Chapter 43 Mycoplasma and Ureaplasma ( 黴漿菌 , 尿漿菌 ) Chapter 46 Chlamydiaceae ( 披衣菌 / 衣源體 )

1. Chapter 43Mycoplasma and Ureaplasma(黴漿菌, 尿漿菌) Chapter 46Chlamydiaceae(披衣菌/衣源體) Chapter 44Rickettsia and OrientiaChapter 45Ehrlichia, Anaplasma, Coxiella Yu Chun-Keung DVM, PhD Department of Microbiology and Immunology

2. Chapter 43 Mycoplasma and Ureaplasma • 200 species; 16 colonize humans • Mycoplasma(黴漿菌) M. pneumoniae M. hominis M. genitalium • Ureaplasma(尿漿菌) U. urealyticum

3. Smallest (0.1-0.3 m) and simplest free-living bacteria (about twice the genome size of certain large viruses) • Small, fried-egg-like colonies (except M. pneumoniae)

4. Lack a cell wall • Highly pleomorphic shapes • Resistant to penicillin, cephalosporins, vancomycin, but sensitive to tetracycline, erythromycin. • Cell membrane contains sterols - rigid • Anaerobic (except M. pneumoniae) • Grow slowly in cell-free media, need sterols, use glucose as a source of energy (ureaplasmas require urea)

5. Epidemiology M. pneumoniae • Strict human pathogen • Worldwide disease with no seasonal incidence • Most common in school-age children and young adults (5-15y), but all age groups are susceptible • Spread by respiratory droplets during coughing episodes in close contact among classmate or family members

6. U. urealyticum, M. hominis, and M. genitalium • Infants (females) are colonized with the agents • Carriage does not persist. Only a small proportion of prepubertal children remains colonized • The incidence of genital mycoplasmas is associated with sexual activity • Sexually active men and women 15% with M. hominis and 45-75% with Ureaplasma

7. Pathogenesis - M. pneumoniae Extracellular pathogen; infect and colonize mucous membrane (nose, throat, trachea, LRT). Adheres to sialated glycoprotein receptor (1) at the base of cilia, (2) on surface of RBC by means of P1 antigen.

8. The mechanism of cellular damage is unknown (produce peroxide and hemolyze RBC?) • Causes ciliostasis, destroy cilia and ciliated epithelial cells; breakdown clearance activity, lead to LRT infection and persistent cough. • M. pneumoniae contains superantigen, can attract inflammatory cells and induce cytokine secretion (TNF, IL-1, IL-6).

9. Clinical disease - M. pneumoniae • Mostly asymptomatic carriage • Cause mild URT disease (acute pharyngitis), low-grade fever, malaise, headache, dry and nonproductive cough, persist for > 2 weeks • Tracheobronchitis with lymphocyte and plasma cell infiltration, atypical (walking) pneumonia • Secondary complication: hemolytic anemia, arthritis, myocarditis, pericarditis, neurologic abnormalities (e.g., meningoencephalitis)

10. Typical pneumonia - bacterial pneumonia • Abrupt, rigorous onset • Productive cough, purulent sputum • High fever, chest pain, stiffness in the neck • Chest consolidation and rales. Murray, et.al: Textbook of Respiratory Medicine

11. Atypical (walking) pneumonia • Chronic in both onset and recovery • Flulike symptomes - generalized aches, discomfort, headache, chill, dry cough, low-grade fever • Chest radiographs: patchy broncho-pneumonia, interstitial pattern, not pneumonia Murray, et.al: Textbook of Respiratory Medicine

12. Diseases caused by U. urealyticum and M. genitalium and M. hominis • M. genitalium : nongonococcal urethritis (NGU), pelvic inflammatory disease • U. urealyticum : NGU, pyelonephritis, abortion, premature birth • M. hominis : pyelonephritis, postpartum fever, systemic infection in immunocompromised patients

13. Lab diagnosis • Culture of mycoplasmas is not routinely attempted, and relatively insensitive • M. pneumoniae can grow in special medium with animal serum (sterols), yeast extract (nucleic acid), glucose, pH indicator, and penicillin. Colonies have a “mulberry-shaped”. • M. hominisrequires arginine for growth. Colonies have a fried-egg appearance. • Ureaplasmarequires urea for growth • Microscope: no cell wall, stain poorly, no value

14. Serology – for M. pneumoniae only • Complement fixation test : high false-positive rate • ELISA for detection of IgM and IgG Abs, more sensitive; need dual serum samples • Cold agglutinins: • IgM Abs that bind the I antigen on human RBC (type O) at 4°C, develop in 65% of the patients – insensitive and nonspecific.

15. Treatment / Prevention / Control • M. pneumoniae: erythromycin, tetracycline (also good for chlamydia) • Ureaplasma: use erythromycin, resistant to tetracycline • M. hominis: resistant to erythromycin and tetracycline, use clindamycin • Avoidance or safe sex for genital mycoplasma • No vaccine available

16. Chapter 46Chlamydia and Chlamydophila Family Chlamydiaceae Genus Chlamydia: C. trachomatis (砂眼披衣菌) Genus Chlamydophilia: C. pneumoniae (肺炎披衣菌)C. psittaci (鸚鵡熱披衣菌)

17. Chlamydiaceae • Obligate intracellular organisms • Were once considered virus, true bacteria • Contain DNA and RNA • Possess ribosomes, synthesize proteins, nucleic acid, and lipids, but cannot synthesize ATP. • Binary fission • Susceptible to numerous antibiotics, but not to penicillin (lack peptidoglycan) • Cell wall: • Major outer membrane protein (MOMP) – serological variants (serovars) • Outer membrane protein 2 (OMP2) – cysteine-rich protein, structure stability of elementary body (EB)

18. Unique development cycle Two morphological distinct forms in cytoplasmic phagosome: (1) elementary body (300-400 nm), resistant to harsh environmental factors; bind to receptors of host cells and stimulate uptake; cannot replicate but infectious, (2) reticulate body (800-1000 nm), reproductive form, metabolically active, noninfectious. Histologic stains can detect phagosome with accumulated RBs (inclusion)

19. 1. Chlamydia trachomatis (砂眼披衣菌) • Infections only occur in humans • Two biovarsand 18 serovars (antigenic differences in MOMP) Biovars Serovars Disease Trachoma A to C Trachoma D to K Urethritis, cervicitis, Inclusion conjunctivitis, Neonatal conjunctivitis, Infant pneumonia LGV L1 to L3 Lymphogranuloma venereum (LGV)

20. Pathogenesis • EBs enter the body via minute abrasions and lacerations • Trachoma serovars primarily infect nonciliatedepithelial cells (urethra, endocervix, endometrium, fallopian tube, anorectum, respiratory tract, conjunctiva) • LGV serovars replicate in mononuclear phagocytes(more invasive); formation of granuloma in lymph nodes draining the site of primary infection, abscesses, or sinus tracts formation

21. Pathogenesis • Direct destruction of cells during replication • Proinflammatory cytokine response stimulates a severe inflammation (accumulations of neutrophils, lymphocytes and plasma cells). • No long-lasting immunity after infection • Re-infection induces a vigorous inflammatory response with subsequent tissue damage (blindness and sterility).

22. Trachoma (砂眼) • A chronic keratoconjuctivitis caused by serovars A, B, Ba, C. • Diffuse follicularconjunctivitis→ eyelid inward → eyelashes abrade cornea → corneal ulceration → pannus formation(invasion of vessels into the cornea) →blindness • Endemic in the Middle East, North Africa, and southern Asia (crowded and poor sanitation regions); predominantly in children. Leading global causes of preventable blindness (>150 million infected, 6 million blinded). • Transmission: eye-to-eye by droplet, hands, contaminated clothing, flies.

24. Urogenital infections • Venereal infections caused by serovars of D to K. • The most common sexually transmitted bacterial disease in U.S. 2.8 million new cases annually (50 million worldwide). • In women: 80% asymptomatic as reservoir; bartholinitis, cervicitis, endometritis, salpingitis, urethritis, which can lead to sterility and ectopic pregnancy. • In men: 25% asymptomatic; nongonococcal urethritis (NGU; urethritis caused by pathogens other than gonococcus)

25. Nongonococcal Urethritis (NGU) • C. trachomatis (35-50% of cases) • Ureaplasma urealyticum(10-30% of cases) • Mycoplasma hominis • Gardnerella vaginalis • Trichomonas vaginalis • Candida albicans

26. Dual infections of C. trachomatis and Neisseria gonorrhoeae are common. • Post-gonococcus urethritis • Symptoms of chlamydial infection develop after successful treatment of gonorrhea. • Reason: longer incubation period + β-lactam antibiotics are ineffective for C. trachomatis • Reiter’s syndrome • Urethritis, conjunctivitis, polyarthritis, mucocutaneous lesion • Usually occurs in young white man • Initiated by genital infection with C. trachomatis.

27. Adult Inclusion Conjunctivitis • Acute follicular conjunctivitis with mucopurulent discharge • Mostly occur in sexually active adults (18-30 yr) with genital infection with serotypes A, B, Ba, D to K. • Acquired by auto-inoculation, oral-genital contact

28. Newborn Inclusion Conjunctivitis • 25% infants acquired from mothers with active genital infections • Swollen and hyperemic eyelids • Long (>12 months) disease course if untreated and are at risk for C. trachomatis pneumonia

29. Infant Pneumonia • A diffuse interstitial pneumonia • Occur in 10-20% infants that exposed to the pathogen at birth • Rhinitis → staccato cough

30. Lymphogranuloma venereum (LGV)花柳性淋巴肉芽腫 • A chronic sexually transmitted disease caused by C. trachomatis L1, L2, L2a, L2b, L3. • More common in men, with male homosexuals being the major reservoir. • Small, painless lesions (papule or ulcer) at site of infection (genitalia). Fever, headache, myalgia. • Inflammation and swelling of regional lymph nodes (inguinal nodes), painful buboes (橫瘻), rupture, fistulas formation. • Proctitis is common in women. • Resolve spontaneously or progress to ulceration or genital elephantiasis (象皮病).

31. Lab diagnosis • Symptomatic infections are easier to diagnosis than asymptomatic infections as more chlamydiae present in specimen. • Cytology – Giemsa-stained cell scrapings • Quality of the specimen is important.Specimens must be obtained from the involved site; pus or exudate is inadequate. • Insensitive, nonspecific • Culture – HeLa, MaCoy, Hep-2 cells • Iodine stain to detect inclusions (=RBs) • The most specific methods for diagnosis. • Sensitivity depends on quality and quantity of specimen.

32. Iodine-stained Chlamydia trachomatis inclusion bodies (arrows)

33. Lab diagnosis • Nucleic acid amplification tests (NAATs) • Test of choice for lab diagnosis of C. trachomatis infection • First-void urine / urethral discharge • Amplification of a specific sequence, then detecting with a species-specific probe • Serologic tests • Limited value for adult urogenital infections, cannot differentiate between current and past infections; good for LGV. • CF test or EIAs: genus-specific LPS as antigen, fourfold increase or >1:256 • MIF test: species- and serovar-specific antigen (MOMPs)

34. T/P/C • Doxycycline for LGV • Azithromycin or doxycycline for ocular and genital infections in adult • Erythromycin for newborn conjunctivitis and pneumonia • Improve sanitary conditions – essential for prevention • Safe sex practices

35. 2. Chlamydophiliapneumoniae • Was first isolated from the conjunctiva of a child in Taiwan - TWAR strain. • An important cause of sinusitis, pharyngitis, bronchitis, and pneumonia. • Infection is common, especially inadults and transmitted person-to-person by respiratory secretions.

36. Clinical disease • Most infections are asymptomatic or mild - persistent cough. • Cannot be differentiated with other atypical pneumonia - Mycoplasma pneumoniae, Legionella pneumophila, and respiratory viruses. • Detected in atherosclerotic lesions in blood vessels. However, the role in the development of atherosclerosis is not clear.

37. Lab diagnosis • Diagnosis is difficult • Do not grow in cell lines used for isolation of C. trachomatis • NAATs are OK with large interlaboratary variation. • Micro-immunofluorescence (MIF) test • The only acceptable serodiagnotic test (specific) • A single IgM titer > 1:16 or a fourfold increase in IgG titer

38. T/P/C • Ubiquitous present, control is difficult • Macrolides (erythromycin), doxycycline

39. 3. Chlamydophilia psittaci (鸚鵡熱披衣菌) • Caused Psittacosis (parrot fever). The natural reservoir is any species of birds (Ornithosis,飼鳥病) • Can infect sheep, goat, cows, and humans (zoonosis) • High risk groups: veterinarians, zookeepers, pet shop workers, employees of poultry industry.

40. Pathogenesis • Inhalation of dried bird excrement, urine, or respiratory secretions; person-to-person transmission is rare. • Bacteriafirstspread to and multiply in reticuloendothelial cells of liver and spleen  necrosis • Thenhematogenous spread to lung and other organs via circulation • Lmphocytic inflammation in lung, edema, necrosis, mucous plugs in bronchioles cyanosis and anoxia

41. Clinical disease • Asymptomaticinfection • Flu-like illness: high fever, headache, chills, myalgia • Serious pneumonia: non-productive cough, rales, consolidation, CNS involvement: common (headache, encephalitis, convulsion, coma) GI symptoms: nausea, vomiting, diarrhea (carditis, hepatomegaly, splenomegaly)

42. Diagnosis and treatment for C. psittaci • Complement fixation test of paired acute and convalescent phase sera • Confirmed by species-specific MIF test • Treatment: tetracyclines or macrolides • No need of isolation of patients and prophylaxia • No vaccine available • Treat birds with chlortetracycline HCl for 45 days.

43. Chapter 44Rickettsia and OrientiaChapter 45Ehrlichia, Anaplasma, Coxiella Rickettsia Howard Ricketts Ehrlichia Paul Ehrlich Coxiella Harold Cox (Historically classified in Rickettsiaceae)

44. Order Rickettsiales Family RickettsiaceaeGenenaRickettsia Orientia Family AnaplasmataceaeGenenaEhrlichia Anaplasma NeorickettsiaWolbachia

45. Chapter 44 Rickettsia and Orientia • Obligate intracellularparasites. • G(-) bacilli, with a minimal peptidoglycan layer (stain poorly with Gram stain) and LPS (weak endotoxin activity) • Maintain inanimal and arthropod reservoirs (by transovarian transmission). • Transmitted to humans by arthropod vectors (ticks, mites, lice, fleas). • Humans are accidental hosts: acquired by arthropod bite or contact of arthropod excreta with abraded skin

46. Rickettsia(also Ehrlichia) is unstable and die quickly outside host cells. • Coxiellahighly resistant to desiccation, remain viable in environment for months to years. After phagocytosis • Rickettsia and Orientia: degrade phagosome membrane by producing phospholipase, multiply in cytoplasm and nucleus of endothelial cells • Ehrlichia and Anaplasma: multiply in cytoplasmic vacuoles (=phagosomes) of hematopoietic cells • Coxiella: multiply in phagolysosome of monocytes and macrophages

47. Important Rickettsial Diseases • Spotted fever group (17 species related to human diseases) R. rickettsii RMSF (>90%) R. akari Rickettsialpox (100%) • Typhus group R. prowazekii Epidemic typhus (40-80%) R. typhi Murine typhus (50%) O. tsutsugamushi Scrub typhus (<50%) (Parentheses: % of rash, 紅斑) • The distribution of rickettsial diseases (restricted area or worldwide) is determined by the distribution of the arthropod hosts/vectors.

48. Pathogenesis • No toxins, no immunopathology • OmpA mediated binding to endothelial cells • Rickettsia replicate in endothelial cells, cause cell damage and blood leakage, vasculitis, skin rash, microthrombi, focal ischemia, hemorrhage. • Hypovolemia, hypoproteinemia, reduced perfusion, organ failure.

49. Rocky mountain spotted fever • Have a restricted geographic and seasonal distribution, corresponding to tick activity. • R. rickettsii is maintained in hard ticks (wood tick and dog tick) by transovarian transmission. • Transmitted to humans by tick bite (need >6h to establish infection). • High fever, chills, headache, skin rash (>90%, extremities to trunk) • Respiratory failure, encephalitis, renal failure.

50. Diagnosis is urgent, the prognosis depends on the duration of illness (identify key clinical signs – rash); fatality 10-25% if untreated • Culture: buffy coat of blood or skin biopsy; tissue culture or embryonated eggs (danger) • Microscopy: Giemsa stain; FA for biopsy tissue specimens (rapid and specific) • Serology: microimmunofluorescence (MIF), detect antibodies against MOMP and LPS antigens; both specific and sensitive • Nucleic acid-based tests: PCR + gene sequencing of a variety of genes • The traditional Weil-Felix test: not recommended for use