1. ChlamydiaeChlamydiae
Mr. Gunjal Prasad NiranjanMr. Gunjal Prasad Niranjan
M.Sc. Medical MicrobiologyM.Sc. Medical Microbiology
PG Dip. CRAPG Dip. CRA
Assistant Prof. Dept. MicrobiologyAssistant Prof. Dept. Microbiology
PDVVPF’s Medical College, AhmednagarPDVVPF’s Medical College, Ahmednagar

2. IntroductionIntroduction
• Obligate intracellular parasites.
• Small, Non - motile, Gram negative.
• Have tropism for squamous epithelial cells & macrophages of
respiratory and gastrointestinal tracts.
• CausesCauses
• 1. Trachoma,1. Trachoma,
• 2. Lymphogranuloma Venerum (LGV),2. Lymphogranuloma Venerum (LGV),
• 3. Psittacosis.3. Psittacosis.
• Can cause diverse diseases in birds and mammals.
• The name ChlamydiaChlamydia – Derived from characteristic appearance
of inclusion bodyinclusion body by these agents.
• The inclusion bodies enclose the nuclei of infected cells as a
cloak or mantle( Chlamys meaning =mantle = symbol of( Chlamys meaning =mantle = symbol of
authority).authority).

3. Difference betweenDifference between ChlamydiaChlamydia and Virusesand Viruses
• Are intercellular parasites, so previously thought to be viruses.
• Also they are filterable through filters retaining bacteria.
• Also due to failure to grow on cell free media.
• Lack the ability to produce own ATP, use host cell ATP. Hence
called Energy parasites.Energy parasites.
• Differ from viruses on following properties -Differ from viruses on following properties -
• They have both DNA and RNA as bacteria.
• Rigid cell walls and ribosomes as bacteria.
• Multiply by binary fission.
• Susceptible to antibiotics and chemotherapeutic agents.
• They do not have an eclipse phase following cellular infection like
viruses.

4. CHLAMYDIACHLAMYDIA
• Order –Order – ChlamydialesChlamydiales
• Family – Chlamydiaceae (only one family)Family – Chlamydiaceae (only one family)
• 4 species4 species in the GenusGenus ChlamydiaChlamydia –
C. trachomatis,C. trachomatis,
C. pneumoniae, affects humansC. pneumoniae, affects humans
C. psittaciC. psittaci andand
C. pecorum affects ruminantsC. pecorum affects ruminants
• All are non-motile, Gram Negative; share antigens, haveAll are non-motile, Gram Negative; share antigens, have
both DNA and RNA, divide by binary fission.both DNA and RNA, divide by binary fission.

5. MorphologyMorphology
• Morphologically Chlamydia exist in two forms asMorphologically Chlamydia exist in two forms as
belowbelow
• Elementary Body / EB: -Elementary Body / EB: -
• Spherical particle measuring 200- 300 nm in size.
• It is an Extracellular infective form.
• Reticulate (Initial) Body/ RB:-Reticulate (Initial) Body/ RB:-
• Non-infectious.
• Intracellular, growing and replicating form.

6. • Elementary Body (EB) the infectious form enters the host cell by
phagocytosis.
• Enlarges its shape and size to develop into Reticulate Body (RB), 500-
1000 nm in size. RB divides repeatedly by binary fusion to form
large number of EB.
• The developing chlamydial microcolony within host cells known as
Inclusion body each mature inclusion body contains about 100 -500
EB.
• These newly formed EB’s, on release may infect the new cells and
the cycle continues.
• Duration of this developmental cycle is about 24- 28 hrs.
MorphologyMorphology

8. C. trachomatisC. trachomatis
• 20 serotypes, classified on the basis of neutralization and
Immunofluorescence tests.
• A, B, Ba, C, D, Da, E, F, G, H, I, Ia, J, Ja, K, L1, L2, L2a, L2b & L3.
• Serotypes A, B, Ba, C – Responsible for causing –
• Hyperendemic Trachoma –Hyperendemic Trachoma – inflammation of the conjunctiva
and cornea and the formation of scar tissue..

9. • Serotype D, Da, E, F, G, H, I, Ia, J, Ja, K – Causes –
• Inclusion Conjunctivitis,Inclusion Conjunctivitis,
• Non - Gonococcal Urethritis (NGU),Non - Gonococcal Urethritis (NGU),
• SalpingitisSalpingitis – inflammation of fallopian tube,
• CervicitisCervicitis,
• PneumoniaPneumonia of new born.
C. trachomatisC. trachomatis

10. • Serotypes L1, L2, L2a, L2b, & L3 –Serotypes L1, L2, L2a, L2b, & L3 – cause LymphogranulomaLymphogranuloma
Venereum (LGV) -Venereum (LGV) -
• A disease caused by Chlamydia trachomatis L1 to L3;
• Transmitted by sexual contactTransmitted by sexual contact; LGV is primarily an infection of
lymphatics and lymph nodes.
• It gains entrance through breaks in the skin, or it can cross the
epithelial cell layer of mucous membranes.
C. trachomatisC. trachomatis

11. Lymphogranuloma Venereum (LGV) -Lymphogranuloma Venereum (LGV) -
• The organism travels from the site of
inoculation down the lymphatic channels
to multiply within mononuclear
phagocytes of the lymph nodes it passes.
• Causes inflammation of and prevents
drainage of the lymph nodes in the genital
area.
• LGV can cause destruction and scarring of
surrounding tissue.

12. C. pneumoniaeC. pneumoniae
• It’s an exclusive human pathogen with no animal host.
• Third most common cause of pneumonia after S.
pneumoniae, & H. influenzae.
• Causes – Acute Respiratory Disease in humans.
• Has only 1 serotype.
• Serological studies shows prevalence rate – 40 -50 %.

13. C. psittaciC. psittaci
• It causes “Psittacosis and Ornithosis”“Psittacosis and Ornithosis” in birds
and humans.
• Human are infected by air born route through
inhalation of dried faeces of infected birds.
• The disease may vary from mild influenza like
illness to severe pneumonia, septicaemia and
Meningoencephalitis.

14. Human DiseasesHuman Diseases
SpeciesSpecies SerotypeSerotype DiseasesDiseases
C. trachomatisC. trachomatis A, B, Ba, CA, B, Ba, C Endemic blinding,Endemic blinding,
TrachomaTrachoma
C. trachomatisC. trachomatis D to KD to K InclusionInclusion
conjunctivitisconjunctivitis
Genital ChlamydiasisGenital Chlamydiasis
C. trachomatisC. trachomatis L1, L2, L3L1, L2, L3 LymphogranulomaLymphogranuloma
Venereum (LGV)Venereum (LGV)
C. psittaciC. psittaci Many serotypesMany serotypes Psittacosis ,Psittacosis ,
OrnithosisOrnithosis
C. pneumoniaeC. pneumoniae Only one serotypeOnly one serotype Acute RespiratoryAcute Respiratory
DiseaseDisease
C. pecorumC. pecorum Primary pathogen ofPrimary pathogen of
ruminantsruminants

15. A. Ocular Infection – TrachomaA. Ocular Infection – Trachoma
• Caused by C. trachomatis serotype A, B, Ba, & C.
• Chronic Kerato-conjunctivities.Chronic Kerato-conjunctivities.
• Major cause of blindness.
• Characterized by follicles, papillary hyperplasia, pannus
formation and in the late stage – Cicatrization.
• Transmission –Transmission – Eye to eye through finger, flies & fomites -
contaminated towel, clothing.
• Incubation period –Incubation period – 3-10 days.
• In endemic area children below 9 yrs age are mostly affected.
• Trachoma has been characterised into I – IV stages, early
stage is most infective one.

16. 2.Inclusion Conjunctivitis2.Inclusion Conjunctivitis
• Caused by C. trachomatis serotype D to K.
• Prevalent in sexually active young people & spread from
genital secretions to the eye by contaminated hands contact.
• Characterised by follicular hypertrophy with scanty non-
purulent discharge.
• This is formerly known as ““ Swimming pool conjunctivitis”Swimming pool conjunctivitis” as
it was associated with swimming in contaminated water.
• Also know as Para-trachoma.Also know as Para-trachoma.

17. 3. Opthalmia neonatorum3. Opthalmia neonatorum
• Neonatal form of Inclusion conjunctivitis - InclusionInclusion
BlenorrheaBlenorrhea
• Infants acquires infection during passage through the infected
birth canal.
• It usually becomes apparent between 5-12 days after birth.
• About 20-50% infants of infected mothers develops the
infection. Considered benign & self limiting few may develop
conjunctival scar.
• Prevented by local antibiotic applications.

18. B. Genital Infections - Genital ChlamydiasisB. Genital Infections - Genital Chlamydiasis
• Chlamydia trachomatis causes “Genital Chlamydiasis” and LGV.“Genital Chlamydiasis” and LGV.
• Both are sexually transmitted diseasesBoth are sexually transmitted diseases..
• 1. Genital Chlamydiasis -1. Genital Chlamydiasis - Caused by D to K serotype.
• Responsible for 40% of cases of NGU, it is a STD.
• In malesIn males
• Urethritis - inflammation of the urethra; results in painful
urination.
• Epididymitis - painful inflammation of the epididymis –
convoluted tubule in each testis, carries sperm to vas difference.
•
• Proctitis - inflammation of the rectum; marked by bloody stools
and a frequent urge to defecate.

19. • In females –In females –
• Urethritis -Urethritis - inflammation of the urethra; results in painful
urination.
• Cervicitis -Cervicitis - inflammation of the uterine cervix.
• Salpingitis -Salpingitis - inflammation of a Fallopian tube (usually the result
of infection spreading from the vagina or uterus).
• Pelvic Inflammatory Disease (PID) -Pelvic Inflammatory Disease (PID) - Inflammation of the female
pelvic organs (especially the Fallopian tubes) caused by infection
chieflychiefly GonococciGonococci andand ChlamydiaChlamydia;; symptoms are abdominal
pain and fever and foul-smelling vaginal discharge.
• Infection may symptomatic or asymptomatic.
• Symptoms –Symptoms – Dysuria –painful or difficulty in urination,
• Non-purulent discharge and frequency of urination.
B. Genital Infections - Genital ChlamydiasisB. Genital Infections - Genital Chlamydiasis

20. 2. Lymphogranuloma Venereum2. Lymphogranuloma Venereum
• LGV is a sexually transmitted disease caused by C.
trachomatis serotypes L1 to L3.
• The primary lesion consist of small painless papule or vesicle
on external genitalia.
• It may ulcerate or healulcerate or heal spontaneously in a few days.
• The regional lymph nodes (inguinal in males and intrpelvic &
para - rectal in female) are enlarged, tender, and may break
open with the formation of sinuses.
• The enlarged inguinal lymph nodes are named bubos.bubos.

22. C. Respiratory Infections – PneumoniaC. Respiratory Infections – Pneumonia
• Third commonest cause of pneumonia following
S. pneumoniae & H. influenzae.
• It is an important risk factor in cardiovascular disease, where
organism is isolated from coronary artery.
• However more studies are required to ascertain its role.

23. 2. Psittacosis2. Psittacosis
• C. psittaci causes “Psittacosis and Ornithosis”“Psittacosis and Ornithosis” in birds and
man.
• Human infection occurs by inhalation of infected dried faeces.
• PsittacosisPsittacosis (Psittacos – parrot) is a disease of parrots.
• Disease acquired from non- psittacine birds known as
“Ornithosis” (Ornithos – birds).
• Incubation periodIncubation period – 1-2 wks.
• Disease may vary from mild influenza to severe illness with
pneumonia, septicaemia and meningoencephalitis.

24. Laboratory DiagnosisLaboratory Diagnosis
• 4 approaches available:
1. Microscopic demonstration of inclusion or
elementary bodies.
2. Isolation of Chlamydia.
3. Demonstration of Chlamydial Ag.
4. Demonstration of Abs or hypersensitivity.

25. 1. Microscopy1. Microscopy
• Gram stain – Gram Negative,.
• Stain better with Castaneda, Mchiavello,
or Giemsa Stain.
• Inclusion bodies are basophilic &
present in cytoplasm.
• Inclusion bodies can be stained with -
Lugol’s iodine – because of presence of
Glycogen matrix.
• Immunofluorescence staining.
IF stainingIF staining

26. 2. Culture2. Culture
• Animal inoculation
• Yolk sac of 6 - 8 days
old chick embryo.
• Tissue culture –
McCoy, HeLa cell lines
Infected cell cytoplasm has a
granular appearance

27. • 1. Animal inoculation –1. Animal inoculation – Mice are inoculated by intranasal or
intraperitoneal or by intracerebral inoculations.
• Mice die within 10 days and smears from various tissue (lung,
peritoneal exudates, spleen or brain) shows elementary
bodies.
• 2. Yolk Sac Inoculation –2. Yolk Sac Inoculation – Yolk sac of chick embryo is
inoculated & the organism can be detected in impression
smears stained by Giemsa stain.
2. Culture2. Culture

28. • 3. Tissue Culture –3. Tissue Culture –
• McCoy cell lines treated with
cycloheximide are mostly used.
• Mouse fibroblast,
• HeLa 229 or
• Monkey kidney cells can also be used.
• Organism growth is detected in tissue
culture by staining for elementary bodies
or inclusion bodies.
2. Culture2. Culture
Infected cell
cytoplasm has a
granular appearance

29. Inclusion bodiesInclusion bodies
• Demonstration of
characteristic inclusion
bodies.
1.1. Trachoma:Trachoma: Halber staedter
Prowazekii or HP bodies in
conjunctival scrapings.
2.2. Psittacosis:Psittacosis: LCL (Levinthal –
Cole - Lillie) bodies in alveolar
macrophages, cell lines.

30. Treatment and PreventionTreatment and Prevention
• Tetracycline, Erythromycin and Sulfonamides.
• Vaccination not proved to be an effective or
practicable method of control.
• Treatment coupled with improved sanitation.
• Safe sexual practices.
• Treatment of patients and their sexual partners.

31. MYCOPLASMAMYCOPLASMA

32. Morphology
• Smallest Free living microorganisms.
• Can pass through bacterial filters.
• Pleomorphic.
• May present as small spherical or branching
filaments.
• Lack rigid cell wall but have triple layered celltriple layered cell
membranemembrane rich in cholesterol and other lipids.
• Resistant to antibiotics acting on cell wall due to
absence of cell wall for e.g. – Penicillin and
Cephalosporins.
• Gram negative.
• Better stained by Giemsa stain.
• Non sporing, Non-flagellated.

33. Mycoplasma of HumansMycoplasma of Humans
• Parasitic
1. Established pathogens: M. pneumoniae.
2. Presumed pathogens: M. hominis, U. urealyticum.
3. Non pathogenic: M. orale, M. buccale, M. genitalium,
M. fermentans.
• Saprophytic –
• Present mainly on skin & in mouth.

34. PathogenicityPathogenicity
• Produce surface infections – Adhere to the
mucosa of respiratory, gastrointestinal &
genitourinary tracts with the help of adhesin.
• Two types of diseases:
1. Atypical Pneumonia
2. Genital infections

35. Mycoplasma pneumoniaMycoplasma pneumonia
• Causes Primary Atypical Pneumonia/ Walking pneumonia.Primary Atypical Pneumonia/ Walking pneumonia.
• Seen in all ages. Self limiting recovery occurs within
two wks.
• Incubation period: 1-3 wks.
• Transmission: Airborne droplets of nasopharyngeal
secretions, close contacts (families, military recruits).
• The Mycoplasma may remain in throat for two or more
months after recovery.

36. Mycoplasma pneumoniaMycoplasma pneumonia

37. Mycoplasma pneumoniaMycoplasma pneumonia
• Gradual onsetGradual onset with fever, malaise - mild sickness or
depression, chills, headache & sore throat.
• Severe coughSevere cough with blood tinged sputum (worsens at
night).
• Complications:Complications:
• Bullous myringitisBullous myringitis – Inflammation of eardrum.
• OtitisOtitis – Inflammation of ear,
• Meningitis –Meningitis – Inflammation of meningis,
• Encephalitis –Encephalitis – Inflammation of brain,
• Hemolytic anemia –Hemolytic anemia – Abnormal breakdown of RBCs.

38. Organ system Symptoms & diseasesOrgan system Symptoms & diseases
caused by mycoplasmacaused by mycoplasma
• Respiratory systemRespiratory system - Pharyngitis - Dry or mildly
productive cough, Wheeze, especially in children, and if
history of asthma, Pneumonia.
• Nervous system -Nervous system - Encephalitis, Aseptic meningitis,
Cerebellar ataxia, Transverse myelitis, Guillain-Barré
syndrome.
• Liver -Liver - Hepatitis - thought to be due to antibody-
mediated inflammation. Mildly abnormal liver function
blood test are common.
• Heart –Heart – Myocarditis, Pericarditis and pericardial effusion.

39. • Blood system -Blood system - Haemolytic anaemia in 60%; usually
mild but may be severe in those with sickle cell
disease. Cold agglutinins may be detected.
• Musculoskeletal system-Musculoskeletal system- Muscle and joint pains are
common. Polyarthritis is rare, thought to be due to
immune response.
• M. pneumoniae has been isolated from joint fluid in
some cases.
• Gastrointestinal system -Gastrointestinal system - Nausea and vomiting,
Abdominal pain, Diarrhoea.
Organ system Symptoms & diseasesOrgan system Symptoms & diseases
caused by mycoplasmacaused by mycoplasma

40. Genital InfectionsGenital Infections
• Caused by M. hominis & U. urealyticum.
• Transmitted by sexual contact.
• Men - Nonspecific urethritis - Inflammation of
Urethra,
• Proctitis - Inflammation of the rectum; marked by
bloody stools and a frequent urge to defecate,

41. • Women -Women -
• Acute Salpingitis – Inflammation of fallopian tube,
• Pelvic Inflammatory Disease -Inflammation of the female
pelvic organs (especially the Fallopian tubes) caused by infection
chiefly Gonococci and Chlamydia; symptoms are abdominal pain
and fever and foul-smelling vaginal discharge.
• Cervicitis, Vaginitis.
• Also associated with infertility, abortion, postpartum
fever – fever immediately after birth,
• Chorioamnionitis – Inflammation of fetal membrane & low
birth weight infants.

42. Laboratory DiagnosisLaboratory Diagnosis
• Specimens –
• Throat swabs, Sputum,
• Nasopharyngeal swabs,
• Respiratory secretions (M. pneumoniae)
• Genital secretions, Urine, cervical
swabs (M. hominis, U. urealyticum)
• Microscopy –
1. Highly pleomorphic, varying from small
spherical shapes to longer branching
filaments.
2. Gram negative, but better stained with
Giemsa, IF staining methods.

43. Laboratory DiagnosisLaboratory Diagnosis
• Isolation of MycoplasmaIsolation of Mycoplasma (Culture) –
1. Semi solid enriched medium containing 20% horse or
human serum, yeast extract & DNA.
Penicillin & Thallium acetate are selective agents.
(serum – source of cholesterol & other lipids)
1. Incubate aerobically for 7 -12 days with 5–10% CO2
at 35-37°C.
Temp range 22- 41°C,
Parasites 35- 37°C,
Saprophytes – lower temp.

44. Laboratory DiagnosisLaboratory Diagnosis
3. Typical “fried egg” appearance of colonies –
Central opaque granular area of growth, surrounded by a
flat, translucent peripheral zone.
Colonies best seen with a hand lens after staining with
Diene’s method –A block of agar on which colony is there
is cut kept on slide and covered with coverslip on which an
alcoholic solution of methylene blue and azure is added
4. Produce beta hemolytic colonies, can agglutinate guinea
pig erythrocytes.

45. Dr Ekta,Microbiology, GMCA
Fried egg coloniesFried egg colonies

46. Mycoplasma & HIV infectionMycoplasma & HIV infection
• Severe & prolonged infections in HIV
infected & other immunodeficient individuals
were reported by many parts of world.

47. TreatmentTreatment
• Tetracycline, Erythromycin & Clarithromycin – drug ofdrug of
choice.choice.
• Resistant to antibiotics which interfere with bacterial
cell wall synthesis as it lacks cell wall.
• Newer Macrolides -Newer Macrolides - They inhibit protein synthesis by
blocking the 50S ribosomal subunit &
• Quinolones –Quinolones – Targets the bacterial enzyme DNA gyrase
in order to inhibit the coiling of bacterial DNA, thus
interfering with bacterial replication being used now.