1. Mycoplasma Introduction:
Nocard and Roux: isolated first member of mycoplasma in 1898 in bovine pleuropneumonia
Old name : pleuropneumonia like organisms [PPLO]

2. 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

3. 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).

4. 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.

5. DIFFERENCES BETWEEN MYCOPLASMAS, BACTERIA, CHLAMYDIA & VIRUSES
CHARACTER
MYCOPLASMA
BACTERIA
CHLAMYDIA
VIRUSES
SIZE µm
1-2µm
0.3µm µm
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

6. 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 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.

7. 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 .

8. 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.

9. GENERAL CHARACTERISTICS
Very small( 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.

10. 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).

11. 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.

12. 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.
The coccus is the basic form of all mycoplasmas in culture.
The diameter of the smallest coccus capable of reproduction is about 300 nm.
In most mycoplasma cultures, elongated or filamentous forms (up to 100 μm long and about μm thick) also occur.
The filaments tend to produce truly branched mycelioid structures, hence the name mycoplasma (myces, a fungus; plasma, a form).
Mycoplasmas reproduce by binary fission, but cytoplasmic division frequently may lag behind genome replication, resulting in formation of multinuclear filaments

13. 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.

14. 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.

15. 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.

16. 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.

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

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

19. 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).

20. 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 , for arginine metabolizing organisms it should be around 7 and for ureaplasmas, range of pH should be

21. 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.

22. 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 107organisms/ml of culture.
Mycoplasma genitalium, another human pathogen, grows so poorly in vitro that only a few successful isolations have been achieved.

23. 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.

24. 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).

25. 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: µm 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.

26. COLONY MORPHOLOGY

27. 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.

28. 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.

30. BIOCHEMICAL PROPERTIES
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.

31. 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.

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

33. 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.

34. Resistance: Destroyed at 450C 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.

35. PATHOGENESIS

36. 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 .

37. 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.

38. 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.

39. 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

40. Transmission electron photomicrograph of a hamster trachea ring infected with M. pneumoniae M, mycoplasma; m, microvillus; C, cilia. 

41. CONTD…. B. Toxic Metabolic Products
The intimate association of the mycoplasma and the host cells provides an environment in which toxic metabolic products accumulate and damage host tissues.
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.
Furthermore, the mycoplasmas have been shown to inhibit host cell catalase by excreting superoxide radicals ( O2– ), thereby increasing the peroxide concentrations.

42. CONTD….

43. CONTD…. C. Immunopathogenesis
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.
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.

44. 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 years of age but disease is most common in children years of age.

45. 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.

46. 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.

47. 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.

48. 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.

49. 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.

50. CONTD….
Pneumonia caused by M. pneumoniae occurs worldwide and no increased seasonal activity is seen.
Epidemics occur every 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%.
In USA, M. pneumoniae causes approximately 20% of community-acquired pneumonias that require hospitalization and probably an even greater proportion of those that do not require hospitalization. M pneumoniae may exist endemically in large urban areas. Disease tends to not be seasonal, except for a slight increase in late summer and early fall.
M .pneumoniae infections occur both endemically and in cyclic epidemics in Japan and several European countries, similar to what occurs in the United States. Less information is available for tropical or polar countries; however, based on seroprevalence studies, the disease also occurs in these regions, suggesting that climate and geography are not important determinants in the epidemiology of M. pneumoniae infections.

51. 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 ( 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.

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

53. GENITAL MYCOPLASMAS
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.

54. 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.

55. 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.

56. 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.

57. 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.

58. 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.

59. 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.

60. 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.

61. 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.

62. 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.

63. LABORATORY DIAGNOSIS SPECIMENS COLLECTED: CULTURE:
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.

64. CONTD…. Identification of colonies: (1) HAEMADSORPTION TEST:
Colonies growing on the agar are flooded with 2ml of % 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.

65. 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.

66. 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.

67. 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.

68. 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.

69. 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 370C. A Titre of 1:20 is suggestive.

70. 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.

72. CONTD….
Serial dilutions of patients serum are mixed with an equal volume of 0.2% washed human O group RBCs & clumping is observed at 40C overnight. The clumping is dissociated at 370C.
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.

73. CONTD…. CFT: Most widely used test.
Complement fixing antibodies appear 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.

74. 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%.

75. 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.

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

77. 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).

78. CONTD…. Microbiology and immunology online
University of South Carolina: Mycoplasma and Ureaplasma by Dr Gene Meyer
Mycoplasmas by Shmuel Razin
Mycoplasma infections

79. Thank You