Mycoplasma: Characteristics, History, and Impact

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

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….



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.

E

LECTRON

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.

C

ONTD

…



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 10

7

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).



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.

            COLONY MORPHOLOGY

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.



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

A

NTIGENIC

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.

R

E

S

I

S

T

A

N

C

E

:



Destroyed at 45

0

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.

PATHOGENESIS

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

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 ( O

2

–

 ), thereby increasing the

peroxide concentrations.

CONTD….

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

CONTD….

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 37

0

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 4

0

C overnight.

The clumping is dissociated at 37

0

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%.

T

REATMENT

 & PREVENTION

:

A

G

E

N

T

D

O

S

E

D

o

x

y

c

y

c

l

i

n

e

1

0

0

m

g

b

i

d

E

r

y

t

h

r

o

m

y

c

i

n

5

0

0

m

g

q

i

d

C

l

a

r

i

t

h

r

o

m

y

c

i

n

5

0

0

m

g

b

i

d

A

z

i

t

h

r

o

m

y

c

i

n

5

0

0

m

g

q

d

L

e

v

o

f

l

o

x

a

c

i

n

5

0

0

m

g

q

d

G

a

t

i

f

l

o

x

a

c

i

n

4

0

0

m

g

q

d

M

o

x

i

f

l

o

x

a

c

i

n

4

0

0

m

g

q

d

N

o

v

a

c

c

i

n

e

s

a

r

e

a

v

a

i

l

a

b

l

e

.

MYCOPLASMAS AS NORMAL FLORA

REFERENCES



Mackie & Mc Cartney Practical Medical Microbiology-

14

th

 edition.



Bailey & Scott’s Diagnostic Microbiology-12

th

 edition.



Color  atlas & textbook of diagnostic

microbiology(Elmer.W.Koneman)-5

th

 edition.



Topley & Wilsons Microbiology  & Microbial

infections- 10

th

 edition



Saunder’s text and review series by: T.Stuart Walker.



Jawetz Medical Microbiology-24

th

 edition.



Textbook of Microbiology by Ananthanarayan and

Paniker-8

th

 edition.



Textbook of Microbiology by D.R. Arora(3

rd

 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