Mycoplasma.pptx pptptptttttttttttttttttt

LAYOUT
 INTRODUCTION
 HISTORY
 GENERAL CHARACTERISTICS
 GENOME
 TAXONOMIC CLASSIFICATION
 CULTURAL CHARACTERISTICS
 BIOCHEMICAL PROPERTIES
 ANTIGENIC PROPERTIES
 RESISTANCE
 PATHOGENESIS
 IMMUNITY
 EPIDEMIOLOGY
 SPECTRUM OF DISEASES
 LABORATORY DIAGNOSIS
 PROPHYLAXIS AND TREATMENT
 MYCOPLASMAS AS NORMAL FLORA

INTRODUCTION
 Mycoplasmas are the smallest and simplest
self-replicating bacteria.
 The mycoplasma cell contains minimum set of
organelles essential for growth and replication: a
plasma membrane, ribosomes, and a genome
consisting of a double-stranded circular DNA
molecule.
 Unlike all other prokaryotes, the mycoplasmas
have no cell walls, and they are
consequently placed in a separate class
Mollicutes(mollis, soft; cutis, skin).

CONTD….
 Mycoplasmas have been nicknamed the
“crabgrass” of cell cultures.
 Contamination of cell cultures by mycoplasmas
presents serious problems in research
laboratories and in biotechnological industries
using cell cultures.
 The origin of contaminating mycoplasmas is
in components of the culture medium,
particularly serum, or in the flora of the
technician's mouth, spread by droplet
infection.

DIFFERENCES BETWEEN MYCOPLASMAS, BACTERIA,
CHLAMYDIA & VIRUSES
CHARACTER MYCOPLASMA BACTERIA CHLAMYDIA VIRUSES
SIZE
0.2-0.3µm 1-2µm 0.3µm 0.01-0.3µm
CELL WALL
- + + -
PRESENCE OF BOTH
DNA & RNA + + + -
MULTIPLICATION IN
CELL-FREE MEDIUM + + - -
MULTIPLICATION
DEPENDENT ON HOST
NUCLEIC ACID
- - - +
CHOLESTEROL
REQUIREMENT + - - -
INTRINSIC ENERGY
METABOLISM + + + -
NARROW HOST RANGE
+ - - +
SENSITIVITY TO
ANTIBIOTICS
INHIBITING CELL
WALL SYNTHESIS
- + + -
SENSITIVITY TO
ANTIBIOTICS
INHIBITING PROTEIN
SYNTHESIS
+ + + -

HISTORY
 The name Mycoplasma is derived from the Greek word
mykes (fungus) and plasma (formed)
[Mycos :Fungus like form of branching filaments &
Plasma :Denoting plasticicity of their shape].It was
first used by Albert Bernhard Frank in 1889. He
thought it was a fungus, due to fungus-like
characteristics.
 An older name for Mycoplasma was
Pleuropneumonia-Like Organisms (PPLO),
referring to organisms similar to the causative agent of
contagious bovine pleuropneumonia (CBPP).It was
later found that the fungus-like growth pattern of
M. mycoides is unique to that species.

CONTD….
 In 1898 Nocard and
Roux reported the
cultivation of the
causative agent of
CBPP, which was at
that time a grave and
widespread disease in
cattle herds. The
disease is caused by
M. mycoides subsp.
mycoides .

CONTD….
 Eaton in 1944 was the first to isolate the
causative agent of PAP(Primary Atypical
Pneumonia) in hamsters and cotton rats.
 He was able to transmit the infection later to
chick embryos by amniotic inoculation.
 Because it was filterable, it was considered to be
a virus(Eaton agent), but was subsequently
shown to be a mycoplasma and named
Mycoplasma pneumoniae.

GENERAL CHARACTERISTICS
 Very small(0.2-0.3 µm) .
 Can pass through bacterial filters.
 Lack a rigid cell wall.
 Bound by a single trilaminar cell membrane that
contains a sterol.
 Extremely pleomorphic varying in shape from
coccoid to filamentous to other bizzare forms.

ELECTRON MICROGRAPH OF THIN-SECTIONED
MYCOPLASMA CELLS
 Cells are bounded by a
single membrane
showing in section the
characteristic
trilaminar shape. The
cytoplasm contains
thin threads
representing sectioned
chromosome and dark
granules representing
ribosomes.
(Courtesy of RM Cole, Bethesda, Maryland).

CONTD….
 Mycoplasma, Ureaplasma, Spiroplasma and
Anaeroplasma cannot synthesize their own cholesterol
and require it as a growth factor in culture medium.
 Acholeplasma synthesizes carotenol as a substitute for
cholesterol, but will incorporate cholesterol if it is
provided.
 Insensitive to cell-wall active antibiotics such as
penicillins and cephalosporins.
 Limited biosynthetic capabilities due to a small
genome.

CONTD….
 Multiply by binary fission. However, cytoplasmic division
may lag behind genome division. This results in the
formation of multinucleate filaments and other shapes.
 Do not possess flagellae or pili
 Non sporing
 Stain poorly with Gram stain. Can be stained with
Giemsa and Dienes methods.
 Are considered as stable L forms by some researchers
but this hypothesis is still not fully accepted.

CONTD…
 Cells may either
divide by binary
fission or first
elongate to
multinucleate
filaments, which
subsequently breakup
to coccoid bodies.
From Razin S: Mycoplasmas: the smallest
pathogenic procaryotes. Isr J Med Sci
17:510, 1981, with permission.

CONTD….
 Membrane proteins & glycolipids exposed on the cell
surface are the major antigenic determinants in
mycoplasmas.
 Antisera containing antibodies to these components
inhibit growth and metabolism of the mycoplasmas and,
in the presence of complement, cause lysis of the
organisms.
 These properties are used in various serologic tests that
differentiate between mycoplasma species and serotypes
and detect antibodies to mycoplasmas in sera of patients.

CONTD….
 Some mycoplasmas possess unique attachment organelles,
which are shaped as a tapered tip in M. pneumoniae and
M. genitalium. Mycoplasma pneumoniae is a pathogen of
the respiratory tract, adhering to the respiratory
epithelium, primarily through the attachment organelle.
 Interestingly, these two human mycoplasmas exhibit
gliding motility on liquid-covered surfaces. The tip
structure always leads, again indicating its importance in
attachment.
 One of the most useful distinguishing features of
mycoplasmas is their peculiar fried-egg colony shape,
consisting of a central zone of growth embedded in the agar
and a peripheral one on the agar surface.

GENOME
 The mycoplasma genome is typically prokaryotic, consisting of a
circular, double stranded DNA molecule.
 The Mycoplasma and Ureaplasma genomes are the smallest
recorded for any self-reproducing prokaryote.
 In some mycoplasmas the number is estimated at fewer than 500,
about one sixth the number of genes in Escherichia coli.
 Mycoplasmas accordingly express a small number of cell
proteins and lack many enzymatic activities and metabolic
pathways.
 Their nutritional requirements are correspondingly complex,
and they are dependent on a parasitic mode of life.

TAXONOMIC CLASSIFICATION
 Kingdom : Bacteria
 Phylum : Firmicutes
 Class: Mollicutes(mollis: soft and cutis: skin)
 Orders: Entomoplasmatales(plants & insects)
Mycoplasmatales
Acholeplasmatales (birds & animals)
Anaeroplasmatales( cattle & birds)

CONTD….
FAMILIES:
 Entomoplasmataceae &
Spiroplasmataceae( order: Entomoplasmatales)
 Mycoplasmataceae( order: Mycoplasmatales)
 Acholeplasmataceae(order: Acholeplasmatales)
 Anaeroplasmataceae(order: Anaeroplasmatales)

CONTD….
 Family Mycoplasmataceae has two genera:
Mycoplasma( approx.100 species) & Ureaplasma
(6 species).
 Family Acholeplasmataceae has only one genus,
Acholeplasma which comprises 10 species of
which one species A.laidlawii may be found from
the specimens of human oral cavity, respiratory
tract and genital tract.
 Spiroplasmataceae(Genus: Spiroplasma) &
Anaeroplasmataceae(Genus: Anaeroplasma).

CULTURAL CHARACTERISTICS
 Aerobes and facultative anaerobes except Anaeroplasma which
is strictly anaerobic.
 For primary isolation, an atmosphere of 95% Nitrogen and
5% Carbon dioxide is preferred.
 They can grow within a temperature range of 22-41°C, the
parasitic species growing optimally at 35-37°C.
 For fermentative organisms, the initial pH of the medium is
adjusted to 7.3-7.8, for arginine metabolizing organisms it should
be around 7 and for ureaplasmas, range of pH should be 6-6.5.

CONTD….
 The dependence of mycoplasmas on their host for
many nutrients explains the great difficulty of
cultivation in the laboratory.
 The complex media for mycoplasma culture
contain serum, which provides fatty acids and
cholesterol for mycoplasma membrane synthesis.
 The requirement of most mycoplasmas for
cholesterol is unique among prokaryotes.
 The consensus is that only a small fraction of
mycoplasmas existing in nature have been
cultivated so far.

CONTD….
 Some of the cultivable mycoplasmas, including
the human pathogen M pneumoniae, grow
very slowly, particularly on primary
isolation.
 Ureaplasma urealyticum, a pathogen of the
human urogenital tract, grows very poorly in
vitro, reaching maximal titers of 107
organisms/ml
of culture.
 Mycoplasma genitalium, another human
pathogen, grows so poorly in vitro that only a few
successful isolations have been achieved.

MEDIA
 PPLO broth:
Bovine heart infusion broth to which are added 20% horse serum
and 10% fresh yeast extract along with glucose and phenol red as
a pH indicator.
Growth of M.pneumoniae is detected by turbidity and colour
change(red to yellow) of phenol red indicator, due to fermentation
of glucose.
Ureaplasma and other mycoplasmas which do not ferment glucose
show only turbidity.
This medium can be solidified by the addition of agar.
Penicillin, polymyxin B and amphotericin B may be added to
inhibit contaminating bacteria and fungi respectively.

CONTD….
Since thallium acetate is inhibitory for U. urealyticum and
M. genitalium and highly poisonous for humans, therefore, it
should not be added to the medium.
A diphasic medium in screw-capped bottle containing an agar
phase that is overlaid with broth medium of similar composition
may also be used.
SP-4 medium: Basal medium( Mycoplasma broth base, tryptone,
peptone, glucose, deionized water) + supplements( CMRL 1066
cell or tissue culture medium, 10X concentrate with glutamine,
25% solution of fresh yeast extract, 2% solution of yeast olate,
fresh bovine serum heated at 56°C for 1 hour, penicillin 100,000
units/ml, phenol red solution, 0.1% w/v).

COLONIES
 Incubation: 2-6 days. Media for isolation of genital
mycoplasmas and M. pneumoniae should be incubated for
1 and 4 weeks respectively, before a final culture report is
made.
 Size: 200-500µm for mycoplasmas and 15-60µm for
ureaplasmas. The colonies of Ureaplasma are extremely
small and thus Ureaplasma are also called T-strains
(tiny strains).
 Platinum loops: Can’t be picked up.
 Subculture is done by cutting out an agar block
with colonies and rubbing it on fresh plates.

DIENES STAIN
 Contains azure II, methylene blue, Na2CO3, benzoic acid
and distilled water.
 The plate containing suspected Mycoplasma colonies is flooded
with Dienes stain diluted 1 in 10 in water. It is then
immediately rinsed with distilled water to remove the stain.
 The medium is decolorized by adding 1ml of 95% ethanol for 1
minute and then removed.
 The wash step is repeated second time, rinsed with distilled
water and allowed to dry.
 The colonies are then observed under low power of a
microscope.

CONTD….
 Mycoplasmas with the FRIED EGG COLONY
morphology appear highly granular and stain
with a dark blue centre and a light blue
periphery.
 The agar background appears clear or slightly
violet.
 Mycoplasmas other than M. pneumoniae remain
stained, but M.pneumoniae reduces the
methylene blue after a period and becomes
colourless.

Mycoplasma.pptx pptptptttttttttttttttttt

 Glucose and other metabolizable carbohydrates
can be used as energy sources by the
fermentative mycoplasmas possessing the
Embden-Meyerhof-Parnas glycolytic
pathway.
 Oxidative phosphorylation does not occur
as an ATP-generating mechanism.
BIOCHEMICAL PROPERTIES

CONTD….
 Breakdown of arginine by the arginine
dihydrolase pathway has been proposed as a
major source of ATP in nonfermentative
mycoplasmas.
 Ureaplasmas have a requirement, unique among
living organisms, for urea. Because they are non-
glycolytic and lack the arginine dihydrolase
pathway, it has been suggested, and later proven
experimentally, that ATP is generated through
an electrochemical gradient produced by
ammonia liberated during the intracellular
hydrolysis of urea by the organism's urease.

BIOCHEMICAL REACTIONS
SPECIES GLUCOSE ARGININE UREA
M. pneumoniae + - -
M. salivarium - + -
M. orale - + -
M. hominis - + -
U. urealyticum - - +
M. fermentans + + -
M. genitalium + - -
A. laidlawii + - -

ANTIGENIC PROPERTIES:
 Surface Antigens: Made up of Glycolipids and proteins.
 Glycolipids antigens are identified by complement
fixation test.
 Glycolipids with similar antigenic structure have been found
in human brain.
 Protein antigens - ELISA.
 Growth inhibition Test: Particular technique useful for
the identification of isolates based on the ability of antisera
to specifically inhibit the growth of the homologous species
on solid media.

RESISTANCE:
 Destroyed at 450
C in 15 min.
 Resistant to lysis by Osmotic shock, penicillin and
Cephalosporins.
 Sensitive: To Surface acting agents, lipolytic
agents(taurocholate, digitonin), Tetracyclines,
Erythromycin.
 Susceptibility to Macrolide antibiotics and
erythromycin is used for species differentiation.
 Growth is inhibited by Gold salts.
 They( M. pneumoniae) can grow in presence of 0.002%
methylene blue in agar while other species are
inhibited.

CONTD….
 Most mycoplasmas that infect humans and other animals
are surface parasites, adhering to the epithelial
linings of the respiratory and urogenital tracts.
 Adherence is firm enough to prevent the
elimination of the parasites by mucous secretions
or urine.
 The intimate association between the adhering
mycoplasmas and their host cells provides an environment
in which local concentrations of toxic metabolites excreted
by the parasite build up and cause tissue damage .

CONTD….
 Moreover, because mycoplasmas lack cell walls, fusion between
the membranes of the parasite and host has been suggested, and
some experimental evidence for it has recently been obtained.
 Membrane fusion would alter the composition and
permeability of the host cell membrane and enable the
introduction of the parasite's hydrolytic enzymes into the
host cell, events expected to cause serious damage.
 Recent studies have indicated the presence in mycoplasmas of
antigenic variability systems. These systems, some of which
are already defined in molecular genetic terms, are responsible for
rapid changes in major surface protein antigens. The change in
the antigenic coat of the parasite helps it to escape
recognition by the immune mechanisms of the host.

CONTD….
A. Adherence factors
 The adherence proteins are one of the major virulence factors.
 The adherence protein in M. pneumoniae has been identified as a
168kDa protein called P1. The P1 adhesin localizes at tips of the
bacterial cells and binds to sialic acid residues on host epithelial
cells. MgPa is a similar adherence protein in M. genitalium.
 The nature of the adhesins in the other species has not been
established.
 Colonization of the respiratory tract by M. pneumoniae results in the
cessation of ciliary movement. The normal clearance mechanisms
of the respiratory tract do not function, resulting in contamination of
the respiratory tract and the development of a dry cough.

CONTD….
Transmission electron
photomicrographs of the
specialized tip organelle of
cytadherence-positive M.
pneumoniae demonstrating:
a) Truncated structure with nap.
b) Clustering of cytadherence-
related proteins (P1, B, C, P30)
at the tip based on
immunolabeling with ferritin
and colloidal gold and
crosslinking studies.
c) Triton X-100-resistant,
cytoskeleton-like, structure
with distinct bleb and parallel
filaments

TRANSMISSION ELECTRON PHOTOMICROGRAPH OF A
HAMSTER TRACHEA RING INFECTED WITH M.
PNEUMONIAE. M, MYCOPLASMA; M,
MICROVILLUS; C, CILIA.

CONTD….
B. Toxic Metabolic Products
o The intimate association of the mycoplasma and the host
cells provides an environment in which toxic metabolic
products accumulate and damage host tissues.
o Both hydrogen peroxide and superoxide, which are
products of mycoplasma metabolism, have been
implicated in pathogenesis since oxidized host lipids ( like
malonyldialdehyde) have been found in infected tissues.
o Furthermore, the mycoplasmas have been shown to
inhibit host cell catalase by excreting superoxide radicals (
O2
–
), thereby increasing the peroxide concentrations.

CONTD….
C. Immunopathogenesis
o Mycoplasmas can activate macrophages and
stimulate cytokine production and lymphocyte
activation (M. pneumoniae is a superantigen).
Thus, it is has been suggested that host factors also
contribute to pathogenesis.
o Experimental evidence in animals supports this
suggestion. Ablation of thymus function before
infection with M. pneumoniae prevents the
development of pneumonia and animals in which
thymic function is restored develop pneumonia at
an exacerbated rate.

CONTD….
 Epidemiologic data in humans suggest that
repeated infections are required before clinical
disease is observed, again suggesting a role for
host related factors in pathogenesis.
 Most children are infected from 2 - 5 years of age
but disease is most common in children 5-15
years of age.

CONTD….
 Recently, M. pneumoniae has been shown to produce
an exotoxin that is also believed to play a major role
in the damage to the respiratory epithelium that
occurs during acute infection. This toxin, named the
community-acquired respiratory disease toxin
(CARDS) is an ADP-ribosylating and
vacuolating cytotoxin similar to pertussis toxin.
 Evidence from animal models of M. pneumoniae
infection have proven that recombinant CARDS toxin
results in significant pulmonary inflammation,
release of proinflammatory cytokines, and airway
dysfunction.

CONTD….
 Variation in CARDS toxin production among M.
pneumoniae strains may be correlated with the
range of severity of pulmonary disease observed
among patients.
 The organism also has the ability to exist and possibly
replicate intracellularly, which may contribute to
chronicity of illness and difficult eradication.
 Additionally, acute mycoplasmal respiratory tract
infection may be associated with exacerbations of
chronic bronchitis and asthma.

CONTD….
 An immunopathologic mechanism also may explain the complications
affecting organs distant from the respiratory tract in some patients
infected with M. pneumoniae.
 Various autoantibodies have been detected in the sera of many of these
patients, including cold agglutinins reacting with the erythrocyte I
antigen, and antibodies reacting with lymphocytes, smooth
muscle cells, and brain and lung antigens.
 Serologic cross-reactions between M. pneumoniae and brain and
lung antigens have been demonstrated, and these antigens are
probably related to the glycolipids of M.
pneumoniae membranes, which are also found in most plants and in
many bacteria.
 Clearly, host reaction varies markedly, as only about half of the
patients develop cold agglutinins and complications are rare,
even among individuals with anti-tissue globulins.

IMMUNITY
 Complement activation via the alternative
pathway and phagocytic cells both play a role in
resistance to infection.
 As the infection proceeds, antibodies play a role
in controlling infection, particularly IgA.
 The development of delayed type
hypersensitivity, however, is associated
with the severity of the disease, which
supports the suggestion that pathogenesis
is at least, in part, immunopathogenesis.

EPIDEMIOLOGY
 All mycoplasmas cultivated and identified thus far
are parasites of humans, animals, plants, or
arthropods.
 The primary habitats of human and animal
mycoplasmas are the mucous surfaces of the
respiratory and urogenital tracts and the joints in
some animals.
 Although some mycoplasmas belong to the normal
flora, many species are pathogens, causing various
diseases that tend to run a chronic course.

CONTD….
 Pneumonia caused by M. pneumoniae occurs worldwide and no increased seasonal activity is seen.
 Epidemics occur every 4 - 8 years.
 Studies have indicated that M. pneumoniae is second only to Streptococcus pneumoniae as a
cause of bacterial pneumonia that requires hospitalization in elderly adults.
 Subclinical infections may occur in 20% of adults infected with Mycoplasma pneumoniae,
suggesting that some degree of immunity may contribute to the failure of appearance of
clinical symptoms in some instances.
 Recent evidence suggests that M. pneumoniae disease is sometimes much more severe than
appreciated, even in otherwise healthy children and adults.
 Severe disease is more common in persons with underlying disease or immunosuppression.
 The overall mortality rate is extremely low, probably less than 0.1%.

CONTD….
AGE:
 The disease is spread by close contact via
aerosolized droplets and thus is most easily
spread in confined populations (e.g., families,
schools, army barracks). The disease is primarily
one of the young (5 - 15 years of age).
 In recent years, M. pneumoniae infection has been
common in persons older than 65 years,
accounting for as much as 15% of community-
acquired pneumonia cases in persons in this
age group.

CONTD….
RACE:
 No racial predilection is apparent.
SEX:
 Available studies indicate no sexual predilection
for M pneumoniae disease.

 Colonization with M. hominis and U.urealyticum
can occur during birth but in most cases the
infection will be cleared.
 Only in a small number of cases does colonization
persist. However, when individuals become
sexually active, colonization rates increase.
 Approximately 15% are colonized with M. hominis
and 45% - 75% with U. urealyticum.
 The carriers are asymptomatic but the organisms
can be opportunistic pathogens.
GENITAL MYCOPLASMAS

CONTD….
 They are present on mucosal surfaces in so many
healthy persons and can be transmitted
venereally suggesting that variation in
prevalence of these organisms in adults is
more likely related to behavioral variables
such as number of sexual partners and
socioeconomic status rather than to
geographic or climatic differences.
 In adults with an intact and functional
immune system, infections associated with
genital mycoplasmal organisms are usually
localized.

CONTD….
 Persons with antibody deficiencies reportedly
have developed severe pulmonary infections,
destructive arthritis and osteomyelitis
associated with subcutaneous abscesses, and
other disseminated infections of various
organ systems.
 Some organisms, such as M. pirum and
M. penetrans, have been primarily isolated
from persons with HIV infection but their
significance as pathogens in this population has
not been established.

CONTD….
 Deaths have occurred in neonates with
bloodstream invasion by Ureaplasma species
and meningitis caused by M.hominis;
however, in some instances, the organisms
spontaneously disappeared from CSF without
treatment.
 Sporadic case reports document fatal infections
caused by Mycoplasma species of animal
origin, including Mycoplasma arginini in
immunosuppressed hosts, but these are
extremely rare.

CONTD….
AGE:
Clinically significant infections may
ensue in individuals who are
sexually active and in neonates but
are rare to nonexistent in older
children and adolescents who are not
sexually active, with the exception of
those with immunodeficiencies.

CONTD….
RACE:
No racial differences have been observed.
SEX:
 No obvious sex predilection is reported for
infections due to genital mycoplasmal species.
 The carriage rate of genital Mycoplasma &
Ureaplasma species in the lower urogenital tract
is somewhat greater for females than for
males.

SPECTRUM OF DISEASES
ORGANISMS CLINICAL MANIFESTATIONS
M. pneumoniae Asymptomatic infections
URTI in children: Mild non
specific symptoms like running
nose, coryza & cough, most without
fever.
LRTI in adults: Mild illness with
non-productive cough, fever,
malaise, pharyngitis & myalgia; 3-
13% of patients develop pneumonia;
complications include rash,
arthritis, encephalitis, myocarditis,
pericarditis and hemolytic anaemia.

CONTD….
Genital Mycoplasmas:
U. Urealyticum & M. hominis
Systemic infections in neonates:
Meningitis, abscess & pneumonia.
U. urealyticum is also associated
with chronic lung disease.
Invasive disease in
immunocompromised patients:
Bacteremia, arthritis(particularly in
agammaglobulinemias), abscesses &
other wound infections, pneumonia ,
peritonitis.
Urogenital tract infections:
Prostatitis, PID, amnionitis, NGU.
M. genitalium NGU in men. Possible cause of
cervicitis & endometritis in females.

PRIMARY ATYPICAL PNEUMONIA
(PAP)
 Caused by M. pneumoniae.
 Other causes of community acquired atypical
pneumonia are L.pneumophila and
C.pneumoniae.
 Typical features of pneumococcal pneumonias like fever
with chills, pleuritic chest pain and rust- coloured
sputum not seen.
 Not recovered on routine sputum cultures, hence,
atypical.
 Penicillins and cephalosporins are ineffective as
mycoplasmas lack cell wall and these agents have
poor intracellular penetration, therefore, not
effective against Chlamydiae and Legionellae.

CONTD….
 IP of PAP is 1-3 weeks and usually recovery
occurs in 3-10 days without antimicrobial
therapy.
 Some of the antibodies that develop are cold
haemagglutinins, Streptococcus MG agglutinins
and antibodies giving biological false positive
Wasserman reaction.

LABORATORY DIAGNOSIS
 SPECIMENS COLLECTED:
Throat swabs, nasopharyngeal swabs, sputum, throat washings, bronchoalveolar lavage, tracheal aspirates and lung
tissue specimens.
Genital mycoplasmas may be isolated from urethral, vaginal and cervical swabs, semen, prostatic secretions, urine,
blood, CSF, amniotic fluid and biopsy specimens from endometrium, fallopian tubes, placentae and aborted foetus.
 CULTURE:
(1) PPLO broth medium dispensed into small vials may be used for transport of swab specimens, while other
specimens may be transported in sterile screw capped containers.
(2) The culture media should be inoculated as soon as possible.
(3) If delay in inoculation is expected, then specimens my be kept at 4°C for 24 hours and at -70°C for more than 24
hours.
(4) Urine specimens should be centrifuged and deposit inoculated into the medium. If inoculation is not immediately
possible, then urine deposit should be diluted with equal volume of transport medium and frozen.
(5) Later the specimens are processed as mentioned earlier.

CONTD….
 Identification of colonies:
(1) HAEMADSORPTION TEST:
Colonies growing on the agar are flooded with 2ml of
0.2-0.4% suspension of washed guinea pig erythrocytes
suspended in Mycoplasma broth medium. The plate is then
incubated at 35°C for 30 minutes. It is occasionally rotated
during this time. It is then washed with 3ml of
Mycoplasma broth medium by gently rotating the plate.
Wash fluid is removed by aspiration with a pipette and
colonies examined under 50X to 100X magnification.
Colonies of M. pneumoniae adsorb erythrocytes to
their surface. Colonies of M. genitalium also share
this property.

CONTD….
(2) TETRAZOLIUM TEST:
M. pneumoniae has the ability to reduce the colourless
compound triphenyl tetrazolium to red compound formazan. The
agar surface bearing the suspected colonies is flooded with a
solution of 2-p-iodophenyl-3-nitrophenyl-5-phenyl tetrazolium
chloride and incubated at 35°C for 1 hour. Colonies of
M.pneumoniae appear reddish after an hour and may
appear purple to black after 3-4 hours. Other mycoplasmas
are negative.
(3) SEROLOGICAL TECHNIQUES:
These include inhibition of colony development around discs
impregnated with specific antiserum or the fluorescence of
colonies treated with such antiserum labelled with a
fluorochrome.

CONTD….
 Biochemical tests may be employed.
 More sophisticated tests, including
electrophoretic analysis of cell proteins,
DNA-DNA hybridization tests, mycoplasmal
DNA cleavage patterns by restriction
endonucleases, and PCR tests employing species-
specific primers for amplification, may be
performed in a research laboratory.

CONTD….
DETECTION OF ANTIGEN:
In respiratory exudates by direct immunofluorescence and
counterimmunoelectrophoresis techniques, immunoblotting with
monoclonal antibodies and ELISA.
DETECTION OF NUCLEIC ACIDS:
Detection of specific DNA by dot blot hybridization and PCR in
respiratory exudates .
Detection of RNA nucleotide sequences by probe hybridization in
respiratory exudates. M. pneumoniae probe hybridizes with the
16s rRNA of the organism and uses an I-125 radioactive label to
generate a detection signal. Sensitivity and specificity are >90%
and results are available in 2 hours.

SEROLOGICAL TESTS
 Serodiagnosis consists of examining serum samples for
antibodies that inhibit the growth and metabolism of
the organism or fix complement with mycoplasmal
antigens.
 A fourfold or greater rise in IgG titer is
considered indicative of recent infection,
whereas a sustained high antibody titer may not
be significant, because a relatively high level of
antibody may persist for at least 1 year after
infection.
 A variety of rapid tests based on indirect
hemagglutination of erythrocytes or latex particles
coated with M .pneumoniae antigens have been
developed, and some are commercially available.

CONTD….
 Specific Tests: Immunoflouresence,
hemagglutination inhibition & metabolic
inhibition.
 Non specific Tests: Streptococcus MG Tests:- Done
by mixing serial dilutions of the patient’s
unheated serum & a heat killed suspension of
Strep MG & observing after overnight incubation
at 370
C. A Titre of 1:20 is suggestive.

CONTD….
COLD AGGLUTININS:
 Approximately 34% - 68% of patients with M. pneumoniae
infection develop cold agglutinins. Cold agglutinins are
antibodies that agglutinate human erythrocytes at 4 degrees C but
not at 37 degrees C.
 The antigen to which the antibodies are directed is the I
antigen. These antibodies arise before the complement
fixing antibodies and they decline faster .
 Cold agglutinins are not specific for M. pneumoniae infections,
they can also appear in other infections and in other diseases (e.g.
Infectious mononucleosis, rubella, adenovirus infections,
psittacosis, tropical eosinophilia, trypanosomiasis, cirrhosis
of liver, paroxysmal haemoglobinuria, haemolytic anaemia,
influenza infections, cold agglutinin disease, leukemia).
 However, if present in a patient with clinical signs of M.
pneumoniae infection, a presumptive diagnosis can be made.

CONTD….
 Serial dilutions of patients serum are mixed
with an equal volume of 0.2% washed human O
group RBCs & clumping is observed at 40
C
overnight. The clumping is dissociated at 370
C.
 Appear about 1 week after infection with a peak
at 4-5 weeks. Thereafter, titres decline rapidly
and the test becomes negative after about five
months.
 A four-fold rise in titre or a single titre of 32
or more is suggestive of M. pneumoniae
infection.

CONTD….
CFT:
 Most widely used test.
 Complement fixing antibodies appear 7-10
days after infection with the organisms and reach
peak titre after 4-6 weeks.
 Such results are obtained in about 80% of the
cases.
 Antigen used is a glycolipid from the organism
that is extracted by chloroform-methanol.
 A recent infection is indicated by a four-fold rise
in antibody titre or a single titre of 64 or more.

CONTD….
ELISA:
EIA for detection of M. pneumoniae specific IgM,
IgG and IgA antibodies have been developed.
More sensitive than CFT.
Sensitivity is 92% and Specificity is 95%.

TREATMENT & PREVENTION:
AGENT DOSE
Doxycycline 100mg bid
Erythromycin 500mg qid
Clarithromycin 500mg bid
Azithromycin 500mg qd
Levofloxacin 500mg qd
Gatifloxacin 400mg qd
Moxifloxacin 400mg qd
No vaccines are available.

MYCOPLASMAS AS NORMAL FLORA
ORGANISM SITE OF COLONIZATION
M. orale Oropharynx
M. salivarium Oropharynx
M. buccale Oropharynx
M. faucium Oropharynx
M. lipophilum Oropharynx
M. primatum Genital tract
M. spermatophilum Genital tract

REFERENCES
 Mackie & Mc Cartney Practical Medical Microbiology-
14th
edition.
 Bailey & Scott’s Diagnostic Microbiology-12th
edition.
 Color atlas & textbook of diagnostic
microbiology(Elmer.W.Koneman)-5th
edition.
 Topley & Wilsons Microbiology & Microbial
infections- 10th
edition
 Saunder’s text and review series by: T.Stuart Walker.
 Jawetz Medical Microbiology-24th
edition.
 Textbook of Microbiology by Ananthanarayan and
Paniker-8th
edition.
 Textbook of Microbiology by D.R. Arora(3rd
edition).

CONTD….
 Microbiology and immunology online
University of South Carolina: Mycoplasma and
Ureaplasma by Dr Gene Meyer
www.pathomicro.sc.edu
 Mycoplasmas by Shmuel Razin
www.ncbi.nlm.nih.gov
 Mycoplasma infections
www.emedicine.medscape.com
 www.wikipedia.com

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