT33 AMT41 AMT75 AMT73 AMT62 AMT36
AMT101 AMT100
7.3
3
7.7
9.1
19.6
19.6
13.2
5.6
12.8
23.4
2.3
9.6
8.6
21.1
15.4
4.42
13.1
6.7
9.6
10.8
Patient Number and age (years old) when strain was isolated
Percent
Survival
1
1.1
7.1
9.2
7.2
15.4
15.2
Fig. 2 A screen of P. aeruginosa clinical isolates for hip mutants: Stationary-phase cultures of clonal early/late
isolate pairs from 14 patients were exposed to ofloxacin (50 MIC) for 8 h, and the surviving cells were
determined by colony count and expressed as percent survival (n ¼ 4). Early isolates are indicated with white
bars, while late isolates are indicated with black bars. The patient number and age at which the tested isolates
were obtained are displayed on the x-axis. A hip mutant emerged in 10 of the 14 patients. A hip mutant did not
emerge in the isolates from the last four patients displayed on the right side of the graph. Taken from Mulcahy
et al. 2010
Persisters: Methods for Isolation and Identifying Contributing Factors—A Review 19](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-36-320.jpg)
![3 Pre-existing and Induced Persisters
The frequency of persisters in hip strains is much higher than in the
wild-type E. coli which facilitates research into the mechanism of
persister formation. The hipA7 mutant was used for single-cell
time-lapse microscopy, which showed that persisters are slow or
non-growing cells, stochastically formed and pre-existing in the
population [22].
In order to distinguish between growing and non-growing
cells, Shah et al. used an unstable GFP variant driven from a ribo-
somal promoter [23]. Cells expressing this plasmid were grown to
mid-exponential phase and analyzed by fluorescent-activated cell
sorting (FACS) (Fig. 3). Two distinct populations were visualized
using forward light scatter, one that fluoresced brightly and one that
did not. Cells from both populations were sorted and visualized by
epifluorescent microscopy and challenged with ofloxacin. The cells
that did not express GFP were much more likely to survive antibiotic
challenge. This study described a new mechanism for identifying
persisters and demonstrated that non-growing cells exist in an
untreated E. coli population [23]. These results suggest that there
is a population of persisters that are pre-existing in the population
and are not formed in a response to antibiotic treatment.
In support of this, it has been demonstrated that an E. coli
culture, if diluted several times and challenged with either ampicil-
lin or ofloxacin, displays a gradual decline and eventual elimination
104
103
102
101
100
0 64
R3
R4
128
Forward Scatter
Green
Fluorescence
Log
%
Survival
Non-Persisters
Persisters
192 256
Bar = 5µm
2
1.6
1.2
0.8
0.4
0
Phase Contrast
a b c
Green Fluorescence
Fig. 3 Isolation of persister cells from an exponentially growing culture. E. coli cells containing a degradable
GFP reporter cassette were grown in LB medium to mid-exponential phase (~1 108
cells/ml) at 37
C with
aeration and sorted using a high-speed cell sorter equipped with a standard GFP filter set. (a) Two populations
were detected using forward light scatter, one that fluoresced brightly (R3), and another that did not (R4). (b)
The sorted populations were visualized by epifluorescent microscopy (bar, 5 μm). (c) Cells were sorted as
described in (a–b). Once sorted both populations were treated with ofloxacin (5 μg/ml) for 3 h, diluted, and
spotted onto LB agar plates for colony counts. Taken from Shah et al. 2006
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![of persisters, even though the initial population size is kept constant
[2]. This indicates that persisters are formed later in the growth
phase, accumulate in stationary phase, and can be diluted out with
serial reinoculation.
Johnson et al. showed that pretreatment of S. aureus with sub-
MIC concentrations of a particular antibiotic significantly increases
the level of multidrug-tolerant persisters [24]. From these results,
the authors conclude that persister formation is the product of
various errors during cell replication. These errors result in tran-
sient periods of slowed metabolism and/or non-replication by
individual cells in growing populations [24].
Although there is mounting evidence to suggest that persisters
are pre-existing and formed stochastically throughout growth,
there is also data to support that persisters can be induced in
response to antibiotics [24, 25] and environmental stress [26].
Dorr et al. demonstrated that pretreatment of E. coli with low levels
of ciprofloxacin induced the formation of persisters to higher doses
of ciprofloxacin (Fig. 4). This study shows that the majority of
persisters to ciprofloxacin are induced in response to that antibiotic
and this is dependent on a functional SOS response [25]. Later
work has demonstrated that persisters can also be formed as a
response to an environmental stress such as DNA damage or oxida-
tive stress [26] and the stringent response [3, 27, 28].
4 Toxin Antitoxins (TA) Modules
The first transcriptome of persisters was generated by taking advan-
tage of the fact that β-lactam antibiotics such as ampicillin are
bacteriolytic, causing lysis of dying cells [29]. An E. coli culture
was treated with a high concentration of ampicillin. A simple cen-
trifugation step was used to collect the surviving persisters and
expression profiles were determined using a microarray. Several
TA modules were among the genes induced in persisters.
TA genes were first identified as addiction modules that play a
role in plasmid maintenance [30]. The toxin and antitoxin genes
make up an operon that is transcribed from the same promoter. In
the case of type II TA systems, the toxin and antitoxins are proteins
which bind together and often repress transcription of the operon
[31]. The antitoxin is less stable and is degraded by cellular pro-
teases; if a daughter cell loses the plasmid, the antitoxin is rapidly
degraded and the toxin prevents cell growth [30]. TAs were subse-
quently discovered on bacterial chromosomes. Many studies have
implicated chromosomal TAs to have a role in persistence which
will be discussed in this section [14, 16, 32–35].
The hipA gene of the hipBA operon is the probably the most
widely studied persister gene. It encodes the toxin HipA which is
neutralized by its cognate antitoxin HipB. Under cellular stress, the
Persisters: Methods for Isolation and Identifying Contributing Factors—A Review 21](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-38-320.jpg)
![Lon protease degrades HipB, releasing HipA and allowing it to
exert its toxic effects on the cell [35]. A recent study revealed that
HipA is a kinase that phosphorylates an aminoacyl-tRNA synthe-
tase, halts protein synthesis, and induces persister formation [36].
There are 11 TA mRNA interferases in E. coli K12, which
are induced under various cellular stresses [3, 37–41].
Upon overexpression, these toxins have been shown to cleave
Fig. 4 Ciprofloxacin-induced persistence: (a) Survival of wild-type cells in exponential phase under different
ciprofloxacin regimes. Two cultures were treated with 0.1 and 1 μg/ml, respectively, for 6 h. Third culture was
treated with 0.1 μg/ml for 3 h after which 1 μg/ml was added (indicated by an arrow). The data are averages of
three independent experiments and error bars indicate standard error. (b) Wild-type cells in exponential phase
were treated for 3 h with increasing concentrations of ciprofloxacin indicated on x-axis. After the initial
treatment, an additional 1 μg/ml of ciprofloxacin was added to the cultures and incubated for another 3 h as in
(a). (Ciprofloxacin MIC is 0.05 μg/ml.) As a control, a parallel culture was exposed to 1 μg/ml for the duration of
the experiment. Bars represent the viability at 0, 3, and 6 h of time course equivalents shown in (a). Open bars:
The initial viability count. Grey bars: The viability after 3-h incubation with ciprofloxacin concentration
indicated on the x-axis. Full bars: The final viability count after additional 3-h incubation with 1 μg/ml
ciprofloxacin. Dashed bars: Viability of the control culture at 3 and 6 h. The data are averages of three
independent experiments and error bars indicate standard error. Taken from Dorr et al. 2009
22 Sarah E. Rowe et al.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-39-320.jpg)
![cellular mRNA and induce persister formation. However, single
mutations of these TA modules did not result in a reduction of
persister levels [34]. Redundancy could explain the lack of a per-
sister phenotype but nevertheless this led to some debate as to their
role in persister formation.
Kenn Gerdes and co-workers serially deleted up to ten of the
recognized type II TA modules (Δ10) in E. coli [34]. They reported
that at least five modules need to be deleted before a significant
reduction in persister levels is observed. A progressive reduction in
persister levels was observed when more TA modules were deleted,
and the Δ10 strain displayed a 100-fold reduction in persister
formation. These results demonstrate the importance of TA mod-
ules in persister formation and their high degree of redundancy for
one another.
It is also a possibility that an individual TA locus may play a role
in persister formation in a specific strain or under specific environ-
mental conditions. Norton et al. reported that a single knockout of
the TA module PasTI had no phenotype when deleted in E. coli lab
strain MG1655 but displayed a 100-fold reduction in persisters
when deleted in a clinical isolate CFT073 [42]. This study high-
lights the limitations of studying the mechanisms of persister for-
mation in lab strains under lab conditions [42].
Similarly, Helaine et al. reported that internalization of Salmo-
nella by macrophages induced persister formation over 100-fold
[43]. Importantly, the group identified two stress conditions
encountered in vivo, vacuolar acidification and nutritional depriva-
tion, which induce persister formation through the 14 TA modules.
Together these studies emphasize the need to study persistence in
an environment that closely resembles in vivo conditions.
5 Genetic Determinants of Persisters
The standard approach for identifying genes which contribute to a
function is to avail of a mutant library and generate a screen. This
does not work well when redundant genes contribute to the same
function, as is the case of the TA modules. An alternative method is
to create an over-expression library and screen for gain-of-function
phenotypes. In this case, even a mild contributor to the phenotype
can be identified. However, this method can be problematic for
persisters as overproduction of many proteins, particular membrane
proteins, can result in toxic protein aggregates. This can cause
growth cessation and artificially emulate an antibiotic tolerant
state. Spoering et al. overexpressed a library in a low-copy vector,
using native promoters for expression and challenged with a lethal
dose of ampicillin [44]. In an attempt to exclude false positives the
authors introduced a growth step in between rounds of antibiotic
selection. Any strains that grew considerably slower than the wild
Persisters: Methods for Isolation and Identifying Contributing Factors—A Review 23](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-40-320.jpg)
![type would be selected against. A particular clone, with elevated
levels of persisters tolerant to ampicillin, carried the gene glpD,
involved in glycerol metabolism. Interestingly, overexpression of
glpD also induced persisters tolerant to ofloxacin suggesting that
these persisters are multidrug tolerant. Deletion of the glpD gene in
a wild-type background reduced levels of persisters to ciprofloxacin
in stationary phase. Recent work has suggested a mechanism by
which the glpD mutant has increased levels of methylglyoxal, a
bacteriostatic metabolite [17].
A screen for novel persister genes utilized the Keio collection
[45], an ordered deletion library of all 3985 nonessential genes in
E. coli. Strains were grown to stationary phase in 96-well plates and
exposed to lethal concentration of ofloxacin [46]. This screen
identified 150 mutants with decreased levels of persisters. Ten
mutants were confirmed to display a decreased level of persisters
to ofloxacin with an unchanged MIC. Most of these genes had
mutations in global regulators, all of which had modest effects on
persister levels [46].
Global regulators RpoS and RelA were also identified to have a
role in P. aeruginosa persistence [6, 28, 47]. An additional nine
genes were identified by a high-throughput screen of an incomplete
P. aeruginosa transposon mutant library [48].
Despite these many screens, a mutant with a complete lack of
persisters has not been identified. The mutants identified to date
can display altered levels of persisters to one or more class of drugs.
This strongly suggests that there is not a central mechanism for
persister formation and persisters surviving different antibiotic
treatments are not identical. It is more likely that persisters can be
formed through different mechanisms and there is a degree of cross
talk between these mechanisms that can lead multidrug-tolerant
persisters.
6 The Importance of Persisters
Many bacteria form biofilms in response to environmental stress
[49]. Biofilms are a surface-attached agglomeration of cells encased
in an exopolymeric and proteinaceous matrix. While most antibio-
tics can penetrate through this matrix, biofilms do protect the cells
from the immune response and can therefore complicate treatment
of an infection leading to persistent and chronic infection [50]. It
was demonstrated that biofilms of P. aeruginosa harbor persister
cells tolerant to antibiotics [7, 27] and there is evidence to suggest
that this complicates treatment for cystic fibrosis patients [20]. A
similar role was recently suggested for the granuloma in MTB
infection, acting as a physical barrier protecting persisters from
the immune system [8]. Kim Lewis suggested a model (Fig. 5)
explaining the recalcitrant nature of biofilm infection [51]. When
24 Sarah E. Rowe et al.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-41-320.jpg)
![a biofilm infection is treated with antibiotics, the majority of the
bacterial cells die and only persisters survive. In the biofilm, persis-
ters are protected from the immune system, and upon removal of
the antibiotic pressure they can repopulate the biofilm matrix. The
combination of persisters and biofilm is the likely culprit of relaps-
ing chronic infections, with persister cells providing protection
from antibiotics while the biofilm provides protection from the
immune system.
Antibiotic resistance represents a huge problem, particularly
with gram-negative bacteria [52, 53]. However, many bacterial
strains isolated from chronic infections are susceptible to the anti-
biotics they were treated with [5, 20]. This indicates that persis-
tence rather than resistance may be a key contributor to the
recalcitrance of some infections. With this in mind, a search for
novel antibiotics which target dormant or stationary cells rather
than fast-growing cells could dramatically reduce treatment
failures.
Bactericidal antibiotics target and corrupt active processes in
cells which are inactive in persister cells, allowing them to survive
the antibiotic treatment. Acyldepsipeptides (ADEPs) are a new class
of antibiotics discovered by Eli Lily in 1985. Brotz-Oesterhelt et al.
found that ADEP targets the ClpP protease, opening the proteo-
lytic core resulting in ATP-independent proteolysis [54]. Despite
impressive efficacy in a variety of animal models of acute infection,
development of the drug was not pursued due to high frequency
of resistance (1 106
) due to null mutation of the nonessential
clpP gene.
Fig. 5 A model of a relapsing biofilm infections. Regular cells and persister cells form in the biofilm and are
shed off into surrounding tissue and the bloodstream. Antibiotics kill regular cells, and the immune system
eliminates escaping persister cells. The matrix protects persister cells from the immune system, and when
the concentration of the antibiotic drops, they repopulate the biofilm, causing a relapse. Taken from
Lewis et al. 2010
Persisters: Methods for Isolation and Identifying Contributing Factors—A Review 25](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-42-320.jpg)
![Importantly, ADEP activates and dysregulates ClpP in an
ATP-independent manner [54]. This suggested that ADEP could
potentially target cells in a low-energy state and may be active
against non-growing cells. Conlon et al. recognized the unique
ability of the drug to target persister cells [5]. They reported that
although a stationary population of S. aureus is highly tolerant to a
range of antibiotics in vitro it remains susceptible to ADEP.
However, following an initial death phase, the high frequency of
resistant mutants allowed the culture to rebound and repopulate.
ADEP paired with rifampicin resulted in eradication of the
population due to increased antibiotic susceptibility of the
ADEP4-resistant clpP null mutants. Importantly, the authors were
able to sterilize a deep-seated murine thigh infection model of
S. aureus. This study highlights the importance of discovering
new antimicrobials that specifically target persister cells.
References
1. Bigger JW (1944) Treatment of staphylococcal
infections with penicillin. Lancet ii:497–500
2. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis
K (2004) Persister cells and tolerance to anti-
microbials. FEMS Microbiol Lett 230
(1):13–18
3. Maisonneuve E, Castro-Camargo M, Gerdes K
(2013) (p)ppGpp controls bacterial persistence
by stochastic induction of toxin-antitoxin activ-
ity. Cell 154(5):1140–1150. doi:10.1016/j.
cell.2013.07.048
4. Amato SM, Brynildsen MP (2014) Nutrient
transitions are a source of persisters in Escher-
ichia coli biofilms. PLoS One 9(3), e93110.
doi:10.1371/journal.pone.0093110
5. Conlon BP, Nakayasu ES, Fleck LE, LaFleur
MD, Isabella VM, Coleman K, Leonard SN,
Smith RD, Adkins JN, Lewis K (2013)
Activated ClpP kills persisters and
eradicates a chronic biofilm infection.
Nature 503(7476):365–370. doi:10.1038/
nature12790
6. Moker N, Dean CR, Tao J (2010) Pseudomo-
nas aeruginosa increases formation of
multidrug-tolerant persister cells in response
to quorum-sensing signaling molecules. J Bac-
teriol 192(7):1946–1955. doi:10.1128/JB.
01231-09
7. Spoering AL, Lewis K (2001) Biofilms and
planktonic cells of Pseudomonas aeruginosa
have similar resistance to killing by antimicro-
bials. J Bacteriol 183(23):6746–6751. doi:10.
1128/JB.183.23.6746-6751.2001
8. Keren I, Minami S, Rubin E, Lewis K (2011)
Characterization and transcriptome analysis of
Mycobacterium tuberculosis persisters. MBio 2
(3):e00100–e00111. doi:10.1128/mBio.
00100-11
9. Slattery A, Victorsen AH, Brown A, Hillman K,
Phillips GJ (2013) Isolation of highly persis-
tent mutants of Salmonella enterica serovar
Typhimurium reveals a new toxin-antitoxin
module. J Bacteriol 195(4):647–657. doi:10.
1128/JB.01397-12
10. Lechner S, Lewis K, Bertram R (2012)
Staphylococcus aureus persisters tolerant to bac-
tericidal antibiotics. J Mol Microbiol Biotech-
nol 22(4):235–244. doi:10.1159/000342449
11. Leung V, Levesque CM (2012) A stress-
inducible quorum-sensing peptide mediates
the formation of persister cells with noninher-
ited multidrug tolerance. J Bacteriol 194
(9):2265–2274. doi:10.1128/JB.06707-11
12. LaFleur MD, Kumamoto CA, Lewis K (2006)
Candida albicans biofilms produce antifungal-
tolerant persister cells. Antimicrob Agents
Chemother 50(11):3839–3846
13. Van Acker H, Sass A, Bazzini S, De Roy K,
Udine C, Messiaen T, Riccardi G, Boon N,
Nelis HJ, Mahenthiralingam E, Coenye T
(2013) Biofilm-grown Burkholderia cepacia
complex cells survive antibiotic treatment by
avoiding production of reactive oxygen species.
PLoS One 8(3), e58943. doi:10.1371/jour
nal.pone.0058943
14. Moyed HS, Bertrand KP (1983) hipA, a newly
recognized gene of Escherichia coli K-12 that
affects frequency of persistence after inhibition
of murein synthesis. J Bacteriol 155
(2):768–775
26 Sarah E. Rowe et al.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-43-320.jpg)
![Chapter 3
A General Method for Measuring Persister Levels
in Escherichia coli Cultures
Niilo Kaldalu, Arvi Jõers, Henri Ingelman, and Tanel Tenson
Abstract
Genetically homogeneous bacterial cultures contain persisters, cells that are not killed by bactericidal
antibiotics. These cells are suggested to be involved in the establishment of chronic infections. Persister
levels depend on growth conditions. Here, we discuss the parameters that have to be considered when
measuring persister levels and provide a sample protocol to do it.
Keywords: Antibiotic, Persister, Antibiotic tolerance, Ampicillin, Fluoroquinolones
1 Introduction
In 1944, Joseph Bigger studied bactericidal action of penicillin on
staphylococcal cultures. He found that some bacteria are able to
evade killing and regrow after the treatment. Dr. Bigger called these
cells persisters and either described or predicted several basic
features that define such surviving minority. Besides, he concluded
that persisters must be in part responsible for the limited success of
antibiotic treatment against infections [1]. Seventy years later,
the main conclusions of his research have been confirmed and
elaborated by many laboratories using several different bacterial
species. It is amazing how many of Bigger’s early observations
hold true, including the complaint that “irregularities and incon-
sistencies occurred in almost all experiments of this type” [1].
Below, we review principal concepts of persister research and try
to look how much some of the researchers’ findings depend on
experimental setup.
1.1 Phenotypic
Tolerance vs.
Phenotypic Resistance
Persisters are defined as bacteria that are tolerant to bactericidal
antibiotics; they are able to survive the drug treatment but are
not able to grow in the presence of the drug [1, 2]. If bacteria
grow in the presence of the drug, they are called resistant.
Jan Michiels and Maarten Fauvart (eds.), Bacterial Persistence: Methods and Protocols,
Methods in Molecular Biology, vol. 1333, DOI 10.1007/978-1-4939-2854-5_3,
© Springer Science+Business Media New York 2016
29](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-46-320.jpg)
![The phenomenon of persistence is phenotypic, a culture that was
started from clonal descendants of a persister cell contains persisters
at the same frequency as the original parental culture [1, 3].
Over several years, persister cells have been considered as non-
replicating cells [2]. According to a central concept of the field,
the non-growing, i.e., nondividing, state is crucial to persistence.
Bacterial cultures contain individual cells that stay non-replicating
in conditions enabling growth [4–6]. These cells can survive treat-
ment with antibiotics and, if they resume growth after the removal
of the drug, these bacteria are called persisters (Fig. 1a). It is a
matter of debate if such heterogeneity of the population is a
survival strategy [7–9] or reflects different extent of unavoidable
damage to the cells [10].
Recently, it has been reported that some persisters might be
actively proliferating: in Mycobacterium, the surviving subset of
bacteria can have highly active efflux pumps [11] or express
5
6
7
8
9
0 2 4 6
persisters
growth curve
1
3
5
7
0 1 2 3 4 5
Time (h)
a b
Time (h)
AMP
OFL
TypeII
TypeI
log
10
CFU/ml
log
10
CFU/ml
Fig. 1 Graphic presentation of results from persister experiments. (a). Typical killing curves of cultures treated
with antibiotics. Killing curves represent kinetics of killing of bacteria during a bactericidal treatment. Cultures
of E. coli MG1655 were grown and treated as described in this chapter. Briefly, both the overnight culture and
the test culture were grown in filter-sterilized LB broth. At the beginning of the experiment, overnight culture
was diluted 1000 and incubated on a shaker at 37
C for 3 h. Then, ampicillin, 100 μg/mL (AMP; dashed
line) or ofloxacin, 5 μg/mL (OFL; solid line) was added and incubation was continued. Killing curves show that
the number of living microbes decreased more than 1000 during the first hour of antibiotic treatment. That
drop is caused by killing of the bulk of phenotypically sensitive bacteria. Starting from the 1-h time point, CFU
numbers decrease more slowly and indicate the number of persisters in the culture. The values represent the
means of five independent AMP treatments and three independent OFL treatments. The error bars indicate the
standard deviation. (b). The number of persisters increases when a growing culture approaches stationary
phase. An overnight culture of E. coli BW25113 was diluted 1:100 and cultured with aeration at 37
C. At the
designated time points, samples were taken and treated with ciprofloxacin (1 μg/mL) for 5 h. Closed symbols,
cell count before the treatment (growth curve); open symbols, cell count after the ciprofloxacin treatment
(persister count). During the first 4 h, the number of persisters does not increase. In this stage, persisters are
the cells that have been in the non-proliferating state since inoculation (type I persisters). After the 4-h time
point, the number of persister increases rapidly and newly formed (type II) persisters make up the majority of
surviving cells. The values are averages of three replicates and the error bars indicate the standard deviation
30 Niilo Kaldalu et al.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-47-320.jpg)
![catalase-peroxidase, which is necessary for killing by isoniazid, in
stochastic pulses [12]. In these instances, surviving cells are repli-
cating, not quiescent. It has also been suggested that some of the
persisters in E. coli are actively dividing [6]. The concept of actively
growing persisters raises terminological discrepancies. Resistance
is usually determined genetically but still, the genetic component
is not part of the definition. Mechanisms of phenotypic antibiotic
resistance have been described recently [13, 14] and occurrence of
noncanonical, phenotypic resistance due to the growth conditions
and metabolic state of bacteria is well known to the microbiological
community [15]. The mycobacterial “dynamic persistence”
[11, 12] would fall under phenotypic resistance as well.
Replicative dormancy of persisters does not necessarily mean
the lack of metabolic activity. It is intuitively reasonable to suggest,
that “shutdown of a target function” (i.e., cell wall synthesis,
protein synthesis, or DNA replication) “will prevent the lethal
action” of the drug [16]. However in dormant cells not all the
targets are equally inactive. For example, the same persister cells of
E. coli log-phase cultures that are tolerant to fluoroquinolones and
cell-wall synthesis inhibitors are killed effectively by aminoglyco-
sides, a group of antibiotics that targets protein synthesis and causes
mistranslation [5, 17, 18]. Such killing is dependent on the mem-
brane potential of these cells, which is required for the uptake of
aminoglycosides. Membrane potential is created due to the active
metabolism and depends on the nutrients provided. Therefore,
whether the non-growing subset of bacteria is killed by aminogly-
cosides, depends on the carbon source of the growth medium [18].
Consequently, persisters may have at least limited activity of protein
synthesis, metabolism, and membrane transport [19].
1.2 Type I
and Type II Persisters
Joseph Bigger acknowledged that some bacteria “are in the
persister phase when inoculated into fresh medium, but the condi-
tion is induced in others by their new environment” [1]. Such
distinction defines two groups of persisters that either retain
dormancy acquired in the stationary phase (type I) or fall into
dormancy during growth (type II) [4]. If antibiotic is supplied at
the very moment of inoculation of the culture [5], in the lag phase,
or in the early logarithmic phase then the surviving persisters
belong to type I. At early stages of growth, the number of persisters
does not increase (Fig. 1b). Type I persisters are lost in the course of
repeated cycles of dilution and growth into early log phase [3].
Type II persisters are formed when a growing culture starts
approaching to the stationary phase. When nutrients are depleting
and growth conditions deteriorate, their number is progressively
increasing and they make up a great majority of the persister
fraction [3] (Fig. 1b). In this state, functional stress response
circuits (e.g., ppGpp synthesis) are important for type II persister
formation but lack of these stress responses still does not fully stop
Measuring Persister Levels in E. coli 31](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-48-320.jpg)
![formation of new persister cells [20, 21]. In addition to the nutrient
depletion, new persister cells are generated in response to many
different stress conditions (see below), including low concentra-
tions of antibiotics [22–24].
1.3 Visualizing
and Isolating
Persisters
The cells with different growth rates can be labeled with various
fluorescent reporters. The reporter can either manifest the physio-
logical activity of the cell [25] or cell division rate that dilutes the
fluorescent protein [26, 27]. Cell division and potential dormancy
can be observed directly in a microfluidics system under the micro-
scope [4, 28]. Alternatively, the cell population can be analyzed
either by flow cytometry [5, 25, 27, 29] or by monitoring growth
of plated cells [30]. For Mycobacterium it has been reported that
cell divisions can be followed from the loss of a very unstable
plasmid [11, 31].
For analyzing the macromolecular content of persisters it is
important to physically isolate this fraction of the population.
Lysis with antibiotics [3, 16, 32] or with alternative agents [33]
has been suggested in the literature as a method to eliminate
growing cells. However, it has to be noted that these methods
pool the cells fulfilling the persister definition with the cells
that will never be able to grow again (see Subheading 1.4). The
nonrecovering population can be considerably higher than the
persister population [5, 6, 27]. Alternatively, cell sorting can be
used for isolating bacteria in different physiological states [6, 25].
Here three complications arise. First, the amount of material that
can be isolated is much smaller compared to the methods based on
cell lysis. Second, and more importantly, the physiological state of
the bacteria might be altered by the sorting procedure. Bacteria are
exposed to high pressure during sorting and carefully planned
experiments are needed to control for dormancy induction by the
sorting procedure. Finally, separation of persisters from perma-
nently nondividing cells remains unresolved. Density gradient
centrifugation has been used for fractionating an E. coli culture
into subpopulations [34, 35]. Although more than 15 fractions
have been described, the connection with the phenomenon of
persisters has not been demonstrated [35].
1.4 Persisters
and VBNC
It has to be noted that not all nondividing cells resume growth in
the usual detection window of 1 or 2 days. In many cases, the cells
preserve membrane integrity and are called viable but non-
culturable (VBNC) [27, 36]. It is currently not clear how many
of these cells might finally resume growth as slow wakeup over
several months has been described [37].
1.5 Stationary-Phase
Persisters
Most of the bactericidal antibiotics are ineffective against
stationary-phase cultures. Certain fluoroquinolones, e.g., ofloxa-
cin, ciprofloxacin, and gatifloxacin against E. coli, are an exception.
32 Niilo Kaldalu et al.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-49-320.jpg)
![If the stationary-phase cells are diluted into the fresh growth
medium and antibiotic is supplied at the same moment, it must
be noted that most of the cells resume growth and become antibi-
otic sensitive soon after dilution. Type I persisters, not stationary-
phase persisters, are assayed in such experiments [5]. For counting
stationary-phase persisters, antibiotics have to be supplied directly
to undiluted non-growing (i.e., stationary phase) cultures, which
are in fact much less sensitive to antibiotic treatment [24, 38].
1.6 Are Persisters
Tolerant to Different
Antibiotics?
High-persistence (hip) mutants were isolated from the original
screens for E. coli mutants with altered persister frequency [39].
Later, it was found that although the screens were made with
ampicillin, increased persister frequency was observed also against
fluoroquinolone antibiotics and vice versa [40]. This suggested that
persisters are equally tolerant to many or all antibiotics. Current
results suggest that the mechanism of antibiotic action considerably
influences the persister levels and different (but probably overlap-
ping) populations survive different antibiotics [24, 41, 42].
1.7 Growth Inhibition
Induces Persistence
Bigger already showed that growth inhibition, either by suboptimal
temperature, nutrient limitation, or bacteriostatic agents, induces
persister formation [1]. In the pioneering work for finding out the
molecular mechanisms of persister formation, Moyed was looking
for mutants with altered persister levels. For avoiding indirect
effects, attempts were made to find mutants with no changes in
minimum inhibitory concentration (MIC) and growth parameters
[39]. Indeed, it was demonstrated later that overexpression of
various toxic proteins can induce increased persister levels [43].
This calls for cautious interpretation of several results where the
growth of bacteria is inhibited or the culture is stressed, as these
might not reveal the biologically relevant mechanisms of persister
formation. The examples include screening for mutants with
altered persister levels, expression of toxins from toxin-antitoxin
systems [11, 44–47], manipulations with ppGpp levels [44], treat-
ing the bacteria with sub MIC antibiotic concentrations [24], etc.
In addition, all strains and mutants with increased MIC values
should be compared with care [38]. Even cell sorting by flow
cytometry [6, 25, 27] might induce persisters from actively grow-
ing cells.
1.8 Introduction
to the Current Protocol
As discussed above, different labs often use different protocols for
the measurement of persister levels. Because the measurement
output is sensitive to conditions, there are several parameters that
are important to control. Here we have assembled a protocol that
contains the most commonly used steps and have highlighted the
essential parameters.
Measuring Persister Levels in E. coli 33](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-50-320.jpg)
![9. If you treat your cultures with fluoroquinolones (ciprofloxacin,
ofloxacin, norfloxacin, gatifloxacin), take 1 mL samples 1, 2, 3,
and 5 h after addition of the drug. Spin down the cells in a
1.5 mL test tube for 5 min at 5000 g and room temperature.
Remove supernatant and resuspend bacteria in 1 mL LB
medium. Repeat centrifugation, remove supernatant, and
resuspend cells in 1 mL of fresh PBS. Continue with serial
dilutions and plating as in the case of the non-treated and
ampicillin-treated samples (see Note 16). Example results of
the persister measurement are shown in Fig. 1. Alternatively,
the results of antibiotic treatment and growth resumption can
be analyzed by flow cytometry (see Note 17).
4 Notes
1. Let the powdered medium hydrate by pouring it slowly on the
surface of the water. This avoids clumping.
2. We strongly recommend using filter-sterilized rich media or
defined minimal media. Autoclaving causes degradation of the
components of rich media and alters the content in an unpredict-
able way. This contributes to inconsistency of results [29, 48].
If you use autoclaved media, minimizing experimental error
would require using always the same model of autoclave, aliquot-
ing identical volumes, loading the same amount of material to
autoclave, and sterilizing your media for the same time at the
same temperature.
3. LB agar can be allowed to solidify and stored at room tempera-
ture. It can be remelted in a microwave oven, allowed to cool to
~55
C, and used for pouring plates. The autoclaved, melted LB
agar should not be stored at 55
C for more than a few hours.
4. If using Na2HPO47H2O, take 21.6 g; for Na2HPO412H2O,
use 28.8 g.
5. Comprehensive guidelines for preparation and storage of anti-
biotic solutions are provided in [49].
6. Ampicillin solution can be routinely frozen and thawed without
any bad consequences if this drug is used for selection of
resistant organisms. However, we have seen that repeated
freezing and thawing cycles of ampicillin solution can change
bactericidal activity [50].
7. Typically, a clonal culture is started from a single fresh colony.
This routine is fully acceptable in the study of persisters.
However, it is practically inevitable that such cultures are
started from an uncertain number of bacteria of uncertain
age. If grown overnight, these cultures may have different
physiology at the moment when they are used for inoculation
36 Niilo Kaldalu et al.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-53-320.jpg)
![of the test cultures. We have seen that persister frequency
depends considerably on exact physiological parameters of the
inoculum [29]. DMSO stocks allow starting all overnight cul-
tures from a similar number of bacteria, which are in a con-
trolled physiological condition, and decrease inconsistency.
8. Regular, autoclaved LB medium may be used for preparation of
DMSO stocks.
9. Besides the strain’s genotype, persister frequency depends on
the growth medium and temperature, aeration, the fold of
dilution of the inoculum, the growth phase of both the
test culture, and the culture that was the source of inoculum
[5, 29, 51]. Even minor details of the procedure such as the
volume of the culture, shape of the culture vessel, angle of the
test tube, and the radius of gyration of the rotary shaker have an
effect on bacterial physiology [52] and may affect the outcome.
We recommend to record and publish these details to enhance
reproducibility of experiments.
10. Bactericidal activity of antibiotics is concentration dependent,
which is manifested by minimum bactericidal activity (MBC)
values and concentration-dependent killing curves. By defini-
tion, persisters can survive high concentrations of antibiotics
for prolonged times. Therefore, high concentrations of anti-
biotics, at least 10 above the MIC, must be used to quantify
the persister fraction [17]. Applying antibiotics at low concen-
trations, those close to MIC, characterizes antibiotic tolerance
of the bulk of sensitive cells in a population, which should not
be confused with the high-level antibiotic tolerance of persis-
ters [53]. The antibiotic concentrations listed in this protocol
have been routinely used in numerous papers in the field.
11. Aeration has a strong effect on bacterial physiology and survival
of bacterial cells [52]. In this protocol, cultures are shaken
during the antibiotic treatment, while typical MBC measure-
ments are carried out in standing liquid cultures. If antibiotics
are added to smaller aliquots taken from a culture, it is impor-
tant whether these samples are further aerated or not.
12. Serial dilutions can be made into individual test tubes but it
is convenient and common to use 96-well microtiter plates.
96-Well plates allow dilution of cultures using either a multi-
channel pipette or a slot pin replicator. In our lab, we routinely
use a 5 μL slot 12-pin replicator strip (VP 451S5; VP Scien-
tific, Inc., San Diego, CA, USA) for making serial dilutions in
the following setup.
(a) 100 μL samples of culture are transferred to the wells of the
first (A) row of a microtiter plate.
(b) All wells of the other rows are filled with 95 μL of the
culture medium.
Measuring Persister Levels in E. coli 37](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-54-320.jpg)
![round, and perceived that his red silk umbrella, which he had laid
aside when he had seated himself by Lenny, was within arm's reach.
Possessing himself of this treasure, he soon expanded its friendly
folds. And thus doubly fortified within and without, under the shade
of the umbrella, and his pipe composedly between his lips, Dr.
Riccabocca gazed on his own incarcerated legs, even with
complacency.
'He who can despise all things,' said he, in one of his native
proverbs, 'possesses all things!'—if one despises freedom, one is
free! This seat is as soft as a sofa! I am not sure, he resumed,
soliloquizing, after a pause, I am not sure that there is not
something more witty than manly and philosophical in that national
proverb of mine which I quoted to the fanciullo, that there are no
handsome prisons! Did not the son of that celebrated Frenchman,
surnamed Bras de Fer, write a book not only to prove that
adversities are more necessary than prosperities, but that among all
adversities a prison is the most pleasant and profitable?[17] But is
not this condition of mine, voluntarily and experimentally incurred, a
type of my life? Is it the first time that I have thrust myself into a
hobble?—and if in a hobble of mine own choosing, why should I
blame the gods?
Upon this, Dr. Riccabocca fell into a train of musing so remote from
time and place, that in a few minutes he no more remembered that
he was in the Parish Stocks, than a lover remembers that flesh is
grass, a miser that mammon is perishable, a philosopher that
wisdom is vanity. Dr. Riccabocca was in the clouds.
CHAPTER X.
The dullest dog that ever wrote a novel (and, entre nous, reader—
but let it go no farther—we have a good many dogs among the
fraternity that are not Munitos),[18] might have seen with half an eye
that the Parson's discourse had produced a very genial and](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-66-320.jpg)
![out of the church, exchanged looks with each other; that ominous
conjunction between Squire and man chilled back all the effects of
the Parson's sermon. The Squire struck his cane violently into the
ground. I would rather you had told me Black Bess had got the
glanders. A young gentleman, coming to visit my son, struck and
insulted in Hazeldean; a young gentleman—'sdeath, sir, a relation—
his grandmother was a Hazeldean. I do believe Jemima's right, and
the world's coming to an end! But Leonard Fairfield in the Stocks!
What will the Parson say? and after such a sermon! 'Rich man,
respect the poor!' And the good widow too; and poor Mark, who
almost died in my arms. Stirn, you have a heart of stone! You
confounded, lawless, merciless miscreant, who the deuce gave you
the right to imprison man or boy in my parish of Hazeldean without
trial, sentence, or warrant? Run and let the boy out before any one
sees him: run, or I shall.—The Squire elevated the cane, and his
eyes shot fire. Mr. Stirn did not run, but he walked off very fast. The
Squire drew back a few paces, and again took his wife's arm. Just
wait a bit for the Parson, while I talk to the congregation. I want to
stop 'em all if I can, from going into the village; but how?
Frank heard, and replied readily—
Give 'em some beer, sir.
Beer! on a Sunday! For shame, Frank! cried Mrs. Hazeldean.
Hold your tongue, Harry. Thank you, Frank, said the Squire, and
his brow grew as clear as the blue sky above him. I doubt if
Riccabocca could have got him out of his dilemma with the same
ease as Frank had done.
Halt there, my men—lads and lasses too—there, halt a bit. Mrs.
Fairfield, do you hear?—halt! I think his reverence has given us a
capital sermon. Go up to the Great House all of you, and drink a
glass to his health. Frank, go with them; and tell Spruce to tap one
of the casks kept for the haymakers. Harry, [this in a whisper] catch
the Parson, and tell him to come to me instantly.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-69-320.jpg)
![These nests are procurable twice every year; the best are found in
deep, damp caves, which, if not injured, will continue to produce
indefinitely. It was once thought that the caves near the sea-coast
were the most productive; but some of the most profitable yet
found, are situated fifty miles in the interior. This fact seems to be
against the opinion, that the nests are composed of the spawn of
fish, or of bêche-de-mer.
The method of procuring these nests is not unattended with danger.
Some of the caves are so precipitous, that no one, but those
accustomed to the employment from their youth, can obtain the
nests, being only approachable by a perpendicular descent of many
hundred feet, by ladders of bamboo and rattan, over a sea rolling
violently against the rocks. When the mouth of the cave is attained,
the perilous task of taking the nests must often be performed by
torch-light, by penetrating into recesses of the rock, where the
slightest slip would be instantly fatal to the adventurers, who see
nothing below them but the turbulent surf, making its way into the
chasms of the rock—such is the price paid to gratify luxury.
After the nests are obtained, they are separated from feathers and
dirt, are carefully dried and packed, and are then fit for the market.
The Chinese, who are the only people that purchase them for their
own use, bring them in junks to this market, where they command
extravagant prices; the best, or white kind, often being worth four
thousand dollars per pecul,[19] which is nearly twice their weight in
silver. The middling kind is worth from twelve to eighteen hundred,
and the worst, or those procured after fledging, one hundred and
fifty to two hundred dollars per pecul. The majority of the best kind
are sent to Pekin, for the use of the court.
It appears, therefore, that this curious dish is only an article of
expensive luxury among the Chinese; the Japanese do not use it at
all, and how the former people acquired the habit of indulging in it,
is only less singular than their persevering in it.](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-83-320.jpg)
![THE PASSION FOR COLLECTING
BOOKS.
Of all the passions to which the human mind can surrender itself,
there is none more absorbing than the mania of book-collecting. Let
those speak honestly who have indulged in it. It is a species of
bulimia—an insatiable appetite, which grows by what it feeds on. I
have purchased my experience of this matter rather dearly, having at
one period occupied much time, and laid out more money than I like
to think of, in forming a select and curious library. My books formed
my chief solace and amusement during many years of an active and
unprofitable professional life. The pressure of pecuniary difficulties
forced me to part with them, and taught me practically, though not
pleasantly, the vast distinction between buying and selling. It was
something to see placarded in imposing type, Catalogue of the
valuable and select library of a gentleman, containing many rare and
curious editions. But, alas! the sum produced was scarcely a third
of the intrinsic value, and less than half of the original cost. There
have been instances—but they are few and far between—where
libraries have been sold at a premium. Take for an example the
collection of Dr. Farmer, of Emmanuel College, Cambridge, singularly
rich in Shakspearian authorities and black-letter lore, which produced
above £2200, and was supposed to have cost the owner not more
than £500. Many were presents. When you get the character of a
collector, a stray gift often drops in, and scarce volumes find their
way to your shelves, which the quondam owners, uninitiated in
bibliomania, know not the worth of. I once purchased an excellent
copy of the quarto Hamlet, of 1611, of an unsuspecting
bibliopolist, for ten shillings; my conscience smote me, but the
temptation was irresistible.[20] The best copy in existence of the
Caxtonian edition of Gower's De Confessione Amantis, fol., 1483,](https://image.slidesharecdn.com/2610556-250703102139-3b157202/85/Bacterial-Persistence-Methods-And-Protocols-Jan-Michiels-Maarten-Fauvart-85-320.jpg)
Bacterial Persistence Methods And Protocols Jan Michiels Maarten Fauvart
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- 5. Bacterial Persistence Jan Michiels Maarten Fauvart Editors Methods and Protocols Methods in Molecular Biology 1333
- 6. M E T H O D S I N M O L E C U L A R B I O L O G Y Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK For further volumes: http://www.springer.com/series/7651
- 8. Bacterial Persistence Methods and Protocols Edited by Jan Michiels and Maarten Fauvart Department of Microbial and Molecular Systems, KU Leuven - University of Leuven, Heverlee, Belgium
- 9. Editors Jan Michiels Department of Microbial and Molecular Systems KU Leuven - University of Leuven Heverlee, Belgium ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-2853-8 ISBN 978-1-4939-2854-5 (eBook) DOI 10.1007/978-1-4939-2854-5 Library of Congress Control Number: 2015945939 Springer New York Heidelberg Dordrecht London # Springer Science+Business Media New York 2016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper Humana Press is a brand of Springer Springer Science+Business Media LLC New York is part of Springer Science+Business Media (www.springer.com) Maarten Fauvart Department of Microbial and Molecular Systems KU Leuven - University of Leuven Heverlee, Belgium
- 10. Preface Antibiotic treatment often fails to clear chronic infections, even in the absence of clinically detectable resistance. This is largely due to the so-called persister cells that can survive exposure to high concentrations of bactericidal antibiotics. It is generally accepted that persisters are responsible for the relapse of infections by notorious pathogens such as Mycobacterium tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella Typhimurium, and Candida albicans. Persisters typically make up only a small part of a cell population. They result from a temporary switch to a state that is insensitive to killing by antibiotics. Their rare and transient nature has long hampered the experimental study of persisters. This volume brings together the most respected researchers in the field of bacterial persistence. It presents a comprehensive collection of methods that have been instrumental to our current understanding of the topic and will likely remain so for years to come. Heverlee, Belgium Jan Michiels Maarten Fauvart v
- 12. Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I INTRODUCTION 1 A Historical Perspective on Bacterial Persistence . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Natalie Verstraeten, Wouter Knapen, Maarten Fauvart, and Jan Michiels PART II QUANTIFICATION OF PERSISTENCE 2 Persisters: Methods for Isolation and Identifying Contributing Factors—A Review . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 Sarah E. Rowe, Brian P. Conlon, Iris Keren, and Kim Lewis 3 A General Method for Measuring Persister Levels in Escherichia coli Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Niilo Kaldalu, Arvi Jõers, Henri Ingelman, and Tanel Tenson 4 Optimized Method for Measuring Persistence in Escherichia coli with Improved Reproducibility. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 F. Goormaghtigh and L. Van Melderen 5 A Microplate-Based System as In Vitro Model of Biofilm Growth and Quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Ilse Vandecandelaere, Heleen Van Acker, and Tom Coenye 6 Protocol for Determination of the Persister Subpopulation in Candida Albicans Biofilms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Katrijn De Brucker, Kaat De Cremer, Bruno P.A. Cammue, and Karin Thevissen PART III SINGLE CELL ANALYSIS OF PERSISTER CELLS 7 Quantitative Measurements of Type I and Type II Persisters Using ScanLag . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Irit Levin-Reisman and Nathalie Q. Balaban 8 Analyzing Persister Physiology with Fluorescence-Activated Cell Sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83 Mehmet A. Orman, Theresa C. Henry, Christina J. DeCoste, and Mark P. Brynildsen 9 Single-Cell Detection and Collection of Persister Bacteria in a Directly Accessible Femtoliter Droplet Array. . . . . . . . . . . . . . . . . . . . . . . . . . 101 Ryota Iino, Shouichi Sakakihara, Yoshimi Matsumoto, and Kunihiko Nishino vii
- 13. PART IV IDENTIFICATION OF PERSISTER MUTANTS AND GENES 10 A Whole-Cell-Based High-Throughput Screening Method to Identify Molecules Targeting Pseudomonas Aeruginosa Persister Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 Veerle Liebens, Valerie Defraine, and Maarten Fauvart 11 Functional Analysis of the Role of Toxin–Antitoxin (TA) Loci in Bacterial Persistence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121 Aaron T. Butt and Richard W. Titball 12 Experimental Evolution of Escherichia coli Persister Levels Using Cyclic Antibiotic Treatments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Bram Van den Bergh, Joran E. Michiels, and Jan Michiels PART V CELLULAR AND ANIMAL MODEL SYSTEMS FOR STUDYING PERSISTENCE 13 In Vitro Models for the Study of the Intracellular Activity of Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Julien M. Buyck, Sandrine Lemaire, Cristina Seral, Ahalieyah Anantharajah, Frédéric Peyrusson, Paul M. Tulkens, and Françoise Van Bambeke 14 A Murine Model for Escherichia coli Urinary Tract Infection . . . . . . . . . . . . . . . 159 Thomas J. Hannan and David A. Hunstad 15 Analysis of Macrophage-Induced Salmonella Persisters. . . . . . . . . . . . . . . . . . . . . 177 Robert A. Fisher, Angela M. Cheverton, and Sophie Helaine 16 Population Dynamics Analysis of Ciprofloxacin-Persistent S. Typhimurium Cells in a Mouse Model for Salmonella Diarrhea . . . . . . . . . . . 189 Patrick Kaiser, Roland R. Regoes, and Wolf-Dietrich Hardt PART VI MATHEMATICAL MODELING OF PERSISTENCE 17 Computational Methods to Model Persistence. . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 Alexandra Vandervelde, Remy Loris, Jan Danckaert, and Lendert Gelens Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 viii Contents
- 14. Contributors AHALIEYAH ANANTHARAJAH Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium NATHALIE Q. BALABAN The Racah Institute of Physics, The Hebrew University of Jerusalem, Jerusalem, Israel MARK P. BRYNILDSEN Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA; Department of Molecular Biology, Princeton University, Princeton, NJ, USA AARON T. BUTT Department of Infection and Immunity, Medical School, University of Sheffield, Sheffield, UK JULIEN M. BUYCK Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium; Focal Area Infection Biology, Biozentrum, University of Basel, Basel, Switzerland BRUNO P.A. CAMMUE Centre of Microbial and Plant Genetics (CMPG), KU Leuven – University of Leuven, Leuven, Belgium; Department of Plant Systems Biology, VIB, Ghent, Belgium ANGELA M. CHEVERTON Section of Microbiology, Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK TOM COENYE Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium BRIAN P. CONLON Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, MA, USA JAN DANCKAERT Applied Physics Research Group (APHY), Vrije Universiteit Brussel, Brussels, Belgium KATRIJN DE BRUCKER Centre of Microbial and Plant Genetics (CMPG), KU Leuven – University of Leuven, Leuven, Belgium KAAT DE CREMER Centre of Microbial and Plant Genetics (CMPG), KU Leuven – University of Leuven, Leuven, Belgium; Department of Plant Systems Biology, VIB, Ghent, Belgium CHRISTINA J. DECOSTE Department of Molecular Biology, Princeton University, Princeton, NJ, USA VALERIE DEFRAINE Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and Molecular Systems, KU Leuven – University of Leuven, Leuven, Belgium MAARTEN FAUVART Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and Molecular Systems, KU Leuven – University of Leuven, Leuven, Belgium ROBERT A. FISHER Section of Microbiology, Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK LENDERT GELENS Applied Physics Research Group (APHY), Vrije Universiteit Brussel, Brussels, Belgium; Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA, USA F. GOORMAGHTIGH Laboratoire de Génétique et Physiologie Bactérienne, IBMM, Faculté des Sciences, Université Libre de Bruxelles (ULB), Gosselies, Belgium ix
- 15. THOMAS J. HANNAN Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MI, USA WOLF-DIETRICH HARDT Institute of Microbiology, Eidgenössische Technische Hochschule ETH, Zurich, Switzerland SOPHIE HELAINE Section of Microbiology, Medical Research Council Centre for Molecular Bacteriology and Infection, Imperial College London, London, UK THERESA C. HENRY Department of Molecular Biology, Princeton University, Princeton, NJ, USA; Rutgers Robert Wood Johnson Medical School, Piscataway, NJ, USA DAVID A. HUNSTAD Department of Pediatrics and Molecular Microbiology, Washington University School of Medicine, St. Louis, MI, USA RYOTA IINO Okazaki Institute for Integrative Bioscience, Institute for Molecular science, National Institutes of Natural Science, Aichi, Japan; Department of Functional Molecular Science, School of Physical Science, The Graduate University of Advanced Studies (SOKENDAI), Kanagawa, Japan HENRI INGELMAN Institute of Technology, University of Tartu, Tartu, Estonia ARVI JÕERS Institute of Technology, University of Tartu, Tartu, Estonia PATRICK KAISER Institute of Microbiology, Eidgenössische Technische Hochschule ETH, Zurich, Switzerland NIILO KALDALU Institute of Technology, University of Tartu, Tartu, Estonia IRIS KEREN Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, MA, USA WOUTER KNAPEN Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and Molecular Systems, KU Leuven – University of Leuven, Leuven, Belgium SANDRINE LEMAIRE Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium; GSK Biologicals, Rixensart, Belgium IRIT LEVIN-REISMAN The Racah Institute of Physics, The Hebrew University of Jerusalem, Jerusalem, Israel KIM LEWIS Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, MA, USA VEERLE LIEBENS Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and Molecular Systems, KU Leuven – University of Leuven, Leuven, Belgium REMY LORIS Structural Biology Research Center, VIB, Brussels, Belgium; Structural Biology Brussels, Department of Biotechnology (DBIT), Vrije Universiteit Brussel, Brussels, Belgium YOSHIMI MATSUMOTO Laboratory of Microbiology and Infectious Diseases, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan JAN MICHIELS Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and Molecular Systems, KU Leuven – University of Leuven, Leuven, Belgium JORAN E. MICHIELS Centre of Microbial and Plant Genetics (CMPG), KU Leuven – University of Leuven, Leuven, Belgium KUNIHIKO NISHINO Laboratory of Microbiology and Infectious Diseases, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan MEHMET A. ORMAN Department of Chemical and Biological Engineering, Princeton University, Princeton, NJ, USA x Contributors
- 16. FRÉDÉRIC PEYRUSSON Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium ROLAND R. REGOES Institute of Integrative Biology, Eidgenössische Technische Hochschule ETH, Zurich, Switzerland SARAH E. ROWE Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, MA, USA SHOUICHI SAKAKIHARA Technical Division, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan CRISTINA SERAL Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium; Department of Microbiology, Hospital Clı́nico Universitario Lozano Blesa, Zaragoza, Spain TANEL TENSON Institute of Technology, University of Tartu, Tartu, Estonia KARIN THEVISSEN Centre of Microbial and Plant Genetics (CMPG), KU Leuven – University of Leuven, Leuven, Belgium RICHARD W. TITBALL Biosciences, College of Life and Environmental Sciences, University of Exeter, Exeter, UK PAUL M. TULKENS Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium HELEEN VAN ACKER Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium FRANÇOISE VAN BAMBEKE Pharmacologie cellulaire et moléculaire, Louvain Drug Research Institute, Université catholique de Louvain, Brussels, Belgium BRAM VAN DEN BERGH Centre of Microbial and Plant Genetics (CMPG), KU Leuven – University of Leuven, Leuven, Belgium L. VAN MELDEREN Laboratoire de Génétique et Physiologie Bactérienne, IBMM, Faculté des Sciences, Université Libre de Bruxelles (ULB), Gosselies, Belgium ILSE VANDECANDELAERE Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium ALEXANDRA VANDERVELDE Structural Biology Research Center, VIB, Brussels, Belgium; Structural Biology Brussels, Department of Biotechnology (DBIT), Vrije Universiteit Brussel, Brussels, Belgium NATALIE VERSTRAETEN Centre of Microbial and Plant Genetics (CMPG), Department of Microbial and Molecular Systems, KU Leuven – University of Leuven, Leuven, Belgium Contributors xi
- 20. Chapter 1 A Historical Perspective on Bacterial Persistence Natalie Verstraeten, Wouter Knapen, Maarten Fauvart, and Jan Michiels Abstract Bactericidal antibiotics quickly kill the majority of a bacterial population. However, a small fraction of cells typically survive through entering the so-called persister state. Persister cells are increasingly being viewed as a major cause of the recurrence of chronic infectious disease and could be an important factor in the emergence of antibiotic resistance. The phenomenon of persistence was first described in the 1940s, but remained poorly understood for decades afterwards. Only recently, a series of breakthrough discoveries has started to shed light on persister physiology and the molecular and genetic underpinnings of persister formation. We here provide an overview of the key studies that have paved the way for the current boom in persistence research, with a special focus on the technological and methodological advances that have enabled this progress. Keywords: Persisters, Persistence, Antibiotic tolerance, Dormancy, Antibiotics, Review 1 The Early Days The first report on the survival of a small fraction of streptococci cells following treatment with penicillin dates from 1942 [1]. Two years later, Joseph Bigger established that addition of penicil- lin to staphylococci does not result in complete sterilization of all cells in a clonal population. One out of a million cells survived even prolonged treatment with antibiotics. He appropriately named the surviving cells persisters [2]. More recently, it was shown that in most bacterial species, the majority of cells are efficiently killed by relatively low concentrations of bactericidal antibiotics. However, killing shows a biphasic pattern and beyond a certain threshold, further increasing the concentration of the antibacterial does not result in complete clearing of the culture (Fig. 1) [3]. For 40 years following its discovery, the persistence phenome- non was largely neglected, at least by molecular geneticists. This was partly due to the fact that the clinical relevance of persister Jan Michiels and Maarten Fauvart (eds.), Bacterial Persistence: Methods and Protocols, Methods in Molecular Biology, vol. 1333, DOI 10.1007/978-1-4939-2854-5_1, © Springer Science+Business Media New York 2016 3
- 21. cells was not clear. In contrast, the threat posed by inherited antibiotic resistance was generally recognized, adding incentives to resistance research. The problem was compounded by technical challenges that inevitably accompany the study of a transient phenotype that is associated with only a very small fraction of cells. A breakthrough discovery came in the early 1980s, from research carried out by Harris Moyed during a sabbatical leave in the lab of Alexander Tomasz [4]. Mutagenesis of Escherichia coli populations with ethyl methanesulfonate (EMS) led to the identification of three highly persistent (hip) mutants exhibiting 10–10,000-fold increased persister fractions upon incubation with penicillin [4, 5]. Moyed’s pioneering work led to the identifi- cation of two mutants hit in the hipA locus that up until now remains the best studied persister gene [6–10]. Furthermore, because of their increased persister fraction, hipA mutants have frequently been used as a tool in persistence research. Crucially and for the first time, hipA mutants enabled the direct observation of persister cells. Using a combination of microfluidics and live cell microscopy, Nathalie Balaban recorded how persisters survived killing by antibiotics through dormancy and subsequent resuscita- tion [11]. In addition, the hipBA locus is a representative for other toxin– antitoxin (TA) loci that are now intensively studied in rela- tion to persistence. TA modules consist of a stable toxin, typically targeting essential cellular functions, and an unstable antitoxin, Fig. 1 Illustration of persistence: The majority of cells in a bacterial culture are efficiently killed by relatively low concentrations of antibiotics. However, beyond a certain threshold, a killing plateau is observed as only persister cells remain viable. When regrown in fresh medium, the surviving cells generate a population as sensitive to the antibiotic as the original population 4 Natalie Verstraeten et al.
- 22. which counteracts the activity of its cognate toxin [12, 13]. TA systems were originally identified on plasmids, where they play a role in plasmid maintenance; yet a significant number of TA loci are chromosomally encoded and these have been implicated in persis- tence [14]. Examples include RelE [6], MqsR [15–17], TisB [18, 19], MazF [20], and YafQ [21]. Interestingly, with the notable exception of Salmonella persisters residing within macrophage vacuoles [22], deletion of a single toxin generally does not affect persistence. This can partly be explained by redundancy of TA systems in most bacteria. Deletion of multiple TA systems, on the other hand, causes a decrease in E. coli persistence [23]. 2 The Rise of Persistence Research Following the discovery of hipA, persistence as a field of study steadily gained attention. This was partly due to the acknowledge- ment of its clinical significance (summarized by [24]). In 1944, Bigger already alluded to the role of persisters in the resuscitation of chronic infections [2]. Decades later, Kim Lewis postulated that persisters might contribute to the recalcitrance of biofilm infections [25, 26]. This is of particular interest as biofilms are known to withstand antibiotic treatment, thereby causing chronic infections [27]. Subsequently, mathematical modeling demonstrated that persistence could extend the duration of antibiotic treatment, thereby causing treatment failure and promoting the emergence of resistance [28]. Finally, two studies have unambiguously demon- strated that prolonged antimicrobial therapy selects for high- persistence strains of Candida albicans during candidiasis and of Pseudomonas aeruginosa during cystic fibrosis lung infections [29, 30]. In addition, the role of persister cells in the development of resistance is becoming increasingly clear [31]. Apart from providing incentives to further intensify persistence research, these findings also promoted the search for anti-persister therapies. At present, several strategies have been described, but their in vivo effectiveness remains to be investigated. Examples include the use of resonant activation [32], electrochemical currents [33], cadaverine [34], metabolites [35, 36], antimicrobial peptides [37], brominated furanones [38–41], and activated ClpP [42] (summarized by [43]). Apart from increased interest due to the clinical importance of persistence, the development of novel techniques also caused per- sistence research to boom. An overview of these novel techniques is provided below. 2.1 Screening Approaches Over the years, several screening procedures have been developed that led to the identification of persister genes. In a first approach, a non-redundant E. coli knockout library was screened for mutants Historical Perspective on Bacterial Persistence 5
- 23. with altered persistence [44]. Persister cells of individual mutants were quantified by treating a stationary-phase culture with oflox- acin and plating the surviving cells on agar medium containing amdinocillin. As the number of spontaneous amdinocillin-resistant mutants is a fraction of the original number of cells, this obviates the need for dilution steps and greatly reduces the laborious task of screening several thousands of strains. A second screening approach employed a P. aeruginosa plaspo- son knockout library. Individual mutants were grown until station- ary phase and treated with either ofloxacin to kill non-persister cells or water, the latter serving as a control. Subsequently, samples were diluted and incubated in an automated plate reader (Bioscreen C, Oy Growth Curves Ab Ltd), allowing the optical density of 200 samples to be measured simultaneously as a function of time. Given the linear relationship between the number of cells in an inoculum and the lag phase, this allowed for the selection of mutants displaying altered persister levels [45]. Both screenings led to the identification of a number of interesting persister genes including some global regulators. In addition, not a single mutant lacking persisters was identified. As a general conclusion, these screenings therefore provided evidence pointing to the multiplicity of persister formation mechanisms. In a final approach, a random overexpression library was generated in E. coli. Cells from the recombinant library were pooled and logarithmically growing cultures of library clones were exposed to multiple rounds of exposure to ampicillin. This led to the enrich- ment of mutants with increased probability of persister formation and ultimately to the identification of glpD as a genuine persister gene [46]. 2.2 Single-Cell Studies As persistence is a phenotypic trait expressed in only a subfraction of a population, advances in single-cell research signified an era of vast new possibilities. First used by the Balaban group [11], transparent microfluidic devices proved instrumental for microscopic examina- tion of persister cells [47–49]. The strength of this technique lies in the possibility to monitor individual cells for prolonged periods of time while adapting growth conditions. For example, normal growth conditions can be alternated with antibiotic treatments in order to kill non-persister cells. This allows to pinpoint persister cells surviving treatment. Subsequently, the history of persister cells can be traced back through the recorded images. Several studies have used this technique to demonstrate preexisting heterogeneity in bacterial populations [11], to characterize the dormant state of single persister cells [47], to monitor persister formation following administration of indole [48], and to correlate high TA expression to cessation of growth [49]. Also developed by the Balaban group, a colony-appearance assay was elaborated to quantify single-cell persister lag phases [50]. 6 Natalie Verstraeten et al.
- 24. Experiments demonstrated that a threshold concentration of toxin molecules is required for induction of persistence. A major drawback of microfluidic devices, or more precisely of microscopy, is the limited number of cells that can be studied simultaneously. This can be circumvented by using flow cytometry, allowing thousands or even millions of cells to be evaluated in a high-throughput manner. A shortcoming of this technique is the inability to continuously monitor individual cells over time. None- theless, flow cytometry has been successfully used to study the kinetics of persister awakening [51]. In addition, while Bigger postulated that persisters are in a dormant, nondividing state [2], flow cytometry has been used to demonstrate that dormancy is not a requirement for entry into the persister state [52]. Finally, a recent study performed by the Holden group showed how to characterize the dynamics of intracellular bacterial replication at the single-cell level. They used a fluorescence dilution technique to quantify the number of replication cycles of internalized Salmonella [53]. This showed the existence of different Salmonella subpopulations in bone marrow-derived macrophages including a non-replicating but metabolically active subpopulation, comprising the persister cells, possibly capable of resuming growth and causing relapsing infections [22]. Similarly, the Bumann group exploited a DsRed variant called TIMERbac , which spontaneously changes color from green to green/orange over time, as a dynamic growth rate reporter to identify persister cells in vivo [54]. 2.3 Transcriptomics Insight into global transcriptional changes in persister cells came from several elegant studies by the Lewis group. To enrich for persisters, all three approaches conveniently employed the meta- bolic inactivity of these cells. In the first report, logarithmically growing populations of the high-persistence E. coli mutant hipA7 [4] were treated with ampicillin, thereby lysing non-persister cells. Isolated RNA was enriched for mRNA, labeled, and hybridized to E. coli GeneChips [6]. Similarly, gene expression profiling of persisters was performed after treating an exponentially growing population of Mycobacterium tuberculosis with D-cycloserine and collecting surviving persister cells by centrifugation. Transcriptome analysis was performed by microarray hybridization [55]. The third study followed a slightly different approach. It was based on the assumption that persisters are dormant cells with low levels of protein synthesis and corresponding low levels of rRNA tran- scription. E. coli persister cells were isolated by linking the rrnB promoter to a gene encoding an unstable fluorescent protein. In so doing, persister cells are dim as compared to normal cells in the population, which allows for the isolation of persisters using fluorescence-activated cell sorting (FACS). cDNA was prepared from purified RNA and hybridized to spotted E. coli DNA micro- arrays [56]. Historical Perspective on Bacterial Persistence 7
- 25. Based on the studies cited above, stress response pathways as well as TA loci were shown to be highly expressed in isolated persister cells. On the other hand, biosynthetic functions including energy production were downregulated [6, 55, 56]. 2.4 Experimental Evolution The use of experimental evolution for elucidating antibacterial resistance mechanisms is a widely used method. A recent study by the Balaban group used this technique for enriching a population with persisters by repeated exposure of a bacterial population to high concentrations of antibiotics. This resulted in evolved strains showing very high persister fractions caused by fixed specific genetic mutations. The increased survival appeared to be the result of an adjustment in the single-cell lag-time distribution, which was correlated with the extent of the antibiotic exposure interval [57]. They implemented the ScanLag method, which allows the simulta- neous measurement of lag times of hundreds of cells [58]. These findings resulted in a new theory regarding persister cells and their ability to adapt to high doses of drugs called tolerance by lag. 2.5 Modeling Apart from these wet lab techniques, mathematical modeling has provided interesting insights [28, 59–62] (summarized by [63]). Briefly, two main strategies can be discerned: the first one relies on estimating the switching rates between persister and non-persister growth states and assumes this process to take place continuously and stochastically (e.g., [11, 28, 59, 61, 64]). The balance between both switching rates provides a straightforward way to model a given persister level, although ignoring exactly what determines the switching rates. The second strategy focuses on the molecular mechanisms of persister formation by TA systems, with the ratio of (free) toxin over antitoxin ultimately determining, at the single-cell level, the decision to switch to the persister state (e.g., [50, 60, 65, 66]). A crucial factor in this type of models is the generation of phenotypic bistability at the population level, typically requiring noisy gene expression and noise amplification through positive feedback mechanisms [67]. Both modeling strategies have their merits, and until a more integrated approach is presented, the choice between both will depend on the goal and specific focus of the study at hand. Mathematical modeling of persistence poses several advantages. Experiments that are not feasible in the lab can be simulated to predict the outcome. It also allows to explain empirically observed persister levels in terms of the parameters encompassed by the model, and why varying some parameters has more impact on persistence than others. Consequently, evolutionary forces that shape persister levels can be identified, which should help to devise strategies to avoid high persister levels emerging in the clinic. 8 Natalie Verstraeten et al.
- 26. 3 State of the Art and Future Perspectives Recently, the field of microbial persistence research has exploded, as evidenced by a host of publications in top-tier journals [10, 22, 23, 35, 48–50, 54, 68–70]. Currently, it is generally accepted that persister cells are present in a bacterial population preceding antibi- otic treatment [71]. It is postulated that their formation results from noisy gene expression [72] as was first suggested by Kim Lewis [6]. However, over the years, several stimuli have been shown to induce persistence. For example, sub-inhibitory concen- trations of fluoroquinolones are known to induce persistence via activation of the tisAB/istR TA locus [18, 19]. Other examples include quorum sensing molecules [73, 74], carbon source transi- tions [70], and nutrient deprivation leading to activation of the stringent response [75]. As was earlier described for HipA [76], a recent model ascribes TA-regulated persistence to stochastic fluctuations in cellular concentrations of the alarmone (p)ppGpp. High (p)ppGpp levels activate TA loci through a regulatory cascade requiring inorganic polyphosphate and Lon protease targeting protein toxins [49]. For an elaborate discussion on the role of these mechanisms in persistence, the reader is referred to some excellent reviews on the topic [3, 77–81]. Adding to the significance of these studies is the recent obser- vation of a phenotypically distinct subpopulation of transiently drug-tolerant persisters in cancer cell populations. These cells are held responsible for “fractional killing” upon chemotherapy [82]. Cell-to-cell variations in protein levels were suggested to contribute to this phenomenon in which each round of therapy kills some but not all of the cells in a tumor [83]. There is a striking analogy between bacterial and cancer cell-derived persistence as both phe- nomena reflect a transiently phenotypic heterogeneity causing mul- tidrug tolerance and recurrence of disease symptoms upon removal of treatment [84]. Added insight into bacterial persistence may therefore impact research areas far beyond infectious disease. Acknowledgements Research in the lab of JM is supported by grants from KU Leuven Research Council (PF/10/010; IDO/09/010; IDO/13/008), IAP-BELSPO, FWO, and IWT. References 1. Hobby GL, Meyer K, Chaffee E (1942) Obser- vations on the mechanism of action of penicil- lin. Exp Biol Med 50(2):281–285 2. Bigger JW (1944) Treatment of staphylococcal infections with penicillin. Lancet 244:497–500 3. Lewis K (2010) Persister cells. Annu Rev Microbiol 64:357–372 4. Moyed HS, Bertrand KP (1983) hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after Historical Perspective on Bacterial Persistence 9
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- 32. Part II Quantification of Persistence
- 34. Chapter 2 Persisters: Methods for Isolation and Identifying Contributing Factors—A Review Sarah E. Rowe, Brian P. Conlon, Iris Keren, and Kim Lewis Abstract Persister cells are phenotypic variants surviving a lethal dose of antibiotic, sufficient to kill the bulk of an exponential phase population. In this chapter we summarize current techniques to isolate persisters and discuss limitations associated with identifying mechanisms of persister formation. Keywords: Hip mutant, Toxin-antitoxin (TA) modules, Antibiotic, Biofilm 1 Introduction Persisters were first described in 1944 by Joseph Bigger, a medical doctor from Trinity College Dublin [1]. Bigger reported that addition of a lethal dose of penicillin to a population of Staphylococ- cus aureus yielded a small subpopulation of surviving cells. Upon reinoculation, these colonies grew into a culture that once again lysed in the presence of penicillin and again yielded a subpopulation of survivors. Bigger referred to these surviving cells as persisters [1]. These cells are contrary to resistant cells as they cannot grow in the presence of the antibiotic. The proportion of persisters in a population varies dramatically depending on growth phase and environmental conditions [2, 3]. Specifically, persister numbers are greatly increased in stationary phase and biofilms [4, 5]. Persisters can be enumerated by adding a lethal concentration of a bactericidal antibiotic to a population of cells. The bulk of the population dies rapidly and the surviving persister cells either die much more slowly or do not die during the experiment (Fig. 1). The persister fraction can be enumerated at various time intervals by removing an aliquot, washing with 1 % NaCl, and plating serial dilutions for colony counting. The surviving persister fraction emerges in a clear biphasic curve in response to the antibiotic. Jan Michiels and Maarten Fauvart (eds.), Bacterial Persistence: Methods and Protocols, Methods in Molecular Biology, vol. 1333, DOI 10.1007/978-1-4939-2854-5_2, © Springer Science+Business Media New York 2016 17
- 35. The time period where the persister fraction remains constant is referred to as the “persister plateau” (Fig. 1). The majority of research has concentrated on persisters in Escherichia coli but persisters have been shown to be produced by many other bacterial species including Pseudomonas aeruginosa, Mycobacterium tuberculosis, Salmonella enterica serovar typhimur- ium, Staphylococcus aureus, and Streptococcus mutans Burkholderia cepacia and even in the fungal pathogen Candida albicans [6–13]. 2 High-Persister (hip) Mutants Although persisters were discovered in the 1940s, they were largely ignored until the early 1980s when Harris Moyed and co-workers isolated high-persistence (hip) mutants [14, 15]. They identified the gene hipA, which was the first gene known to contribute to persister formation; this gene was later identified as the toxin of an antitoxin/toxin (TA) module. The hipA7 allele of the gene pro- duced 1000 times more persisters to two classes of antibiotics: beta- lactams and fluoroquinolones [16]. This was the first indication that some persisters are multidrug tolerant. Importantly the hipA7 mutant has the same minimal inhibitory concentration (MIC) as the parent strain [16], indicating that the increased level of persis- ters is not due to acquisition of resistance. Time [h] Log CFU/ml 3 6 Antibiotic added here 108 Persister plateau Drug susceptible cells Resistant mutant Fig. 1 Typical biphasic kill curve of a bacterial population in exponential phase of growth: A culture of E. coli grown to mid-exponential phase is challenged with 10 MIC of a bactericidal antibiotic (such as ciprofloxa- cin). The majority of cells die rapidly. The persister fraction is enumerated by removing an aliquot of cells after 3 and 6 h of exposure to the antibiotic. Cells are washed with 1 % NaCl and serial dilutions are plated for counting. Persister cells differ from resistant cells as they can tolerate the antibiotic but cannot grow in its presence 18 Sarah E. Rowe et al.
- 36. Hip mutants can be identified in vitro by generating a mutant library and exposing it to several rounds of a lethal dose of an antibiotic [17]. The bacterial mutant library can be obtained either by chemical mutagenesis (ethyl methanesulfonate) [18] or by trans- poson mutagenesis [19]. Following several rounds of antibiotic challenge, the genetic basis for hip mutant phenotypes is identified by subjecting surviving cells to whole-genome sequencing or micro-array-based genetic footprinting [17]. Persisters represent transient phenotypic variants that are genetically identical to their drug-susceptible kin. Such traits have proved taxing for in vivo investigations as these persisters tend to resuscitate. Mulcahy et al. took a different approach [20]. They hypothesized that if hip mutants had an advantage then they will be selected for in vivo during infection or in response to antibiotic treatment. Longitudinal isolates of Pseudomonas aeruginosa were obtained from patients throughout the course of a cystic fibrosis infection. The study demonstrated that strains developed a hip phenotype over the course of the infection (Fig. 2). A similar finding was observed in Candida albicans with patients suffering from chronic oral thrush [21]. These studies indicate that persisters are clinically relevant and may contribute to treatment failures. 0.8 0.00001 0.0001 0.001 0.01 0.1 1 AMT47 AMT60 AMT66 AMT71 AMT74 AMT76 AMT33 AMT41 AMT75 AMT73 AMT62 AMT36 AMT101 AMT100 7.3 3 7.7 9.1 19.6 19.6 13.2 5.6 12.8 23.4 2.3 9.6 8.6 21.1 15.4 4.42 13.1 6.7 9.6 10.8 Patient Number and age (years old) when strain was isolated Percent Survival 1 1.1 7.1 9.2 7.2 15.4 15.2 Fig. 2 A screen of P. aeruginosa clinical isolates for hip mutants: Stationary-phase cultures of clonal early/late isolate pairs from 14 patients were exposed to ofloxacin (50 MIC) for 8 h, and the surviving cells were determined by colony count and expressed as percent survival (n ¼ 4). Early isolates are indicated with white bars, while late isolates are indicated with black bars. The patient number and age at which the tested isolates were obtained are displayed on the x-axis. A hip mutant emerged in 10 of the 14 patients. A hip mutant did not emerge in the isolates from the last four patients displayed on the right side of the graph. Taken from Mulcahy et al. 2010 Persisters: Methods for Isolation and Identifying Contributing Factors—A Review 19
- 37. 3 Pre-existing and Induced Persisters The frequency of persisters in hip strains is much higher than in the wild-type E. coli which facilitates research into the mechanism of persister formation. The hipA7 mutant was used for single-cell time-lapse microscopy, which showed that persisters are slow or non-growing cells, stochastically formed and pre-existing in the population [22]. In order to distinguish between growing and non-growing cells, Shah et al. used an unstable GFP variant driven from a ribo- somal promoter [23]. Cells expressing this plasmid were grown to mid-exponential phase and analyzed by fluorescent-activated cell sorting (FACS) (Fig. 3). Two distinct populations were visualized using forward light scatter, one that fluoresced brightly and one that did not. Cells from both populations were sorted and visualized by epifluorescent microscopy and challenged with ofloxacin. The cells that did not express GFP were much more likely to survive antibiotic challenge. This study described a new mechanism for identifying persisters and demonstrated that non-growing cells exist in an untreated E. coli population [23]. These results suggest that there is a population of persisters that are pre-existing in the population and are not formed in a response to antibiotic treatment. In support of this, it has been demonstrated that an E. coli culture, if diluted several times and challenged with either ampicil- lin or ofloxacin, displays a gradual decline and eventual elimination 104 103 102 101 100 0 64 R3 R4 128 Forward Scatter Green Fluorescence Log % Survival Non-Persisters Persisters 192 256 Bar = 5µm 2 1.6 1.2 0.8 0.4 0 Phase Contrast a b c Green Fluorescence Fig. 3 Isolation of persister cells from an exponentially growing culture. E. coli cells containing a degradable GFP reporter cassette were grown in LB medium to mid-exponential phase (~1 108 cells/ml) at 37 C with aeration and sorted using a high-speed cell sorter equipped with a standard GFP filter set. (a) Two populations were detected using forward light scatter, one that fluoresced brightly (R3), and another that did not (R4). (b) The sorted populations were visualized by epifluorescent microscopy (bar, 5 μm). (c) Cells were sorted as described in (a–b). Once sorted both populations were treated with ofloxacin (5 μg/ml) for 3 h, diluted, and spotted onto LB agar plates for colony counts. Taken from Shah et al. 2006 20 Sarah E. Rowe et al.
- 38. of persisters, even though the initial population size is kept constant [2]. This indicates that persisters are formed later in the growth phase, accumulate in stationary phase, and can be diluted out with serial reinoculation. Johnson et al. showed that pretreatment of S. aureus with sub- MIC concentrations of a particular antibiotic significantly increases the level of multidrug-tolerant persisters [24]. From these results, the authors conclude that persister formation is the product of various errors during cell replication. These errors result in tran- sient periods of slowed metabolism and/or non-replication by individual cells in growing populations [24]. Although there is mounting evidence to suggest that persisters are pre-existing and formed stochastically throughout growth, there is also data to support that persisters can be induced in response to antibiotics [24, 25] and environmental stress [26]. Dorr et al. demonstrated that pretreatment of E. coli with low levels of ciprofloxacin induced the formation of persisters to higher doses of ciprofloxacin (Fig. 4). This study shows that the majority of persisters to ciprofloxacin are induced in response to that antibiotic and this is dependent on a functional SOS response [25]. Later work has demonstrated that persisters can also be formed as a response to an environmental stress such as DNA damage or oxida- tive stress [26] and the stringent response [3, 27, 28]. 4 Toxin Antitoxins (TA) Modules The first transcriptome of persisters was generated by taking advan- tage of the fact that β-lactam antibiotics such as ampicillin are bacteriolytic, causing lysis of dying cells [29]. An E. coli culture was treated with a high concentration of ampicillin. A simple cen- trifugation step was used to collect the surviving persisters and expression profiles were determined using a microarray. Several TA modules were among the genes induced in persisters. TA genes were first identified as addiction modules that play a role in plasmid maintenance [30]. The toxin and antitoxin genes make up an operon that is transcribed from the same promoter. In the case of type II TA systems, the toxin and antitoxins are proteins which bind together and often repress transcription of the operon [31]. The antitoxin is less stable and is degraded by cellular pro- teases; if a daughter cell loses the plasmid, the antitoxin is rapidly degraded and the toxin prevents cell growth [30]. TAs were subse- quently discovered on bacterial chromosomes. Many studies have implicated chromosomal TAs to have a role in persistence which will be discussed in this section [14, 16, 32–35]. The hipA gene of the hipBA operon is the probably the most widely studied persister gene. It encodes the toxin HipA which is neutralized by its cognate antitoxin HipB. Under cellular stress, the Persisters: Methods for Isolation and Identifying Contributing Factors—A Review 21
- 39. Lon protease degrades HipB, releasing HipA and allowing it to exert its toxic effects on the cell [35]. A recent study revealed that HipA is a kinase that phosphorylates an aminoacyl-tRNA synthe- tase, halts protein synthesis, and induces persister formation [36]. There are 11 TA mRNA interferases in E. coli K12, which are induced under various cellular stresses [3, 37–41]. Upon overexpression, these toxins have been shown to cleave Fig. 4 Ciprofloxacin-induced persistence: (a) Survival of wild-type cells in exponential phase under different ciprofloxacin regimes. Two cultures were treated with 0.1 and 1 μg/ml, respectively, for 6 h. Third culture was treated with 0.1 μg/ml for 3 h after which 1 μg/ml was added (indicated by an arrow). The data are averages of three independent experiments and error bars indicate standard error. (b) Wild-type cells in exponential phase were treated for 3 h with increasing concentrations of ciprofloxacin indicated on x-axis. After the initial treatment, an additional 1 μg/ml of ciprofloxacin was added to the cultures and incubated for another 3 h as in (a). (Ciprofloxacin MIC is 0.05 μg/ml.) As a control, a parallel culture was exposed to 1 μg/ml for the duration of the experiment. Bars represent the viability at 0, 3, and 6 h of time course equivalents shown in (a). Open bars: The initial viability count. Grey bars: The viability after 3-h incubation with ciprofloxacin concentration indicated on the x-axis. Full bars: The final viability count after additional 3-h incubation with 1 μg/ml ciprofloxacin. Dashed bars: Viability of the control culture at 3 and 6 h. The data are averages of three independent experiments and error bars indicate standard error. Taken from Dorr et al. 2009 22 Sarah E. Rowe et al.
- 40. cellular mRNA and induce persister formation. However, single mutations of these TA modules did not result in a reduction of persister levels [34]. Redundancy could explain the lack of a per- sister phenotype but nevertheless this led to some debate as to their role in persister formation. Kenn Gerdes and co-workers serially deleted up to ten of the recognized type II TA modules (Δ10) in E. coli [34]. They reported that at least five modules need to be deleted before a significant reduction in persister levels is observed. A progressive reduction in persister levels was observed when more TA modules were deleted, and the Δ10 strain displayed a 100-fold reduction in persister formation. These results demonstrate the importance of TA mod- ules in persister formation and their high degree of redundancy for one another. It is also a possibility that an individual TA locus may play a role in persister formation in a specific strain or under specific environ- mental conditions. Norton et al. reported that a single knockout of the TA module PasTI had no phenotype when deleted in E. coli lab strain MG1655 but displayed a 100-fold reduction in persisters when deleted in a clinical isolate CFT073 [42]. This study high- lights the limitations of studying the mechanisms of persister for- mation in lab strains under lab conditions [42]. Similarly, Helaine et al. reported that internalization of Salmo- nella by macrophages induced persister formation over 100-fold [43]. Importantly, the group identified two stress conditions encountered in vivo, vacuolar acidification and nutritional depriva- tion, which induce persister formation through the 14 TA modules. Together these studies emphasize the need to study persistence in an environment that closely resembles in vivo conditions. 5 Genetic Determinants of Persisters The standard approach for identifying genes which contribute to a function is to avail of a mutant library and generate a screen. This does not work well when redundant genes contribute to the same function, as is the case of the TA modules. An alternative method is to create an over-expression library and screen for gain-of-function phenotypes. In this case, even a mild contributor to the phenotype can be identified. However, this method can be problematic for persisters as overproduction of many proteins, particular membrane proteins, can result in toxic protein aggregates. This can cause growth cessation and artificially emulate an antibiotic tolerant state. Spoering et al. overexpressed a library in a low-copy vector, using native promoters for expression and challenged with a lethal dose of ampicillin [44]. In an attempt to exclude false positives the authors introduced a growth step in between rounds of antibiotic selection. Any strains that grew considerably slower than the wild Persisters: Methods for Isolation and Identifying Contributing Factors—A Review 23
- 41. type would be selected against. A particular clone, with elevated levels of persisters tolerant to ampicillin, carried the gene glpD, involved in glycerol metabolism. Interestingly, overexpression of glpD also induced persisters tolerant to ofloxacin suggesting that these persisters are multidrug tolerant. Deletion of the glpD gene in a wild-type background reduced levels of persisters to ciprofloxacin in stationary phase. Recent work has suggested a mechanism by which the glpD mutant has increased levels of methylglyoxal, a bacteriostatic metabolite [17]. A screen for novel persister genes utilized the Keio collection [45], an ordered deletion library of all 3985 nonessential genes in E. coli. Strains were grown to stationary phase in 96-well plates and exposed to lethal concentration of ofloxacin [46]. This screen identified 150 mutants with decreased levels of persisters. Ten mutants were confirmed to display a decreased level of persisters to ofloxacin with an unchanged MIC. Most of these genes had mutations in global regulators, all of which had modest effects on persister levels [46]. Global regulators RpoS and RelA were also identified to have a role in P. aeruginosa persistence [6, 28, 47]. An additional nine genes were identified by a high-throughput screen of an incomplete P. aeruginosa transposon mutant library [48]. Despite these many screens, a mutant with a complete lack of persisters has not been identified. The mutants identified to date can display altered levels of persisters to one or more class of drugs. This strongly suggests that there is not a central mechanism for persister formation and persisters surviving different antibiotic treatments are not identical. It is more likely that persisters can be formed through different mechanisms and there is a degree of cross talk between these mechanisms that can lead multidrug-tolerant persisters. 6 The Importance of Persisters Many bacteria form biofilms in response to environmental stress [49]. Biofilms are a surface-attached agglomeration of cells encased in an exopolymeric and proteinaceous matrix. While most antibio- tics can penetrate through this matrix, biofilms do protect the cells from the immune response and can therefore complicate treatment of an infection leading to persistent and chronic infection [50]. It was demonstrated that biofilms of P. aeruginosa harbor persister cells tolerant to antibiotics [7, 27] and there is evidence to suggest that this complicates treatment for cystic fibrosis patients [20]. A similar role was recently suggested for the granuloma in MTB infection, acting as a physical barrier protecting persisters from the immune system [8]. Kim Lewis suggested a model (Fig. 5) explaining the recalcitrant nature of biofilm infection [51]. When 24 Sarah E. Rowe et al.
- 42. a biofilm infection is treated with antibiotics, the majority of the bacterial cells die and only persisters survive. In the biofilm, persis- ters are protected from the immune system, and upon removal of the antibiotic pressure they can repopulate the biofilm matrix. The combination of persisters and biofilm is the likely culprit of relaps- ing chronic infections, with persister cells providing protection from antibiotics while the biofilm provides protection from the immune system. Antibiotic resistance represents a huge problem, particularly with gram-negative bacteria [52, 53]. However, many bacterial strains isolated from chronic infections are susceptible to the anti- biotics they were treated with [5, 20]. This indicates that persis- tence rather than resistance may be a key contributor to the recalcitrance of some infections. With this in mind, a search for novel antibiotics which target dormant or stationary cells rather than fast-growing cells could dramatically reduce treatment failures. Bactericidal antibiotics target and corrupt active processes in cells which are inactive in persister cells, allowing them to survive the antibiotic treatment. Acyldepsipeptides (ADEPs) are a new class of antibiotics discovered by Eli Lily in 1985. Brotz-Oesterhelt et al. found that ADEP targets the ClpP protease, opening the proteo- lytic core resulting in ATP-independent proteolysis [54]. Despite impressive efficacy in a variety of animal models of acute infection, development of the drug was not pursued due to high frequency of resistance (1 106 ) due to null mutation of the nonessential clpP gene. Fig. 5 A model of a relapsing biofilm infections. Regular cells and persister cells form in the biofilm and are shed off into surrounding tissue and the bloodstream. Antibiotics kill regular cells, and the immune system eliminates escaping persister cells. The matrix protects persister cells from the immune system, and when the concentration of the antibiotic drops, they repopulate the biofilm, causing a relapse. Taken from Lewis et al. 2010 Persisters: Methods for Isolation and Identifying Contributing Factors—A Review 25
- 43. Importantly, ADEP activates and dysregulates ClpP in an ATP-independent manner [54]. This suggested that ADEP could potentially target cells in a low-energy state and may be active against non-growing cells. Conlon et al. recognized the unique ability of the drug to target persister cells [5]. They reported that although a stationary population of S. aureus is highly tolerant to a range of antibiotics in vitro it remains susceptible to ADEP. However, following an initial death phase, the high frequency of resistant mutants allowed the culture to rebound and repopulate. ADEP paired with rifampicin resulted in eradication of the population due to increased antibiotic susceptibility of the ADEP4-resistant clpP null mutants. Importantly, the authors were able to sterilize a deep-seated murine thigh infection model of S. aureus. This study highlights the importance of discovering new antimicrobials that specifically target persister cells. References 1. Bigger JW (1944) Treatment of staphylococcal infections with penicillin. Lancet ii:497–500 2. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K (2004) Persister cells and tolerance to anti- microbials. FEMS Microbiol Lett 230 (1):13–18 3. Maisonneuve E, Castro-Camargo M, Gerdes K (2013) (p)ppGpp controls bacterial persistence by stochastic induction of toxin-antitoxin activ- ity. Cell 154(5):1140–1150. doi:10.1016/j. cell.2013.07.048 4. Amato SM, Brynildsen MP (2014) Nutrient transitions are a source of persisters in Escher- ichia coli biofilms. PLoS One 9(3), e93110. doi:10.1371/journal.pone.0093110 5. Conlon BP, Nakayasu ES, Fleck LE, LaFleur MD, Isabella VM, Coleman K, Leonard SN, Smith RD, Adkins JN, Lewis K (2013) Activated ClpP kills persisters and eradicates a chronic biofilm infection. Nature 503(7476):365–370. doi:10.1038/ nature12790 6. Moker N, Dean CR, Tao J (2010) Pseudomo- nas aeruginosa increases formation of multidrug-tolerant persister cells in response to quorum-sensing signaling molecules. J Bac- teriol 192(7):1946–1955. doi:10.1128/JB. 01231-09 7. Spoering AL, Lewis K (2001) Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicro- bials. J Bacteriol 183(23):6746–6751. doi:10. 1128/JB.183.23.6746-6751.2001 8. Keren I, Minami S, Rubin E, Lewis K (2011) Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2 (3):e00100–e00111. doi:10.1128/mBio. 00100-11 9. Slattery A, Victorsen AH, Brown A, Hillman K, Phillips GJ (2013) Isolation of highly persis- tent mutants of Salmonella enterica serovar Typhimurium reveals a new toxin-antitoxin module. J Bacteriol 195(4):647–657. doi:10. 1128/JB.01397-12 10. Lechner S, Lewis K, Bertram R (2012) Staphylococcus aureus persisters tolerant to bac- tericidal antibiotics. J Mol Microbiol Biotech- nol 22(4):235–244. doi:10.1159/000342449 11. Leung V, Levesque CM (2012) A stress- inducible quorum-sensing peptide mediates the formation of persister cells with noninher- ited multidrug tolerance. J Bacteriol 194 (9):2265–2274. doi:10.1128/JB.06707-11 12. LaFleur MD, Kumamoto CA, Lewis K (2006) Candida albicans biofilms produce antifungal- tolerant persister cells. Antimicrob Agents Chemother 50(11):3839–3846 13. Van Acker H, Sass A, Bazzini S, De Roy K, Udine C, Messiaen T, Riccardi G, Boon N, Nelis HJ, Mahenthiralingam E, Coenye T (2013) Biofilm-grown Burkholderia cepacia complex cells survive antibiotic treatment by avoiding production of reactive oxygen species. PLoS One 8(3), e58943. doi:10.1371/jour nal.pone.0058943 14. Moyed HS, Bertrand KP (1983) hipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis. J Bacteriol 155 (2):768–775 26 Sarah E. Rowe et al.
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- 46. Chapter 3 A General Method for Measuring Persister Levels in Escherichia coli Cultures Niilo Kaldalu, Arvi Jõers, Henri Ingelman, and Tanel Tenson Abstract Genetically homogeneous bacterial cultures contain persisters, cells that are not killed by bactericidal antibiotics. These cells are suggested to be involved in the establishment of chronic infections. Persister levels depend on growth conditions. Here, we discuss the parameters that have to be considered when measuring persister levels and provide a sample protocol to do it. Keywords: Antibiotic, Persister, Antibiotic tolerance, Ampicillin, Fluoroquinolones 1 Introduction In 1944, Joseph Bigger studied bactericidal action of penicillin on staphylococcal cultures. He found that some bacteria are able to evade killing and regrow after the treatment. Dr. Bigger called these cells persisters and either described or predicted several basic features that define such surviving minority. Besides, he concluded that persisters must be in part responsible for the limited success of antibiotic treatment against infections [1]. Seventy years later, the main conclusions of his research have been confirmed and elaborated by many laboratories using several different bacterial species. It is amazing how many of Bigger’s early observations hold true, including the complaint that “irregularities and incon- sistencies occurred in almost all experiments of this type” [1]. Below, we review principal concepts of persister research and try to look how much some of the researchers’ findings depend on experimental setup. 1.1 Phenotypic Tolerance vs. Phenotypic Resistance Persisters are defined as bacteria that are tolerant to bactericidal antibiotics; they are able to survive the drug treatment but are not able to grow in the presence of the drug [1, 2]. If bacteria grow in the presence of the drug, they are called resistant. Jan Michiels and Maarten Fauvart (eds.), Bacterial Persistence: Methods and Protocols, Methods in Molecular Biology, vol. 1333, DOI 10.1007/978-1-4939-2854-5_3, © Springer Science+Business Media New York 2016 29
- 47. The phenomenon of persistence is phenotypic, a culture that was started from clonal descendants of a persister cell contains persisters at the same frequency as the original parental culture [1, 3]. Over several years, persister cells have been considered as non- replicating cells [2]. According to a central concept of the field, the non-growing, i.e., nondividing, state is crucial to persistence. Bacterial cultures contain individual cells that stay non-replicating in conditions enabling growth [4–6]. These cells can survive treat- ment with antibiotics and, if they resume growth after the removal of the drug, these bacteria are called persisters (Fig. 1a). It is a matter of debate if such heterogeneity of the population is a survival strategy [7–9] or reflects different extent of unavoidable damage to the cells [10]. Recently, it has been reported that some persisters might be actively proliferating: in Mycobacterium, the surviving subset of bacteria can have highly active efflux pumps [11] or express 5 6 7 8 9 0 2 4 6 persisters growth curve 1 3 5 7 0 1 2 3 4 5 Time (h) a b Time (h) AMP OFL TypeII TypeI log 10 CFU/ml log 10 CFU/ml Fig. 1 Graphic presentation of results from persister experiments. (a). Typical killing curves of cultures treated with antibiotics. Killing curves represent kinetics of killing of bacteria during a bactericidal treatment. Cultures of E. coli MG1655 were grown and treated as described in this chapter. Briefly, both the overnight culture and the test culture were grown in filter-sterilized LB broth. At the beginning of the experiment, overnight culture was diluted 1000 and incubated on a shaker at 37 C for 3 h. Then, ampicillin, 100 μg/mL (AMP; dashed line) or ofloxacin, 5 μg/mL (OFL; solid line) was added and incubation was continued. Killing curves show that the number of living microbes decreased more than 1000 during the first hour of antibiotic treatment. That drop is caused by killing of the bulk of phenotypically sensitive bacteria. Starting from the 1-h time point, CFU numbers decrease more slowly and indicate the number of persisters in the culture. The values represent the means of five independent AMP treatments and three independent OFL treatments. The error bars indicate the standard deviation. (b). The number of persisters increases when a growing culture approaches stationary phase. An overnight culture of E. coli BW25113 was diluted 1:100 and cultured with aeration at 37 C. At the designated time points, samples were taken and treated with ciprofloxacin (1 μg/mL) for 5 h. Closed symbols, cell count before the treatment (growth curve); open symbols, cell count after the ciprofloxacin treatment (persister count). During the first 4 h, the number of persisters does not increase. In this stage, persisters are the cells that have been in the non-proliferating state since inoculation (type I persisters). After the 4-h time point, the number of persister increases rapidly and newly formed (type II) persisters make up the majority of surviving cells. The values are averages of three replicates and the error bars indicate the standard deviation 30 Niilo Kaldalu et al.
- 48. catalase-peroxidase, which is necessary for killing by isoniazid, in stochastic pulses [12]. In these instances, surviving cells are repli- cating, not quiescent. It has also been suggested that some of the persisters in E. coli are actively dividing [6]. The concept of actively growing persisters raises terminological discrepancies. Resistance is usually determined genetically but still, the genetic component is not part of the definition. Mechanisms of phenotypic antibiotic resistance have been described recently [13, 14] and occurrence of noncanonical, phenotypic resistance due to the growth conditions and metabolic state of bacteria is well known to the microbiological community [15]. The mycobacterial “dynamic persistence” [11, 12] would fall under phenotypic resistance as well. Replicative dormancy of persisters does not necessarily mean the lack of metabolic activity. It is intuitively reasonable to suggest, that “shutdown of a target function” (i.e., cell wall synthesis, protein synthesis, or DNA replication) “will prevent the lethal action” of the drug [16]. However in dormant cells not all the targets are equally inactive. For example, the same persister cells of E. coli log-phase cultures that are tolerant to fluoroquinolones and cell-wall synthesis inhibitors are killed effectively by aminoglyco- sides, a group of antibiotics that targets protein synthesis and causes mistranslation [5, 17, 18]. Such killing is dependent on the mem- brane potential of these cells, which is required for the uptake of aminoglycosides. Membrane potential is created due to the active metabolism and depends on the nutrients provided. Therefore, whether the non-growing subset of bacteria is killed by aminogly- cosides, depends on the carbon source of the growth medium [18]. Consequently, persisters may have at least limited activity of protein synthesis, metabolism, and membrane transport [19]. 1.2 Type I and Type II Persisters Joseph Bigger acknowledged that some bacteria “are in the persister phase when inoculated into fresh medium, but the condi- tion is induced in others by their new environment” [1]. Such distinction defines two groups of persisters that either retain dormancy acquired in the stationary phase (type I) or fall into dormancy during growth (type II) [4]. If antibiotic is supplied at the very moment of inoculation of the culture [5], in the lag phase, or in the early logarithmic phase then the surviving persisters belong to type I. At early stages of growth, the number of persisters does not increase (Fig. 1b). Type I persisters are lost in the course of repeated cycles of dilution and growth into early log phase [3]. Type II persisters are formed when a growing culture starts approaching to the stationary phase. When nutrients are depleting and growth conditions deteriorate, their number is progressively increasing and they make up a great majority of the persister fraction [3] (Fig. 1b). In this state, functional stress response circuits (e.g., ppGpp synthesis) are important for type II persister formation but lack of these stress responses still does not fully stop Measuring Persister Levels in E. coli 31
- 49. formation of new persister cells [20, 21]. In addition to the nutrient depletion, new persister cells are generated in response to many different stress conditions (see below), including low concentra- tions of antibiotics [22–24]. 1.3 Visualizing and Isolating Persisters The cells with different growth rates can be labeled with various fluorescent reporters. The reporter can either manifest the physio- logical activity of the cell [25] or cell division rate that dilutes the fluorescent protein [26, 27]. Cell division and potential dormancy can be observed directly in a microfluidics system under the micro- scope [4, 28]. Alternatively, the cell population can be analyzed either by flow cytometry [5, 25, 27, 29] or by monitoring growth of plated cells [30]. For Mycobacterium it has been reported that cell divisions can be followed from the loss of a very unstable plasmid [11, 31]. For analyzing the macromolecular content of persisters it is important to physically isolate this fraction of the population. Lysis with antibiotics [3, 16, 32] or with alternative agents [33] has been suggested in the literature as a method to eliminate growing cells. However, it has to be noted that these methods pool the cells fulfilling the persister definition with the cells that will never be able to grow again (see Subheading 1.4). The nonrecovering population can be considerably higher than the persister population [5, 6, 27]. Alternatively, cell sorting can be used for isolating bacteria in different physiological states [6, 25]. Here three complications arise. First, the amount of material that can be isolated is much smaller compared to the methods based on cell lysis. Second, and more importantly, the physiological state of the bacteria might be altered by the sorting procedure. Bacteria are exposed to high pressure during sorting and carefully planned experiments are needed to control for dormancy induction by the sorting procedure. Finally, separation of persisters from perma- nently nondividing cells remains unresolved. Density gradient centrifugation has been used for fractionating an E. coli culture into subpopulations [34, 35]. Although more than 15 fractions have been described, the connection with the phenomenon of persisters has not been demonstrated [35]. 1.4 Persisters and VBNC It has to be noted that not all nondividing cells resume growth in the usual detection window of 1 or 2 days. In many cases, the cells preserve membrane integrity and are called viable but non- culturable (VBNC) [27, 36]. It is currently not clear how many of these cells might finally resume growth as slow wakeup over several months has been described [37]. 1.5 Stationary-Phase Persisters Most of the bactericidal antibiotics are ineffective against stationary-phase cultures. Certain fluoroquinolones, e.g., ofloxa- cin, ciprofloxacin, and gatifloxacin against E. coli, are an exception. 32 Niilo Kaldalu et al.
- 50. If the stationary-phase cells are diluted into the fresh growth medium and antibiotic is supplied at the same moment, it must be noted that most of the cells resume growth and become antibi- otic sensitive soon after dilution. Type I persisters, not stationary- phase persisters, are assayed in such experiments [5]. For counting stationary-phase persisters, antibiotics have to be supplied directly to undiluted non-growing (i.e., stationary phase) cultures, which are in fact much less sensitive to antibiotic treatment [24, 38]. 1.6 Are Persisters Tolerant to Different Antibiotics? High-persistence (hip) mutants were isolated from the original screens for E. coli mutants with altered persister frequency [39]. Later, it was found that although the screens were made with ampicillin, increased persister frequency was observed also against fluoroquinolone antibiotics and vice versa [40]. This suggested that persisters are equally tolerant to many or all antibiotics. Current results suggest that the mechanism of antibiotic action considerably influences the persister levels and different (but probably overlap- ping) populations survive different antibiotics [24, 41, 42]. 1.7 Growth Inhibition Induces Persistence Bigger already showed that growth inhibition, either by suboptimal temperature, nutrient limitation, or bacteriostatic agents, induces persister formation [1]. In the pioneering work for finding out the molecular mechanisms of persister formation, Moyed was looking for mutants with altered persister levels. For avoiding indirect effects, attempts were made to find mutants with no changes in minimum inhibitory concentration (MIC) and growth parameters [39]. Indeed, it was demonstrated later that overexpression of various toxic proteins can induce increased persister levels [43]. This calls for cautious interpretation of several results where the growth of bacteria is inhibited or the culture is stressed, as these might not reveal the biologically relevant mechanisms of persister formation. The examples include screening for mutants with altered persister levels, expression of toxins from toxin-antitoxin systems [11, 44–47], manipulations with ppGpp levels [44], treat- ing the bacteria with sub MIC antibiotic concentrations [24], etc. In addition, all strains and mutants with increased MIC values should be compared with care [38]. Even cell sorting by flow cytometry [6, 25, 27] might induce persisters from actively grow- ing cells. 1.8 Introduction to the Current Protocol As discussed above, different labs often use different protocols for the measurement of persister levels. Because the measurement output is sensitive to conditions, there are several parameters that are important to control. Here we have assembled a protocol that contains the most commonly used steps and have highlighted the essential parameters. Measuring Persister Levels in E. coli 33
- 51. 2 Materials Prepare all media using deionized water. Store liquid media at room temperature and agar plates at 4 C, agar side up, in closed bags. 1. LB medium: Dissolve premixed powder for LB broth in water according to the manufacturer’s instructions (see Note 1). Filter-sterilize (see Note 2). Prepare fresh and use within 1 week. 2. LB agar plates: Prepare LB medium as described in step 1, and add agar to a final concentration of 1.5 % (w/v). Pour powder on the top of water in a beaker, stir to dissolve the soluble components, bring to boil in a microwave oven, and boil for 1 min to dissolve agar. Transfer by 200 mL into 250 mL flasks; autoclave at 121 C for 15 min. Allow to cool to ~55 C and pour plates. Pour ~20 mL of LB agar per 10 cm polystyrene Petri dish (see Note 3). 3. Phosphate-buffered saline (PBS): For 1 L of 10 PBS, dissolve 2 g KCl, 2.4 g KH2PO4, 80 g NaCl, and 11.45 g Na2HPO4 (see Note 4) in 800 mL water, and fill up to 1000 mL. Dilute 10 in water to prepare 1 PBS. Autoclave and store at room temperature. 4. Stock solutions of antibiotics (see Note 5). (a) Ampicillin: Dissolve ampicillin sodium salt in water at a concentration of 100 mg/mL. Do not freeze and melt. Store at 4 C for up to 1 week (see Note 6). (b) Ciprofloxacin, norfloxacin, gatifloxacin, ofloxacin: All these fluoroquinolone antibiotics are soluble in 0.1 M NaOH. Dissolve, dilute with water to a concentration of 5 mg/mL, aliquot, and freeze. Store at 20 C for up to 3 months. 5. Dimethyl sulfoxide (DMSO). 3 Methods We provide a procedure for characterization of persistence of E. coli, which is suitable for comparison of different isolates and strains. Altering incubation times and temperatures, media for growth of the inoculum and the test culture, aeration regimens, and other experimental details have an impact on persister formation and must explicitly stated when publishing results. 3.1 Preparation of E. coli Culture DMSO Stocks We recommend starting overnight cultures from frozen DMSO stocks. This helps to standardize cultures and reduce variability between individual experiments (see Note 7). 34 Niilo Kaldalu et al.
- 52. 1. Inoculate a test tube containing 3 mL LB medium with cells from a freshly grown colony on an LB agar plate. Grow on a shaker at 37 C overnight (see Note 8). 2. Make a 100 dilution of the overnight culture into 20 mL of LB medium. Incubate on a shaker at 37 C. Sample over time to measure the optical density at λ ¼ 600 nm. 3. When the OD600 of the culture has reached ~0.6, add DMSO to 8 % (v/v) by mixing 9.2 mL of the culture with 0.8 mL of DMSO. Dispense in 100 μl aliquots in cluster tubes and store frozen at 80 C. DMSO stocks can be stored for up to 3 months. 3.2 Growing Overnight Cultures for Inoculum A standardized procedure for preparation of inocula makes experi- ments reproducible and is required for consistent results (see Note 9). 1. Transfer 3 mL of filter-sterilized LB medium into a sterile test tube. 2. Melt an aliquot of the DMSO stock and use 50 μL for starting a culture. DMSO stocks should not be refrozen and reused. 3. Grow on a shaker at 220 rpm and 37 C for 16 h. 3.3 Growing Experimental Cultures and Performing Antibiotic Treatments 1. Transfer 20 mL of filter-sterilized LB medium into a sterile 100 mL Erlenmeyer flask. 2. Inoculate with 20 μL of overnight culture (1000 dilution). Put on a shaker at 220 rpm and 37 C. 3. Incubate for 3 h, and then take a 100 μL pretreatment sample of the culture to determine the number of culturable bacteria at the start of the antibiotic treatment. 4. Add antibiotic solution to the culture. Use ampicillin at a concentration of 100 μg/mL, ciprofloxacin, gatifloxacin, norfloxacin, and ofloxacin at concentrations of 5 μg/mL (see Note 10). If using antibiotic stock solutions listed in Subheading 2, add 20 μL of an antibiotic solution. Continue incubation on a shaker at 220 rpm and 37 C (see Note 11). 5. Make serial dilutions of the pretreatment sample. Use LB medium for dilution. Alternatively, serial dilutions can be made using sterile PBS or 0.9 % solution of NaCl (see Note 12). 6. Plate dilutions onto LB agar plates (see Note 13). Bacteria can be either spot plated or spread plated (see Note 14). 7. Place plates at 37 C for overnight incubation (see Note 15). 8. If you treat your cultures with ampicillin (Amp) or any other cell-wall synthesis inhibitor, take 100 μL samples 1, 2, 3, and 5 h after addition of the drug. Make serial dilutions just like you did of the pretreatment sample and plate. Incubate at 37 C for 24 h (see Note 15) and count colonies. Measuring Persister Levels in E. coli 35
- 53. 9. If you treat your cultures with fluoroquinolones (ciprofloxacin, ofloxacin, norfloxacin, gatifloxacin), take 1 mL samples 1, 2, 3, and 5 h after addition of the drug. Spin down the cells in a 1.5 mL test tube for 5 min at 5000 g and room temperature. Remove supernatant and resuspend bacteria in 1 mL LB medium. Repeat centrifugation, remove supernatant, and resuspend cells in 1 mL of fresh PBS. Continue with serial dilutions and plating as in the case of the non-treated and ampicillin-treated samples (see Note 16). Example results of the persister measurement are shown in Fig. 1. Alternatively, the results of antibiotic treatment and growth resumption can be analyzed by flow cytometry (see Note 17). 4 Notes 1. Let the powdered medium hydrate by pouring it slowly on the surface of the water. This avoids clumping. 2. We strongly recommend using filter-sterilized rich media or defined minimal media. Autoclaving causes degradation of the components of rich media and alters the content in an unpredict- able way. This contributes to inconsistency of results [29, 48]. If you use autoclaved media, minimizing experimental error would require using always the same model of autoclave, aliquot- ing identical volumes, loading the same amount of material to autoclave, and sterilizing your media for the same time at the same temperature. 3. LB agar can be allowed to solidify and stored at room tempera- ture. It can be remelted in a microwave oven, allowed to cool to ~55 C, and used for pouring plates. The autoclaved, melted LB agar should not be stored at 55 C for more than a few hours. 4. If using Na2HPO47H2O, take 21.6 g; for Na2HPO412H2O, use 28.8 g. 5. Comprehensive guidelines for preparation and storage of anti- biotic solutions are provided in [49]. 6. Ampicillin solution can be routinely frozen and thawed without any bad consequences if this drug is used for selection of resistant organisms. However, we have seen that repeated freezing and thawing cycles of ampicillin solution can change bactericidal activity [50]. 7. Typically, a clonal culture is started from a single fresh colony. This routine is fully acceptable in the study of persisters. However, it is practically inevitable that such cultures are started from an uncertain number of bacteria of uncertain age. If grown overnight, these cultures may have different physiology at the moment when they are used for inoculation 36 Niilo Kaldalu et al.
- 54. of the test cultures. We have seen that persister frequency depends considerably on exact physiological parameters of the inoculum [29]. DMSO stocks allow starting all overnight cul- tures from a similar number of bacteria, which are in a con- trolled physiological condition, and decrease inconsistency. 8. Regular, autoclaved LB medium may be used for preparation of DMSO stocks. 9. Besides the strain’s genotype, persister frequency depends on the growth medium and temperature, aeration, the fold of dilution of the inoculum, the growth phase of both the test culture, and the culture that was the source of inoculum [5, 29, 51]. Even minor details of the procedure such as the volume of the culture, shape of the culture vessel, angle of the test tube, and the radius of gyration of the rotary shaker have an effect on bacterial physiology [52] and may affect the outcome. We recommend to record and publish these details to enhance reproducibility of experiments. 10. Bactericidal activity of antibiotics is concentration dependent, which is manifested by minimum bactericidal activity (MBC) values and concentration-dependent killing curves. By defini- tion, persisters can survive high concentrations of antibiotics for prolonged times. Therefore, high concentrations of anti- biotics, at least 10 above the MIC, must be used to quantify the persister fraction [17]. Applying antibiotics at low concen- trations, those close to MIC, characterizes antibiotic tolerance of the bulk of sensitive cells in a population, which should not be confused with the high-level antibiotic tolerance of persis- ters [53]. The antibiotic concentrations listed in this protocol have been routinely used in numerous papers in the field. 11. Aeration has a strong effect on bacterial physiology and survival of bacterial cells [52]. In this protocol, cultures are shaken during the antibiotic treatment, while typical MBC measure- ments are carried out in standing liquid cultures. If antibiotics are added to smaller aliquots taken from a culture, it is impor- tant whether these samples are further aerated or not. 12. Serial dilutions can be made into individual test tubes but it is convenient and common to use 96-well microtiter plates. 96-Well plates allow dilution of cultures using either a multi- channel pipette or a slot pin replicator. In our lab, we routinely use a 5 μL slot 12-pin replicator strip (VP 451S5; VP Scien- tific, Inc., San Diego, CA, USA) for making serial dilutions in the following setup. (a) 100 μL samples of culture are transferred to the wells of the first (A) row of a microtiter plate. (b) All wells of the other rows are filled with 95 μL of the culture medium. Measuring Persister Levels in E. coli 37
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- 56. the credit of the chief; but he was inexorable to that worst of diplomatic offenses—an ill-timed, stupid, over-zealous obedience to orders, which, if it established the devotion of the employé, got the employer into what is popularly called a scrape! And though, by those unversed in the intricacies of the human heart, and unacquainted with the especial hearts of prime-ministers and Right- hand men, it might have seemed natural that Mr. Stirn, as he stood still, hat in hand, in the middle of the road, stung, humbled, and exasperated by the mortification he had received from the lips of Randal Leslie, would have felt that that young gentleman was the proper object of his resentment; yet such a breach of all the etiquette of diplomatic life as resentment toward a superior power was the last idea that would have suggested itself to the profound intellect of the Premier of Hazeldean. Still, as rage like steam must escape somewhere, Mr. Stirn, on feeling—as he afterward expressed it to his wife—that his buzzom was a-burstin, turned with the natural instinct of self preservation to the safety-valve provided for the explosion; and the vapor within him rushed into vent upon Lenny Fairfield. He clapped his hat on his head fiercely, and thus relieved his buzzom. You young willain! you howdacious wiper! and so all this blessed Sabbath afternoon, when you ought to have been in church on your marrow bones, a-praying for your betters, you has been a-fitting with a young gentleman, and a wisiter to your master, on the werry place of the parridge hinstitution that you was to guard and pertect; and a-bloodying it all over, I declares, with your blaggard little nose! Thus saying, and as if to mend the matter, Mr. Stirn aimed an additional stroke at the offending member; but Lenny mechanically putting up both his arms to defend his face, Mr. Stirn struck his knuckles against the large brass buttons that adorned the cuff of the boy's coat-sleeve—an incident which considerably aggravated his indignation. And Lenny, whose spirit was fairly roused at what the narrowness of his education conceived to be a signal injustice, placing the trunk of the tree between Mr. Stirn and himself, began that task of self-justification which it was equally impolitic to
- 57. conceive and imprudent to execute, since, in such a case, to justify was to recriminate. I wonder at you, Master Stirn—if mother could hear you! You know it was you who would not let me go to church; it was you who told me to— Fit a young gentleman, and break the Sabbath, said Mr. Stirn, interrupting him with a withering sneer. O yes! I told you to disgrace his honor the Squire, and me, and the parridge, and bring us all into trouble. But the Squire told me to make an example, and I will! With those words, quick as lightning flashed upon Mr. Stirn's mind the luminous idea of setting Lenny in the very Stocks which he had too faithfully guarded. Eureka! the example was before him! Here, he could gratify his long grudge against the pattern boy; here, by such a selection of the very best lad in the parish, he could strike terror into the worst; here he could appease the offended dignity of Randal Leslie; here was a practical apology to the Squire for the affront put upon his young visitor; here, too, there was prompt obedience to the Squire's own wish that the Stocks should be provided as soon as possible with a tenant. Suiting the action to the thought, Mr. Stirn made a rapid plunge at his victim, caught him by the skirt of his jacket, and, in a few seconds more, the jaws of the Stocks had opened, and Lenny Fairfield was thrust therein—a sad spectacle of the reverses of fortune. This done, and while the boy was too astounded, too stupefied by the suddenness of the calamity for the resistance he might otherwise have made—nay, for more than a few inaudible words—Mr. Stirn hurried from the spot, but not without first picking up and pocketing the half-crown designed for Lenny, and which, so great had been his first emotions, he had hitherto even almost forgotten. He then made his way toward the church, with the intention to place himself close by the door, catch the Squire as he came out, whisper to him what had passed, and lead him, with the whole congregation at his heels, to gaze upon the sacrifice offered up to the joint Powers of Nemesis and Themis.
- 58. CHAPTER VII. Unaffectedly I say it—upon the honor of a gentleman, and the reputation of an author, unaffectedly I say it—no words of mine can do justice to the sensations experienced by Lenny Fairfield, as he sat alone in that place of penance. He felt no more the physical pain of his bruises; the anguish of his mind stifled and over-bore all corporeal suffering—an anguish as great as the childish breast is capable of holding. For first and deepest of all, and earliest felt, was the burning sense of injustice. He had, it might be with erring judgment, but with all honesty, earnestness, and zeal, executed the commission intrusted to him; he had stood forth manfully in discharge of his duty; he had fought for it, suffered for it, bled for it. This was his reward! Now, in Lenny's mind there was pre-eminently that quality which distinguishes the Anglo-Saxon race—the sense of justice. It was perhaps the strongest principle in his moral constitution; and the principle had never lost its virgin bloom and freshness by any of the minor acts of oppression and iniquity which boys of higher birth often suffer from harsh parents, or in tyrannical schools. So that it was for the first time that that iron entered into his soul, and with it came its attendant feeling—the wrathful galling sense of impotence. He had been wronged, and he had no means to right himself. Then came another sensation, if not so deep, yet more smarting and envenomed for the time—shame! He, the good boy of all good boys—he, the pattern of the school, and the pride of the parson—he, whom the Squire, in sight of all his contemporaries, had often singled out to slap on the back, and the grand Squire's lady to pat on the head, with a smiling gratulation on his young and fair repute—he, who had already learned so dearly to prize the sweets of an honorable name—he, to be made, as it were, in the twinkling of an eye, a mark for opprobrium, a butt of scorn, a jeer, and a byword! The streams of his life were poisoned at the fountain. And then came a tenderer thought of his mother! of the shock this would be to her—she who had already begun to look up to him as her stay
- 59. and support: he bowed his head, and the tears, long suppressed, rolled down. Then he wrestled and struggled, and strove to wrench his limbs from that hateful bondage; for he heard steps approaching. And he began to picture to himself the arrival of all the villagers from church, the sad gaze of the Parson, the bent brow of the Squire, the idle, ill- suppressed titter of all the boys, jealous of his unblotted character— character of which the original whiteness could never, never be restored! He would always be the boy who had sat in the Stocks! And the words uttered by the Squire came back on his soul, like the voice of conscience in the ears of some doomed Macbeth. A sad disgrace Lenny—you'll never be in such a quandary. Quandary, the word was unfamiliar to him; it must mean something awfully discreditable. The poor boy could have prayed for the earth to swallow him. CHAPTER VIII. Kettles and frying-pans! what has us here? cried the tinker. This time Mr. Sprott was without his donkey; for, it being Sunday, it is to be presumed that the donkey was enjoying his Sabbath on the Common. The tinker was in his Sunday's best, clean and smart, about to take his lounge in the park. Lenny Fairfield made no answer to the appeal. You in the wood, my baby! Well that's the last sight I should ha' thought to see. But we all lives to larn, added the tinker, sententiously. Who gave you them leggins? Can't you speak, lad? Nick Stirn. Nick Stirn! Ay, I'd ha' ta'en my davy on that: and cos vy? 'Cause I did as he told me, and fought a boy as was trespassing on these very Stocks; and he beat me—but I don't care for that; and
- 60. that boy was a young gentleman, and going to visit the Squire; and so Nick Stirn— Lenny stopped short, choked by rage and humiliation. Augh, said the tinker, staring, you fit with a young gentleman, did you? Sorry to hear you confess that, my lad! Sit there, and be thankful you ha' got off so cheap. 'Tis salt and battery to fit with your betters, and a Lunnon justice o' peace would have given you two months o' the treadmill. But vy should you fit cos he trespassed on the Stocks? It ben't your natural side for fitting, I takes it. Lenny murmured something not very distinguishable about serving the Squire, and doing as he was bid. Oh, I sees, Lenny, interrupted the tinker, in a tone of great contempt, you be one o' those who would rayther 'unt with the 'ounds than run with the 'are! You be's the good pattern boy, and would peach agin your own horder to curry favor with the grand folks. Fie, lad! you be sarved right: stick by your horder, then you'll be 'spected when you gets into trouble, and not be 'varsally 'espised —as you'll be arter church-time! Vell, I can't be seen 'sorting with you, now you are in this here drogotary fix; it might hurt my cracter, both with them as built the Stocks, and them as wants to pull 'em down. Old kettles to mend! Vy, you makes me forgit the Sabbath. Sarvent, my lad, and wish you well out of it; 'specks to your mother, and say we can deal for the pan and shovel all the same for your misfortin. The tinker went his way. Lenny's eye followed him with the sullenness of despair. The tinker, like all the tribe of human comforters, had only watered the brambles to invigorate the prick of the thorns. Yes, if Lenny had been caught breaking the Stocks, some at least would have pitied him; but to be incarcerated for defending them, you might as well have expected that the widows and orphans of the Reign of Terror would have pitied Dr. Guillotin when he slid through the grooves of his own deadly machine. And even the tinker, itinerant, ragamuffin vagabond as he was, felt ashamed to be found
- 61. with the pattern boy! Lenny's head sank again on his breast, heavily as if it had been of lead. Some few minutes thus passed, when the unhappy prisoner became aware of the presence of another spectator to his shame: he heard no step, but he saw a shadow thrown over the sward. He held his breath, and would not look up, with some vague idea that if he refused to see he might escape being seen. CHAPTER IX. Per Bacco! said Dr. Riccabocca, putting his hand on Lenny's shoulder, and bending down to look into his face—Per Bacco! my young friend, do you sit here from choice or necessity? Lenny slightly shuddered, and winced under the touch of one whom he had hitherto regarded with a sort of superstitious abhorrence. I fear, resumed Riccabocca, after waiting in vain for an answer to his question, that, though the situation is charming, you did not select it yourself. What is this?—and the irony of the tone vanished —what is this, my poor boy? You have been bleeding, and I see that those tears which you try to check come from a deep well. Tell me, povero fanciullo mio, (the sweet Italian vowels, though Lenny did not understand them, sounded softly and soothingly),—tell me, my child, how all this happened. Perhaps I can help you—we have all erred; we should all help each other. Lenny's heart, that just before had seemed bound in brass, found itself a way as the Italian spoke thus kindly, and the tears rushed down; but he again stopped them, and gulped out sturdily— I have not done no wrong; it ben't my fault—and 'tis that which kills me! concluded Lenny, with a burst of energy. You have not done wrong? Then, said the philosopher, drawing out his pocket handkerchief with great composure, and spreading it on
- 62. the ground—then I may sit beside you. I could only stoop pityingly over sin, but I can lie down on equal terms with misfortune. Lenny Fairfield did not quite comprehend the words, but enough of their general meaning was apparent to make him cast a grateful glance on the Italian. Riccabocca resumed, as he adjusted the pocket-handkerchief, I have a right to your confidence, my child, for I have been afflicted in my day; yet I too say with thee, 'I have not done wrong.' Cospetto! (and here the Dr. seated himself deliberately, resting one arm on the side column of the Stocks, in familiar contact with the captive's shoulder, while his eye wandered over the lovely scene around)—Cospetto! my prison, if they had caught me, would not have had so fair a look-out as this. But, to be sure, it is all one: there are no ugly loves, and no handsome prisons! With that sententious maxim, which, indeed, he uttered in his native Italian, Riccabocca turned round and renewed his soothing invitations to confidence. A friend in need is a friend indeed, even if he come in the guise of a Papist and wizard. All Lenny's ancient dislike to the foreigner had gone, and he told him his little tale. Dr. Riccabocca was much too shrewd a man not to see exactly the motives which had induced Mr. Stirn to incarcerate his agent (barring only that of personal grudge, to which Lenny's account gave him no clew). That a man high in office should make a scape-goat of his own watch-dog for an unlucky snap, or even an indiscreet bark, was nothing strange to the wisdom of the student of Machiavelli. However, he set himself to the task of consolation with equal philosophy and tenderness. He began by reminding, or rather informing, Leonard Fairfield of all the instances of illustrious men afflicted by the injustice of others that occurred to his own excellent memory. He told him how the great Epictetus, when in slavery, had a master whose favorite amusement was pinching his leg, which, as the amusement ended in breaking that limb, was worse than the Stocks. He also told him the anecdote of Lenny's own gallant countryman, Admiral Byng, whose execution gave rise to Voltaire's
- 63. celebrated witticism, En Angleterre on tue un admiral pour encourager les autres. (In England they execute one admiral in order to encourage the others.) Many more illustrations, still more pertinent to the case in point, his erudition supplied from the stores of history. But on seeing that Lenny did not seem in the slightest degree consoled by these memorable examples, he shifted his ground, and reducing his logic to the strict argumentum ad rem, began to prove, 1st, that there was no disgrace at all in Lenny's present position, that every equitable person would recognize the tyranny of Stirn and the innocence of its victim; 2dly, that if even here he were mistaken, for public opinion was not always righteous, what was public opinion, after all? A breath—a puff, cried Dr. Riccabocca, a thing without matter—without length, breadth, or substance—a shadow—a goblin of our own creating. A man's own conscience is his sole tribunal, and he should care no more for that phantom 'opinion' than he should fear meeting a ghost if he cross the church-yard at dark. Now, as Lenny did very much fear meeting a ghost if he crossed the church-yard at dark, the simile spoiled the argument, and he shook his head very mournfully. Dr. Riccabocca was about to enter into a third course of reasoning, which, had it come to an end, would doubtless have settled the matter, and reconciled Lenny to sitting in the Stocks till doomsday, when the captive, with the quick ear and eye of terror and calamity, became conscious that church was over, that the congregation in a few seconds more would be flocking thitherward. He saw visionary hats and bonnets through the trees, which Riccabocca saw not, despite all the excellence of his spectacles—heard phantasmal rustlings and murmurings which Riccabocca heard not, despite all that theoretical experience in plots, stratagems, and treasons, which should have made the Italian's ear as fine as a conspirator's or a mole's. And with another violent but vain effort at escape, the prisoner exclaimed, Oh, if I could but get out before they come! Let me out—let me out. O, kind sir, have pity—let me out!
- 64. Diavolo! said the philosopher, startled, I wonder that never occurred to me before. After all, I believe he has hit the right nail on the head; and looking close, he perceived that though the partition wood had hitched firmly into a sort of spring-clasp, which defied Lenny's unaided struggles, still it was not locked (for, indeed, the padlock and key were snug in the justice-room of the Squire, who never dreamt that his orders would be executed so literally and summarily as to dispense with all formal appeal to himself). As soon as Dr. Riccabocca made that discovery, it occurred to him that all the wisdom of all the schools that ever existed can't reconcile man or boy to a bad position, the moment there is a fair opportunity of letting him out of it. Accordingly, without more ado, he lifted up the creaking board, and Lenny Fairfield darted forth like a bird from a cage—halted a moment as if for breath, or in joy; and then, taking at once to his heels, fled, fast as a hare to its form—fast to his mother's home. Dr. Riccabocca dropped the yawning-wood into its place, picked up his handkerchief, and restored it to his pocket; and then, with some curiosity, began to examine the nature of that place of duresse, which had caused so much painful emotion to its rescued victim. Man is a very irrational animal at best, quoth the sage, soliloquizing, and is frightened by strange buggabooes! 'Tis but a piece of wood!—how little it really injures; and, after all, the holes are but rests to the legs, and keep the feet out of the dirt. And this green bank to sit upon—under the shade of the elm-tree—verily the position must be more pleasant than otherwise! I've a great mind— Here the Doctor looked around, and, seeing the coast still clear, the oddest notion imaginable took possession of him; yet not indeed a notion so odd, considered philosophically—for all philosophy is based upon practical experiment—and Dr. Riccabocca felt an irresistible desire practically to experience what manner of thing that punishment of the Stocks really was. I can but try!—only for a moment, said he, apologetically, to his own expostulating sense of dignity. I have time to do it before any one comes. He lifted up the
- 65. partition again: but Stocks are built on the true principle of English law, and don't easily allow a man to criminate himself—it was hard to get into them without the help of a friend. However, as we before noticed, obstacles only whetted Dr. Riccabocca's invention. He looked round and saw a withered bit of stick under the tree—this he inserted in the division of the Stocks, somewhat in the manner in which boys place a stick under a sieve for the purpose of ensnaring sparrows: the fatal wood thus propped, Dr. Riccabocca sat gravely down on the bank, and thrust his feet through the apertures. Nothing in it! cried he, triumphantly, after a moment's deliberation. The evil is only in idea. Such is the boasted reason of mortals! With that reflection, nevertheless, he was about to withdraw his feet from their voluntary dilemma, when the crazy stick suddenly gave way, and the partition fell back into its clasp. Doctor Riccabocca was fairly caught—Facilis descensus—sed revocare gradum! True, his hands were at liberty, but his legs were so long that, being thus fixed, they kept the hands from the rescue; and as Dr. Riccabocca's form was by no means supple, and the twin parts of the wood stuck together with that firmness of adhesion which things newly painted possess, so, after some vain twists and contortions, in which he succeeded at length (not without a stretch of the sinews that made them crack again) in finding the clasp and breaking his nails thereon, the victim of his own rash experiment resigned himself to his fate. Dr. Riccabocca was one of those men who never do things by halves. When I say he resigned himself, I mean not only Christian but philosophical resignation. The position was not quite so pleasant as, theoretically, he had deemed it; but he resolved to make himself as comfortable as he could. And first, as is natural in all troubles to men who have grown familiar with that odoriferous comforter which Sir Walter Raleigh is said first to have bestowed upon the Caucasian races, the Doctor made use of his hands to extract from his pocket his pipe, match-box, and tobacco-pouch. After a few whiffs he would have been quite reconciled to his situation, but for the discovery that the sun had shifted its place in the heavens, and was no longer shaded from his face by the elm-tree. The Doctor again looked
- 66. round, and perceived that his red silk umbrella, which he had laid aside when he had seated himself by Lenny, was within arm's reach. Possessing himself of this treasure, he soon expanded its friendly folds. And thus doubly fortified within and without, under the shade of the umbrella, and his pipe composedly between his lips, Dr. Riccabocca gazed on his own incarcerated legs, even with complacency. 'He who can despise all things,' said he, in one of his native proverbs, 'possesses all things!'—if one despises freedom, one is free! This seat is as soft as a sofa! I am not sure, he resumed, soliloquizing, after a pause, I am not sure that there is not something more witty than manly and philosophical in that national proverb of mine which I quoted to the fanciullo, that there are no handsome prisons! Did not the son of that celebrated Frenchman, surnamed Bras de Fer, write a book not only to prove that adversities are more necessary than prosperities, but that among all adversities a prison is the most pleasant and profitable?[17] But is not this condition of mine, voluntarily and experimentally incurred, a type of my life? Is it the first time that I have thrust myself into a hobble?—and if in a hobble of mine own choosing, why should I blame the gods? Upon this, Dr. Riccabocca fell into a train of musing so remote from time and place, that in a few minutes he no more remembered that he was in the Parish Stocks, than a lover remembers that flesh is grass, a miser that mammon is perishable, a philosopher that wisdom is vanity. Dr. Riccabocca was in the clouds. CHAPTER X. The dullest dog that ever wrote a novel (and, entre nous, reader— but let it go no farther—we have a good many dogs among the fraternity that are not Munitos),[18] might have seen with half an eye that the Parson's discourse had produced a very genial and
- 67. humanizing effect upon his audience. When all was over, and the congregation stood up to let Mr. Hazeldean and his family walk first down the aisle, (for that was the custom at Hazeldean,) moistened eyes glanced at the Squire's sun-burned, manly face with a kindness that bespoke revived memory of many a generous benefit and ready service. The head might be wrong now and then—the heart was in the right place, after all. And the lady, leaning on his arm, came in for a large share of that gracious good feeling. True, she now and then gave a little offense when the cottages were not so clean as she fancied they ought to be—and poor folks don't like a liberty taken with their houses any more than the rich do; true, that she was not quite so popular with the women as the Squire was, for, if the husband went too often to the alehouse, she always laid the fault on the wife, and said, No man would go out of doors for his comforts, if he had a smiling face and a clean hearth at his home; whereas the Squire maintained the more gallant opinion, that if Gill was a shrew, it was because Jack did not, as in duty bound, stop her mouth with a kiss! Still, notwithstanding these more obnoxious notions on her part, and a certain awe inspired by the stiff silk gown and the handsome aquiline nose, it was impossible, especially in the softened tempers of that Sunday afternoon, not to associate the honest, comely, beaming countenance of Mrs. Hazeldean with comfortable recollections of soups, jellies, and wine in sickness, loaves and blankets in winter, cheering words and ready visits in every little distress, and pretexts afforded by improvement in the grounds and gardens (improvements which, as the Squire, who preferred productive labor, justly complained, would never finish) for little timely jobs of work to some veteran grandsire, who still liked to earn a penny, or some ruddy urchin in a family that came too fast. Nor was Frank, as he walked a little behind, in the whitest of trowsers and the stiffest of neckcloths—with a look of suppressed roguery in his bright hazel eye, that contrasted his assumed stateliness of mien—without his portion of the silent blessing. Not that he had done any thing yet to deserve it; but we all give youth so large a credit in the future. As for Miss Jemima, her trifling foibles only rose from too soft and feminine a susceptibility, too ivy-like a
- 68. yearning for some masculine oak, whereon to entwine her tendrils; and so little confined to self was the natural lovingness of her disposition, that she had helped many a village lass to find a husband, by the bribe of a marriage gift from her own privy purse; notwithstanding the assurances with which she accompanied the marriage gift,—viz., that the bridegroom would turn out like the rest of his ungrateful sex; but that it was a comfort to think that it would be all one in the approaching crash. So that she had her warm partisans, especially among the young; while the slim Captain, on whose arm she rested her forefinger, was at least a civil-spoken gentleman, who had never done any harm, and who would doubtless do a deal of good if he belonged to the parish. Nay, even the fat footman, who came last with the family Prayer-book, had his due share in the general association of neighborly kindness between hall and hamlet. Few were there present to whom he had not extended the right-hand of fellowship, with a full horn of October in the clasp of it: and he was a Hazeldean man, too, born and bred, as two-thirds of the Squire's household (now letting themselves out from their large pew under the gallery) were. On his part, too, you could see that the Squire was 'moved withal,' and a little humbled moreover. Instead of walking erect, and taking bow and courtesy as matter of course, and of no meaning, he hung his head somewhat, and there was a slight blush on his cheek; and as he glanced upward and round him—shyly, as it were—and his eye met those friendly looks, it returned them with an earnestness that had in it something touching as well as cordial—an eye that said, as well as eye could say, I don't quite deserve it, I fear, neighbors; but I thank you for your good-will with my whole heart. And so readily was that glance of the eye understood that I think, if that scene had taken place out of doors instead of in the church, there would have been an hurrah as the Squire passed out of sight. Scarcely had Mr. Hazeldean got well out of the church-yard, ere Mr. Stirn was whispering in his ear. As Stirn whispered the Squire's face grew long, and his color changed. The congregation, now flocking
- 69. out of the church, exchanged looks with each other; that ominous conjunction between Squire and man chilled back all the effects of the Parson's sermon. The Squire struck his cane violently into the ground. I would rather you had told me Black Bess had got the glanders. A young gentleman, coming to visit my son, struck and insulted in Hazeldean; a young gentleman—'sdeath, sir, a relation— his grandmother was a Hazeldean. I do believe Jemima's right, and the world's coming to an end! But Leonard Fairfield in the Stocks! What will the Parson say? and after such a sermon! 'Rich man, respect the poor!' And the good widow too; and poor Mark, who almost died in my arms. Stirn, you have a heart of stone! You confounded, lawless, merciless miscreant, who the deuce gave you the right to imprison man or boy in my parish of Hazeldean without trial, sentence, or warrant? Run and let the boy out before any one sees him: run, or I shall.—The Squire elevated the cane, and his eyes shot fire. Mr. Stirn did not run, but he walked off very fast. The Squire drew back a few paces, and again took his wife's arm. Just wait a bit for the Parson, while I talk to the congregation. I want to stop 'em all if I can, from going into the village; but how? Frank heard, and replied readily— Give 'em some beer, sir. Beer! on a Sunday! For shame, Frank! cried Mrs. Hazeldean. Hold your tongue, Harry. Thank you, Frank, said the Squire, and his brow grew as clear as the blue sky above him. I doubt if Riccabocca could have got him out of his dilemma with the same ease as Frank had done. Halt there, my men—lads and lasses too—there, halt a bit. Mrs. Fairfield, do you hear?—halt! I think his reverence has given us a capital sermon. Go up to the Great House all of you, and drink a glass to his health. Frank, go with them; and tell Spruce to tap one of the casks kept for the haymakers. Harry, [this in a whisper] catch the Parson, and tell him to come to me instantly.
- 70. My dear Hazeldean, what has happened? you are mad. Don't bother—do what I tell you. But where is the Parson to find you? Where, gad zooks, Mrs. H., at the Stocks to be sure! CHAPTER XI. Dr. Riccabocca, awakened out of his reverie by the sound of footsteps—was still so little sensible of the indignity of his position, that he enjoyed exceedingly and with all the malice of his natural humor, the astonishment and stupor manifested by Stirn, when that functionary beheld the extraordinary substitute which fate and philosophy had found for Lenny Fairfield. Instead of the weeping, crushed, broken-hearted captive whom he had reluctantly come to deliver, he stared, speechless and aghast, upon the grotesque but tranquil figure of the Doctor, enjoying his pipe and cooling himself under his umbrella, with a sang-froid that was truly appalling and diabolical. Indeed, considering that Stirn always suspected the Papisher of having had a hand in the whole of that black and midnight business, in which the Stocks had been broken, bunged up, and consigned to perdition, and that the Papisher had the evil reputation of dabbling in the Black Art, the hocus-pocus way in which the Lenny he incarcerated was transformed into the Doctor he found, conjoined with the peculiarly strange, eldritch, and Mephistophelean physiognomy and person of Riccabocca, could not but strike a thrill of superstitious dismay into the breast of the parochial tyrant. While to his first confused and stammered exclamations and interrogatories, Riccabocca replied with so tragic an air, such ominous shakes of the head, such mysterious, equivocating, long-worded sentences, that Stirn every moment felt more and more convinced that the boy had sold himself to the Powers of Darkness; and that he himself, prematurely, and in the flesh, stood face to face with the Arch-Enemy.
- 71. Mr. Stirn had not yet recovered his wonted intelligence, which, to do him justice, was usually prompt enough—when the Squire, followed hard by the Parson, arrived at the spot. Indeed, Mrs. Hazeldean's report of the Squire's urgent message, disturbed manner, and most unparalleled invitation to the parishioners, had given wings to Parson Dale's ordinarily slow and sedate movements. And while the Squire, sharing Stirn's amazement, beheld indeed a great pair of feet projecting from the stocks, and saw behind them the grave face of Doctor Riccabocca, under the majestic shade of the umbrella, but not a vestige of the only being his mind could identify with the tenancy of the Stocks, Mr. Dale, catching him by the arm, and panting hard, exclaimed with a petulance he had never before been known to display—except at the whist-table— Mr. Hazeldean, Mr. Hazeldean, I am scandalized—I am shocked at you. I can bear a great deal from you, sir, as I ought to do; but to ask my whole congregation, the moment after divine service, to go up and guzzle ale at the Hall, and drink my health, as if a clergyman's sermon had been a speech at a cattle-fair! I am ashamed of you, and of the parish! What on earth has come to you all? That's the very question I wish to Heaven I could answer, groaned the Squire, quite mildly and pathetically—What on earth has come to us all! Ask Stirn: (then bursting out) Stirn, you infernal rascal, don't you hear?—what on earth has come to us all? The Papisher is at the bottom of it, sir, said Stirn, provoked out of all temper. I does my duty, but I is but a mortal man, arter all. A mortal fiddlestick—where's Leonard Fairfield, I say? Him knows best, answered Stirn, retreating mechanically, for safety's sake, behind the Parson, and pointing to Dr. Riccabocca. Hitherto, though both the Squire and Parson had indeed recognized the Italian, they had merely supposed him to be seated on the bank. It never entered into their heads that so respectable and dignified a
- 72. man could by any possibility be an inmate, compelled or voluntary, of the Parish Stocks. No, not even though, as I before said, the Squire had seen, just under his nose, a very long pair of soles inserted in the aperture—that sight had only confused and bewildered him, unaccompanied as it ought to have been with the trunk and face of Lenny Fairfield. Those soles seemed to him optical delusions, phantoms of the overheated brain; but now, catching hold of Stirn, while the Parson in equal astonishment caught hold of him —the squire faltered out, Well, this beats cock-fighting! The man's as mad as a March hare, and has taken Dr. Rickeybockey for little Lenny! Perhaps, said the Doctor, breaking silence, with a bland smile, and attempting an inclination of the head as courteous as his position would permit—perhaps, if it be quite the same to you, before you proceed to explanations—you will just help me out of the Stocks. The Parson, despite his perplexity and anger, could not repress a smile, as he approached his learned friend, and bent down for the purpose of extricating him. Lord love your reverence, you'd better not! cried Mr. Stirn. Don't be tempted—he only wants to get you into his claws. I would not go a-near him for all the— The speech was interrupted by Dr. Riccabocca himself, who now, thanks to the Parson, had risen into his full height, and half a head taller than all present—even than the tall Squire—approached Mr. Stirn, with a gracious wave of the hand. Mr. Stirn retreated rapidly toward the hedge, amidst the brambles of which he plunged himself incontinently. I guess whom you take me for, Mr. Stirn, said the Italian, lifting his hat with his characteristic politeness. It is certainly a great honor; but you will know better one of these days, when the gentleman in question admits you to a personal interview in another and—a hotter world.
- 73. CHAPTER XII. But how on earth did you get into my new Stocks? asked the Squire, scratching his head. My dear sir, Pliny the elder got into the crater of Mount Etna. Did he, and what for? To try what it was like, I suppose, answered Riccabocca. The Squire burst out a-laughing. And so you got into the Stocks to try what it was like. Well, I can't wonder—it is a very handsome pair of Stocks, continued the Squire, with a loving look at the object of his praise. Nobody need be ashamed of being seen in those Stocks—I should not mind it myself. We had better move on, said the Parson drily, or we shall be having the whole village here presently, gazing on the lord of the manor in the same predicament as that from which we have just extricated the Doctor. Now pray what is the matter with Lenny Fairfield? I can't understand a word of what has passed. You don't mean to say that good Lenny Fairfield (who was absent from church by-the-by) can have done any thing to get into disgrace? Yes, he has though, cried the Squire. Stirn, I say—Stirn. But Stirn had forced his way through the hedge and vanished. Thus left to his own powers of narrative at second-hand, Mr. Hazeldean now told all he had to communicate: the assault upon Randal Leslie, and the prompt punishment inflicted by Stirn; his own indignation at the affront to his young kinsman, and his good-natured merciful desire to save the culprit from the addition of public humiliation. The Parson, mollified toward the rude and hasty invention of the beer-drinking, took the Squire by the hand. Ah, Mr. Hazeldean, forgive me, he said repentantly; I ought to have known at once
- 74. that it was only some ebullition of your heart that could stifle your sense of decorum. But this is a sad story about Lenny, brawling and fighting on the Sabbath-day. So unlike him, too—I don't know what to make of it. Like or unlike, said the Squire, it has been a gross insult to young Leslie; and looks all the worse because I and Audley are not just the best friends in the world. I can't think what it is, continued Mr. Hazeldean, musingly, but it seems that there must be always some association of fighting connected with that prim half-brother of mine. There was I, son of his own mother—who might have been shot through the lungs, only the ball lodged in the shoulder—and now his wife's kinsman—my kinsman, too—grandmother a Hazeldean—a hard-reading sober lad, as I am given to understand, can't set his foot into the quietest parish in the three kingdoms, but what the mildest boy that ever was seen—makes a rush at him like a mad bull. It is Fatality! cried the Squire solemnly. Ancient legend records similar instances of totality in certain houses, observed Riccabocca. There was the House of Pelops—and Polynices and Eteocles—the sons of Œdipus! Pshaw, said the Parson; but what's to be done? Done? said the Squire; why, reparation must be made to young Leslie. And though I wished to spare Lenny, the young ruffian, a public disgrace—for your sake, Parson Dale, and Mrs. Fairfield's; yet a good caning in private— Stop, sir! said Riccabocca mildly, and hear me. The Italian then, with much feeling and considerable tact, pleaded the cause of his poor protégé, and explained how Lenny's error arose only from mistaken zeal for the Squire's service, and in the execution of the orders received from Mr. Stirn. That alters the matter, said the Squire, softened: and all that is necessary now will be for him to make a proper apology to my kinsman.
- 75. Yes, that is just, rejoined the Parson; but I still don't learn how he got out of the Stocks. Riccabocca then resumed his tale; and, after confessing his own principal share in Lenny's escape, drew a moving picture of the boy's shame and honest mortification. Let us march against Philip! cried the Athenians when they heard Demosthenes— Let us go at once and comfort the child! cried the Parson, before Riccabocca could finish. With that benevolent intention, all three quickened their pace, and soon arrived at the widow's cottage. But Lenny had caught sight of their approach through the window; and not doubting that, in spite of Riccabocca's intercession, the Parson was come to upbraid, and the Squire to re-imprison, he darted out by the back way, got among the woods, and lay there perdu all the evening. Nay, it was not till after dark that his mother—who sate wringing her hands in the little kitchen, and trying in vain to listen to the Parson and Mrs. Dale, who (after sending in search of the fugitive) had kindly come to console the mother—heard a timid knock at the door and a nervous fumble at the latch. She started up, opened the door, and Lenny sprang to her bosom, and there buried his face, sobbing loud. No harm, my boy, said the Parson, tenderly; you have nothing to fear—all is explained and forgiven. Lenny looked up, and the veins on his forehead were much swollen. Sir, said he, sturdily, I don't want to be forgiven—I ain't done no wrong. And—I've been disgraced—and I won't go to school, never no more. Hush, Carry! said the Parson to his wife, who, with the usual liveliness of her little temper, was about to expostulate. Good-night, Mrs. Fairfield. I shall come and talk to you to-morrow, Lenny; by that time you will think better of it.
- 76. The Parson then conducted his wife home, and went up to the Hall to report Lenny's safe return; for the Squire was very uneasy about him, and had even in person shared the search. As soon as he heard Lenny was safe—Well, said the Squire, let him go the first thing in the morning to Rood Hall, to ask Master Leslie's pardon, and all will be right and smooth again. A young villain! cried Frank, with his cheeks the color of scarlet; to strike a gentleman and an Etonian, who had just been to call on me! But I wonder Randal let him off so well—any other boy in the sixth form would have killed him! Frank, said the Parson, sternly, if we all had our deserts, what should be done to him who not only lets the sun go down on his own wrath, but strives with uncharitable breath to fan the dying embers of another's? The clergyman here turned away from Frank, who bit his lip, and seemed abashed—while even his mother said not a word in his exculpation; for when the Parson did reprove in that stern tone, the majesty of the Hall stood awed before the rebuke of the Church. Catching Riccabocca's inquisitive eye, Mr. Dale drew aside the philosopher, and whispered to him his fears that it would be a very hard matter to induce Lenny to beg Randal Leslie's pardon, and that the proud stomach of the pattern-boy would not digest the Stocks with as much ease as a long regimen of philosophy had enabled the sage to do. This conference Miss Jemima soon interrupted by a direct appeal to the Doctor respecting the number of years (even without any previous and more violent incident) that the world could possibly withstand its own wear and tear. Ma'am, said the Doctor, reluctantly summoned away, to look at a passage in some prophetic periodical upon that interesting subject —ma'am, it is very hard that you should make one remember the end of the world, since, in conversing with you, one's natural temptation is to forget its existence.
- 77. Miss Jemima blushed scarlet. Certainly that deceitful, heartless compliment justified all her contempt for the male sex; and yet, such is human blindness, it went far to redeem all mankind in her credulous and too confiding soul. He is about to propose, sighed Miss Jemima. Giacomo, said Riccabocca, as he drew on his nightcap, and stepped majestically into the four-posted bed. I think we shall get that boy for the garden now! Thus each spurred his hobby, or drove her car, round the Hazeldean whirligig. (To be continued.)
- 78. ON BIRDS, BALLOONS, AND BOLUSES. The bird of Æsculapius ought, certainly, to have been a goose; for Quack, quack, quack, should be the great motto of medicine. One professor invents an ointment for other people's bad legs, which keeps him comfortably on his own, while another makes a harvest of every body's corn, and a third publishes a pill to smooth the pillow of every invalid, or a bolus to render his bolster bearable. In another phase of quackery, we find specifics for the hair recommended to those who are ready to take any nonsense into their heads, and will boldly stand the hazard of the dye, in the vain hope that the gray, indicating the twilight or winter time of life, may be exchanged for the dark, brown tints of summer, or autumn at the latest; and we are constantly being invited to remove our baldness in advertisements, which we know to be the very essence of balderdash. Quackery, however, seems to be successful in some cases, for the public will swallow any thing from a puff to a pill, from music to medicine, from a play to a plaster, and there is no doubt that (to paraphrase Macbeth, when speaking of the possibility that Birnam Wood being come to Dunsinane:) If Barnum would but come to Drury Lane, he would, by his force of quackery, make that pay him which has paid no one else during the last quarter of a century. Such is the spirit of the age, that, reading the accounts from America relative to our own protégée, Jenny Lind, we are disposed to think that the nightingale is being made a goose of in the United States—so vast is the amount of quackery with which her name is just now identified.
- 79. As there is good to be got from every evil, we are justified in expecting that the puff and quack malady will cure itself, and if things are likely to mend when they get to the worst, we may congratulate ourselves upon humbug having reached almost the antipodes of sense and propriety. The balloon mania has already nearly exhausted the utmost resources of absurdity; for M. Poitevin on a donkey—how very like putting butter upon bacon! has failed to attract, and three or four women suspended in the air are now necessary to tempt the curiosity of the Parisian public when a balloon ascends from the Hippodrome. We expect to hear next that Poitevin intends going up attached to the balloon by the hair of his head, for he seems quite silly enough to become the victim of such a very foolish attachment.—Punch.
- 80. CAROL FOR THE NEW YEAR. BY ALFRED TENNYSON. Ring out, wild bells, to the wild sky, The flying cloud, the frosty light. The year is dying in the night; Ring out, wild bells, and let him die. Ring out the old, ring in the new, Ring, happy bells, across the snow, The Year is going, let him go; Ring out the false, ring in the true. Ring out the grief that saps the mind, For those that here we see no more; Ring out the feud of rich and poor, Ring in redress to all mankind. Ring out a slowly dying cause, And ancient forms of party strife; Ring in the nobler modes of life, With sweeter manners, purer laws. Ring out the want, the care, the sin, The faithless coldness of the times; Ring out, ring out my mournful rhymes, But ring the fuller minstrel in. Ring out false pride in place and blood, The civic slander and the spite; Ring in the love of truth and right, Ring in the common love of God.
- 81. Ring out the shapes of foul disease, Ring out the narrowing lust of gold; Ring out the thousand wars of old, Ring in the thousand years of peace.
- 82. THE EDIBLE BIRDS'-NESTS OF CHINA. Among the various articles exposed for sale to the natives, in the innumerable streets of Canton, the edible birds'-nests deserve especial notice. They owe their celebrity only to the whimsical luxury of the Chinese, and are brought principally from Java and Sumatra, though they are found on most of the rocky islets of the Indian Archipelago. The nest is the habitation of a small swallow, named (from the circumstance of having an edible house) hirundo esculenta. They are composed of a mucilaginous substance, but as yet have never been analyzed with sufficient accuracy to show the constituents. Externally, they resemble ill-concocted, fibrous isinglass, and are of a white color, inclining to red. Their thickness is little more than that of a silver spoon, and the weight from a quarter to half an ounce. When dry, they are brittle, and wrinkled; the size is nearly that of a goose's egg. Those that are dry, white, and clean, are the most valuable. They are packed in bundles, with split rattans run through them to preserve the shape. Those procured after the young are fledged are not salable in China. The quality of the nests, varies according to the situation and extent of the caves, and the time at which they are taken. If procured before the young are fledged, the nests are of the best kind; if they contain eggs only, they are still valuable; but, if the young are in the nests, or have left them, the whole are then nearly worthless, being dark-colored, streaked with blood, and intermixed with feathers and dirt.
- 83. These nests are procurable twice every year; the best are found in deep, damp caves, which, if not injured, will continue to produce indefinitely. It was once thought that the caves near the sea-coast were the most productive; but some of the most profitable yet found, are situated fifty miles in the interior. This fact seems to be against the opinion, that the nests are composed of the spawn of fish, or of bêche-de-mer. The method of procuring these nests is not unattended with danger. Some of the caves are so precipitous, that no one, but those accustomed to the employment from their youth, can obtain the nests, being only approachable by a perpendicular descent of many hundred feet, by ladders of bamboo and rattan, over a sea rolling violently against the rocks. When the mouth of the cave is attained, the perilous task of taking the nests must often be performed by torch-light, by penetrating into recesses of the rock, where the slightest slip would be instantly fatal to the adventurers, who see nothing below them but the turbulent surf, making its way into the chasms of the rock—such is the price paid to gratify luxury. After the nests are obtained, they are separated from feathers and dirt, are carefully dried and packed, and are then fit for the market. The Chinese, who are the only people that purchase them for their own use, bring them in junks to this market, where they command extravagant prices; the best, or white kind, often being worth four thousand dollars per pecul,[19] which is nearly twice their weight in silver. The middling kind is worth from twelve to eighteen hundred, and the worst, or those procured after fledging, one hundred and fifty to two hundred dollars per pecul. The majority of the best kind are sent to Pekin, for the use of the court. It appears, therefore, that this curious dish is only an article of expensive luxury among the Chinese; the Japanese do not use it at all, and how the former people acquired the habit of indulging in it, is only less singular than their persevering in it.
- 84. They consider the edible bird's-nest as a great stimulent, tonic, and aphrodisiac, but its best quality, perhaps, is its being perfectly harmless. The labor bestowed to render it fit for the table is enormous; every feather, stick, or impurity of any kind, is carefully removed; and then, after undergoing many washings and preparations, it is made into a soft, delicious jelly. The sale of birds'- nests is a monopoly with all the governments in whose dominions they are found. About two hundred and fifty thousand peculs, at a value of one million four hundred thousand dollars, are annually brought to Canton. These come from the islands of Java, Sumatra, Macassar, and those of the Sooloo group. Java alone sends about thirty thousand pounds, mostly of the first quality, estimated at seventy thousand dollars. I am indebted for much information on this curious article of commerce, to the captain of a Java ship, a very well informed man, trading regularly to China, who had large quantities on board, and whose wife, a native of that country, to satisfy my curiosity, prepared a dinner for me of Chinese dishes, including the bird's-nest and the sea-slug, both of which I partook of, and found them very palatable. —Berncastle's Voyage to China.
- 85. THE PASSION FOR COLLECTING BOOKS. Of all the passions to which the human mind can surrender itself, there is none more absorbing than the mania of book-collecting. Let those speak honestly who have indulged in it. It is a species of bulimia—an insatiable appetite, which grows by what it feeds on. I have purchased my experience of this matter rather dearly, having at one period occupied much time, and laid out more money than I like to think of, in forming a select and curious library. My books formed my chief solace and amusement during many years of an active and unprofitable professional life. The pressure of pecuniary difficulties forced me to part with them, and taught me practically, though not pleasantly, the vast distinction between buying and selling. It was something to see placarded in imposing type, Catalogue of the valuable and select library of a gentleman, containing many rare and curious editions. But, alas! the sum produced was scarcely a third of the intrinsic value, and less than half of the original cost. There have been instances—but they are few and far between—where libraries have been sold at a premium. Take for an example the collection of Dr. Farmer, of Emmanuel College, Cambridge, singularly rich in Shakspearian authorities and black-letter lore, which produced above £2200, and was supposed to have cost the owner not more than £500. Many were presents. When you get the character of a collector, a stray gift often drops in, and scarce volumes find their way to your shelves, which the quondam owners, uninitiated in bibliomania, know not the worth of. I once purchased an excellent copy of the quarto Hamlet, of 1611, of an unsuspecting bibliopolist, for ten shillings; my conscience smote me, but the temptation was irresistible.[20] The best copy in existence of the Caxtonian edition of Gower's De Confessione Amantis, fol., 1483,
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