Enterobacteriaceae

Laboratory diagnosis of infections
produced by germs of the family
Enterobateriaceae

Family Enterobacteriaceae
- Clinical significance -
• Intestinal and extraintestinal infections
• Highly pathogenic species of the genera:
– Yersinia
– Salmonella
– Shigella
• Facultatively pathogenic species of the genera:
– Escherichia coli
– Klebsiella
– Enterobacter
– Proteus
– Serratia
– Citrobacter

- Common characters -
• Gram negative bacilli, nonsporulating, non-fastidious
• Glucose-fermenters;
• Lactose is only fermented by some genera – good
differential criterion
• Oxidase-negative
• Catalase-positive
• Habitat:
– soil, plants, human & animal intestines, mucous membranes;
– Particular situation: Salmonella typhi (causative agent of typhoid
fever) – only present in humans (diseased / asymptomatic
carriers)

- Collection of specimens -
• Extraintestinal infections:
– urine, respiratory/digestive samples, wound secretions, blood,
CSF, etc)
• Intestinal infections:
– Faeces: collection close to onset / depending on pathogenesis of
infection
– Transport media: Stuart, Cary-Blair, Amies

Collection of urine
When?:
- in the morning (first miction)
How?:
- clean uro-genital area
- eliminate first flow
- collect middle flow in
sterile container
Send to lab immediately or store
at 2-8°C

Collection of stool (faeces)
• Disposable stool collection containers (simple / with
transportation medium Carry Blair: non-nutritive medium
which prevents overgrowth of Enterobacteriaceae but
preserves viable enteric pathogens (Salmonella,
Shigella, etc)

- Isolation (inoculation of culture media) -
• Extraintestinal specimens from normally sterile sites:
– Blood agar
• Extraintestinal specimens with moderate bacterial load
(e.g. pus, sputum):
– Blood agar + MacConkey
• Highly contaminated specimens (faeces):
– MacConkey (low selectivity)
– ADCL, Hektoen agar, XLD agar (medium selectivity)
– High selective media e.g. S-S (for Salmonella and Shigella),
Wilson-Blair (for Salmonella)

e.g. Hektoen agar
(developed at the Hektoen Institute in Chicago)
• indicators of:
– lactose fermentation
– H2S production;
• inhibitors (bile salts)
to prevent the growth
of Gram positive
bacteria
Lactose H2S
Salmonella (-); alkaline
reaction: blue-
green colonies
(+);
black
centre
colonies
Shigella (-); alkaline
reaction: blue-
green colonies
(-)
E.coli and
others
(+); acid
reaction:
yellow-orange
colonies
(-)

Left: lactose (+) = yellow-orange
Right: lactose (-) = blue-green, H2S (+) = black
centre
E.coli / others (definitely not
Salmonella, not Shigella)
• Salmonella (not Shigella
because of H2S production –
black centre colonies)

- Identification -
• Biochemical tests:
– TSI (triple sugar iron) agar
– MIU (motility, indol, urea) agar
– Simmons agar (use of citrate as unique carbon source)
– PAD (phenylalanine deaminase) test
– Fermentation of sugars
• Antigenic structure-based identification:
– Agglutination with antisera

Biochemical tests = testing for enzyme systems
• characterization of bacterial isolate by testing for
characteristic enzyme systems
• Method: re-inoculation of isolated colony (primary
culture) into a series tubes with culture media containing
specific substrates and chemical indicators
• Principle: detection of
– pH changes produced by utilization of substrates /
– colour / other changes produced by specific by-
products

Biochemical tests (continued)
TSI (triple sugar iron) agar – assessment of bacterial
capacity to:
a. metabolize lactose and/or sucrose
b. conduct fermentation to produce acid
c. produce gas during fermentation
d. generate H2S

TSI agar:
sucrose, lactose, glucose + mehyl red + ferrous
sulfate
IF:
• only glucose fermented →acid production in the butt of tube →
yellow, but insufficient acid to affect the methyl red in the slant
• either sucrose or lactose fermented → sufficient fermentation
products → both the butt and the slant yellow
• gas during fermentation → gas bubbles/cracking of agar
• no fermentation → slant and butt remain red
• If bacterium forms H2S, this chemical will react with the iron to form
ferrous sulfide = black precipitate in the butt (black butt)

TSI agar (continued)
• R = red = no fermentation
(obligate aerobe)
• Y = yellow = some
fermentation (facultative
anaerobe)
• YG = fermentation + gas
• ”+” = Black = H2S

Identification – biochemical tests
• API (Analytical Profile Index)

Escherichia coli
• Gram negative, short bacilli, rounded ends,
nonsporulating, motile (peritrichous cilia)
• Normal microbial flora of human and animal intestines;
involved in vitamin synthesis and balance of intestinal
microbiota
• Facultatively pathogenic

Escherichia coli
• Enteral infections (5 groups):
– EPEC (enteropathogenic E.coli)
– ETEC (enterotoxigenic E.coli)
– EIEC (enteroinvasive E.coli)
– EHEC (enterohemorrhagic E.coli) – produce verotoxins* (bloody
severe diarrhoea resembling dysentery!)
– EAEC (enteroadherent E.coli)
• Extraenteral infections:
– Urinary, respiratory, wounds & burns, sepsis , meningitis, etc.

*Verotoxins (Shiga-like toxins)
• toxins produced by certain strains of E.coli (EHEC)
which disrupt the function of the ribosome
• Action similar to the toxin produced by Shigella
disenteriae strains (Sh.shiga) – see below
• causes the hemolytic uremic syndrome
• Term “verotoxin” related to the effect on “vero” cell
cultures (lineages of kidney epithelial cells extracted from
African green monkeys; name: abbreviation from “verda
reno” = green kidney in Esperanto)

E.coli – Enteral infections
Specimen: stool
• Culture media: MacConkey:
pH indicator = neutral red (red
in acid medium; colourless in
basic medium):
• RED colonies (lactose-
positive), round, shiny, 2-3 mm

E.coli colonies on MacConkey:
lactose positive (red) colonies

E.coli on medium containing lactose and bile
salts: lactose positive (red) colonies

E.coli on Hektoen agar: yellow-orange
colonies (lactose acidification)

Identification: biochemical tests

Identification on antigenic structure
• Slide agglutination with Ab
against O and B antigens:
– 5 lactose-positive colonies –
pick up with loop and emulsify
with polyvalent anti-EPEC
serum
– If positive test (agglutination)
continue with monovalent
antisera (standard set)
• Similar procedure for EIEC,
ETEC, EHEC (antisera for
antigens O and H)

Escherichia coli
• Enteral infections (5 groups):
– EPEC (enteropathogenic E.coli)
– ETEC (enterotoxigenic E.coli)
– EIEC (enteroinvasive E.coli)
– EHEC (enterohemorrhagic E.coli) – produce verotoxins (bloody
severe diarrhoea resembling disenteria!)
– EAEC (enteroadherent E.coli)
• Extraenteral infections:
– Urinary, respiratory, wounds & burns, sepsis , meningitis, etc.

E.coli – Urinary tract infections (UTI)
• Collection of urine for bacterial culture (see above)
• Gram stained smear from urine sediment (after
centrifugation): high no of PMNs + Gram negative bacilli
• Quantitative urine culture:
– A. Dilutions technique
– B. Calibrated loop technique

E.coli – Urinary tract infections (continued)
Quantitative urine culture
• Colony counts: method to determine number of viable
bacterial cells in urine specimen
• 1 colony = 1 viable bacterial cell (colony-forming unit =
CFU) → after inoculation: division by binary fission i.e. 1
cell to 2; 2 to 4; 4 to 8; 8 to 16....and so on (at least a
million cells must be present in order to be seen as a
colony with the naked eye!)
• Results reported as: number of bacterial cells per mL of
urine

Quantitative urine culture (continued)
A. The dilutions technique:
• 2 dilutions (1/100 and 1/1000) in sterile saline solution
• Inoculate 0.1 mL of each dilution onto blood agar and
MacConkey (lactose containing medium)
• Spread inoculum to obtain isolated colonies (L-shaped
loop); Incubate overnight at 37°C
• Calculate the no of germs / mL urine i.e. multiply:
– No of colonies (CFU) x
– dilution factor (100 or 1000) x
– 10 (we inoculated 0.1 mL of each dilution)

B. The calibrated loop technique: (nondiluted sample)
• 5 mm loop → 0.01 mL urine (dilution: 1/100) →
MacConkey
• 2.5 mm loop → 0.001 mL urine (dilution: 1/1000) →
blood agar
• Incubate overnight at 37°C
• Calculation: (for each plate)
• No. of colonies (CFU) x dilution = no of germs / mL
• Calculate the arithmetic mean between the 2 counts
(on the 2 plates)

• Interpretation of results:
• Under 10,000 germs/mL = nonsignificant bacteriuria
(probably contamination from lower urethra)
• 10,000–100,000 germs/mL = nonconclusive; repeat test
• Over 100,000 germs/mL = UTI
• Next steps: identification of causative agent i.e.
– colonial characters, biochemical tests
– Antimicrobial susceptibility testing

Genus Salmonella
• The most complex of all Enterobacteriaceae
• over 2400 serotypes – Classification: Kauffmann-White
scheme based on bacterial antigens:
– O (somatic)
– H (flagellar)
– Vi (virulence) – derived from the K (capsular) antigen
• Clinical significance:
– Food poisoning
– Systemic infections (the germs cross the intestinal barrier) –
”enteric fevers”
• Transmission: fecal-oral (via contaminated water, foods)

Genus Salmonella
- The Kauffmann-White classification -
• E.g. Based on the “O”
antigens: groups A – I (+
others)
• some examples given in
table →
“O” group A S.paratyphi A
“O” group B S.paratyphi B
S.typhimurium
“O” group C S.paratyphi C
“O” group D S.typhi
S.enteritidis

Salmonella typhi
• Prototype agent of ”enteric fevers” (other enteric fevers
caused by Salomnella paratyphi A, B, C – less severe)
• Typhoid fever - fever in plateau (39-40°C), headache,
muscle ache, vomit, diarrhoea /constipation, skin rush
(lenticular maculae; “rose spots”), mental confusion,
hepato-splenomegaly
• Complications: internal bleeding, intestinal perforation +
peritonitis
• Laboratory diagnosis:
– Bacteriology
– Serology

Salmonella typhi (+ paratyphi)
- Bacteriological diagnosis -
• Collection of specimens depends on:
– Patient status: diseased / chronic carrier
– Clinical stage (time from onset)
• Secimens:
– Blood for blood culture (best collected during 1st week; percent
of isolation decreases to 25% in the 4th week)
– Bone marrow (in late stages) – lower patient compliance
– Faeces for coproculture (low isolation during 1st week; percent
increases progressively to ~75% in 4th week); also collected
from suspected chronic carriers
– Urine for culture (same isolation curve as for faeces)

- Bacteriological diagnosis - continued
Blood culture:
• inoculated media examined daily for 7-10 days; negative
result only if liquid media remain sterile for 10 days!
• Gram stained smear from turbid tubes: Gram negative
bacilli
• Subculture on agar slant
• Identification based on:
– colonial characters,
– biochemical tests,
– antigenic structure – slide agglutination with anti-O and anti-H
sera (Kauffmann-White classification)

Coproculture:
• Both in diseased patients and in chronic carrires
• Inoculation in liquid enrichment media (favour
multiplication of salmonellae and inhibit other microbial
flora) e.g.
– Leifson (nutrient broth with acid sodium selenite);
– Muller-Kauffmann (broth with tetrationate and bile)
• Incubate overnight at 37°C
• Reinoculate on selective solid media (nutrients + sugars
+ pH indicator + substances which inhibit other germs)

Coproculture: (continued)
Colonial characters on selective solid media:
• Wilson-Blair (high selectivity; indicator: brilliant green)
– Black, flat colonies, 1-2 mm, metallic halo
• S-S (selective for Salmonella and Shigella):
– Fine, semitransparent colonies, with black centre (H2S)
• Hektoen enteric agar :
– Fine, green colonies, with black centre (H2S)
• MacConkey (medium selectivity):
– Semitransparent, lactose-negative (colourless) colonies

Salmonella on S-S agar: fine, semitransparent
colonies, black centre (H2S)

Salmonella on MacConkey agar
• Semitransparent, lactose-
negative (colourless)
colonies

Salmonella on agar with lactose and bile salts:
lactose negative (colourless) colonies with black
centre (H2S)

Salmonella on Hektoen agar
• black centre
colonies (H2S)

Salmonella – Coproculture - continued
• Identification based on:
– colonial characters (see above),
– biochemical tests (API 20E),
– antigenic structure – slide agglutination with anti-O and anti-H
sera (Kauffmann-White classification)
– Phage typing – see next slide

(Bacterio)phage typing for Salmonella
• Bacteriophage = virus which specifically attacks bacteria
• Banks of phages developed for Salmonella serotypes
Procedure:
• agar plates flooded with liquid culture of the bacterial
isolate; remove excess liquid; leave culture film to dry;
• inoculate set of phage suspensions onto plate surface;
incubate overnight at 37°C
• Interpretation: phage lysis reactions recorded and
compared to a set of standards (developed by Public
Health England, formerly PHLS, Colindale, England)

Phage typing Salmonella enteritidis

Salmonella typhi
- Serological diagnosis -
The Widal test:
• Principle: reaction between antibodies in patient serum
and specific antigens of S. typhi → clumping
(agglutination) visible to the naked eye
• easy to perform BUT less reliable than bacteriology:
– cross-reactivity with other Salmonella species,
– the test cannot distinguish between a current infection
and a previous infection or vaccination status
• Still used in low resource areas with high
prevalence of typhoid fever (endemic areas e.g.
India, Pakistan)

Genus Shigella
Clinical significance:
• dysentery – diarrhoea with multiple stools with mucus
and blood + general symptoms: dehydration, fever,
abdominal pain + neurologic symptoms (neurotoxin
secreted by Shigella shiga)
Common characters:
• Gram negative bacilli, nonmotile, nonsporulating

Genus Shigella - classification
4 serological subgroups (based on biochemical characters
and antigenic structure):
• Subgroup A: Shigella dysenteriae
– Types: Sh. shiga, Sh. Schmitzi, Sh. Large-Sachs
• Subgroup B: Shigella flexneri
• Subgroup C: Shigella boydii
• Subgroup D: Shigella sonnei
_______________
• Shigella shiga: the most severe disease (secretion of
neurotoxin)

Kiyoshi Shiga (1871-1957)
• Discovered Shigella dysenteriae during severe
epidemic in 1897: over 90,000 cases
• “shiga” toxin named after him

Genus Shigella – Bacteriological diagnosis
• Collection of specimens: faeces (especially portions with
mucus and blood)
• Transport: Cary Blair medium
• Cultivation and isolation: endo-agar, MacConkey, S-S,
Hektoen, XLD
• Colonial characters:
– small, 1-2 mm, transparent, round / irregular contour, convex,
lactose-negative (colourless i.e. colour of the culture medium)
– No H2S production

Hektoen agar:
Shigella – colourless colonies;
Salmonella - green colonies with black centre

Shigella – identification, continued
• Biochemical characters: API 20E

Genus Shigella – Bacteriological diagnosis
- continued -
• Antigenic structure based
identification:
– Agglutination with sets of anti-
sera (polyvalent + monovalent:
subgroups + typing)

Genus Klebsiella
• Comensal/Facultatively pathogenic: Colonizes the
respiratory mucosa and the intestine
• In immunosuppressed patients (premature infants,
elderly people) – potential for severe infections
(pneumonia, sepsis, meningitis)
• Hospital acquired infections: surgical wound infections,
urinary infections, sepsis
• Involvement in diarrhoeic diseasae - debated

Genus Klebsiella
4 species important for human pathology:
• Klebsiella pneumoniae – comensal of human airways
and intestinal mucosa; facultatively pathogenic
• Klebsiella oxytoca - idem
• Klebsiella ozenae – ozena = chronic inflammatory
infection of the nasal mucosa; mucosal atrophia, crusts
& purulent secretions with unpleasant odour
• Klebsiella rhinoscleromatis – rhinoscleroma = chronic
hypertrophic rhinitis with granulomatous lesions

Genus Klebsiella
Common characters:
• Gram negative, short bacilli,
rounded ends, nonsporulating,
arranged in diplo (in pairs) on
the long axis, enacpsulated
• Sometimes bipolar staining
(ends more intensly stained
than middle of rod)

Genus Kelbsiella
Isolation:
• Specimens from normally sterile sites (blood, CSF, etc):
– Nutrient broth + reinoculation on blood agar
• Specimens from highly contaminates sites (e.g. faeces):
– Media for enterobacteria: MacConkey, XLD
• Identification:
– Blood agar – large, white-grey colonies, mucous, aspect of
”pouring culture” – in time the colour changes to brown
(”chameleoning” phenomenon)
– MacConkey – large, mucoid colonies, red/pink (lactose positive)
– in time colour changes to yellow (lactose negative) –
“chameleoning” by alkalinisation of the medium

Klebsiella – blood agar (non hemolytic
mucoid colonies)

Klebsiella – pink colonies (Lactose positive)
on MacConkey agar

Klebsiella colonies on MacConkey: colour starts to
change - Lactose positive (red/pink) colonies start
to change to lactose negative (yellow) –
alkalinisation

Klebsiella – identification – continued
Biochemical characters

Genus Proteus
• 4 species:
– P.vulgaris, P.mirabilis, P.penneri, P. myxofaciens
• Common characters:
– Gram negative, short bacilli, rounded ends, high polymorphism,
high motility (peritrichous cili), nonsporulating, nonencapsulated
• Habitat:
– soil, trash, sewage, altered meat, etc. – involved in putrefaction
processes
• Clinical significance:
– comensal of human and animal digestive flora;
– facultatively pathogenic: UTI, otitis, synusitis, meningitis, sepsis
(community or hospital acquired infections)

Genus Proteus
• Collection of specimens:
– urine, faeces, pus, sputum, CSF, blood, etc
• Direct microscopy:
– only for naturally sterile specimens e.g. CSF
– PMNs + Gram negative bacilli, noncharacteristic arrangement +
filamentous bacilli (high polymorphism of Proteus spp)
• Isolation and identification:
– Blood agar: swarming phenomenon (concentric growth waves
invading the entire plate after overnight incubation); invades
other bacterial colonies; no isolated colonies
– Selective media: round colonies, same colour as the
medium/transparent, black centre (”cat‘s eye”) – H2S production

Proteus
• Swarming
phenomenon on
tryptic soy agar

Genera Morganella and Providencia
• Previously classified as species of the genus Proteus
• Morganella morganii (formerly: Proteus morganii)
• Providencia (formerly: Proteus rettgeri)
• Involved in UTI especially in urinary catheterized patients

Genera Proteus, Morganella, Providencia

Genus Yersinia
3 species important for human pathology:
1. Y. enterocolitica:
– intestinal pathogen (some strains produce an enterotoxin similar
to E.coli; may infect abdominal lymph nodes – apendicitis-like
symptoms)
• Isolation: faeces inoculated on selective media:
– MacConkey – colonies much smaller than of other
enterobacteria
– CIN (cefsulodin, irgasan, novobiocin): overnight incubation at
32°C/48 hours at 25°C: transparent colonies→ (at 48 hours):
larger, pink colonies (increased motility at room temperature)

Genus Yersinia
3 species important for human pathology (continued):
2. Y.pseudotuberculosis
• Enteric infection involving also abdominal lymph vessels
and nodes
• Collection of specimens, Isolation and identification
similar with Y.enterocolitica
• Differential diagnosis based on biochemcal tests

Genus Yersinia
3 species important for human pathology (continued):
3. Y.pestis – Plague:
– reservoir of germs: rodents (rats)
– interpersonal transmission (human to human)
– Routes of infection:
• Vectors: Flea bites →skin lesions (inflammation,
necrosis, purulent secretion) + swollen lymph
nodes (buboes) = bubonic plague →sepsis
• Airborne: Inhalation →pneumonia = pulmonary
plague

Left: Oriental rat flea (vector of Y.pestis)
Right, upper image: Y.pestis infected flea bite
Right, lower image: swollen lymph nodes (buboes)

Yersinia – medium with lactose and bile
salts

Yersinia – identification: API 20E gallery

Hektoen agar inoculated with stool sample
• E.coli – red arrow
• Salmonella – blue
arrow
• Proteus – yellow
arrow

S-S agar:
A = Klebsiella; B = E.coli; C = Salmonella; D = Proteus;
E = Ps.aeruginosa
• Klebsiella and E.coli –
ferment sugars (red
colonies)
• Salmonella and
Proteus – H2S
production (black
centre)
• Pseudomonas
aeruginosa –
colourless colonies

Enterobacteriaceae

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