Mycoplasma and Ureaplasma Chlamydiaceae Rickettsia and Orientia Ehrlichia, Anaplasma, Coxiella

Comparison of Bacteria and Intermediate Forms of Bacteria with Viruses   BACTERIA MYCOPLASMA CHLAMYDIA RICKETTSIAE VIRUSES Growth outside host cell Yes No No* Independent protein synthesis Generation of metabolic energy Rigid cell envelope EB-yes RB-no Reproduction by fission Nucleic acids DNA & RNA DNA or RNA Antibiotic susceptibility

Mycoplasma and Ureaplasma

5 families, 200 species; 16 colonize humans and 5 associated with diseases Mycoplasma M. pneumoniae M. hominis M. genitalium M. fermentans Ureaplasma U. urealyticum

Smallest (0.1-0.3 m) and simplest free-living bacteria (about twice the genome size of certain large viruses)

Lack a cell wall (highly pleomorphic), resistant to penicillin, cephalosporins, vancomycin, 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)

Small, fried-egg-like colonies (except M. pneumoniae)

Epidemiology M. pneumoniae Worldwide disease with no seasonal incidence Most common in school-age children and young adults (5-10y), but all age groups are susceptible Spread by nasal secretions in close contact among classmate or family members

U. urealyticum, M. hominis, and M. genitalium Infants (females) are colonized with the agents at birth Only a small proportion of prepubertal children remain 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

Pathogenesis Extracellular pathogen; infect and colonize mucous membrane; do not invade other tissues. M. pneumoniae adheres to sialated glycoprotein receptor at the base of cilia (and on surface of RBC) by means of P1 antigen.

The mechanism of cellular damage is unknown (produce peroxidase 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).

Clinical disease Cause mild URT disease, low-grade fever, malaise, headache, dry and nonproductive cough, persist for > 2 weeks 10% patients develop LRT symptoms: tracheobronchitis, atypical (walking) pneumonia Secondary complication: otitis media, erythema multiforme (Stevens-Johnson syndrome), myocarditis, pericarditis

U. urealyticum and M. genitalium: cause nongonococcal urethritis M. hominis: implicated as a cause of pyelonephritis, pelvic inflammatory disease, and postpartum fever

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, glucose, and penicillin. Colonies have a “mulberry-shaped”. M. hominis requires arginine for growth. Colonies have a fried-egg appearance. Ureaplasma requires urea for growth Microscope: no cell well, stain poorly, no value

Serology - M. pneumoniae Complement fixation test : high false-positive rate ELISE for detection of IgM and IgG Abs Nonspecific reaction to outer membrane glycolipids : cold agglutinins: IgM Abs that bind the I antigen on human RBC at 4°C) develop in 65% of the patients - a test frequently used to confirm the diagnosis. False-positive seen in infections with Epstein-Barr virus, cytomegalovirus, and adenovirus.

Treatment M. pneumoniae: erythromycin, tetracycline (also good for chlamydia) Ureaplasma: use erythromycin, resistant to tetracycline M. hominis: resistant to erythromycin and tetracycline, use clindamycin

Chlamydiaceae

Family Chlamydiaceae Genus Chlamydia: C. trachomatis Genus Chlamydophilia: C. pneumoniae C. psittaci

Chlamydiaceae Obligate intracellular organisms, were once considered virus. Possess inner and outer membranes Contain DNA and RNA Possess ribosomes Synthesize proteins, nucleic acid, and lipids, but cannot synthesize ATP. Respond to wide-spectrum antibiotics, but not to penicillin (lack peptidoglycan)

Unique development cycle Two morphological distinct forms in cytoplasmic phagosome: (1) elementary body (300-400 nm), resistant to harsh environmental factor; bind to receptors of host cells and stimulate uptake; metabolically inactive but infectious, (2) reticulate body (800-1000 nm), reproductive form, divide by binary fission, noninfectious. Histologic stains can detect phagosome with accumulated RBs (inclusion)

1. Chlamydia trachomatis Infections only occur in humans Two biovars (trachoma and LGV) and 19 serotypes (antigen differences in MOMP) Serotypes Disease A to C Trachoma D to K Urethritis, cervicitis Inclusion conjunctivitis Neonatal conjunctivitis Infant pneumonia L1 to L3 Lymphogranuloma venereum

Pathogenesis EBs enter the body via minute abrasions and lacerations Primarily infect nonciliated columnar, cuboidal, or transitional epithelial cells (urethra, endocervix, endometrium, fallopian tube, anorectum, respiratory tract, conjunctiva) LGV biovar replicate in mononuclear phagocytes in lymphatic system (formation of granuloma, abscesses, or sinus tracts in LN draining the site of primary infection)

Pathogenesis Destruct cells during replication Infection stimulates a severe inflammatory response (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).

Trachoma A chronic suppurative eye disease caused by serotypes A,B,Ba,C. Follicular conjunctivitis →scar →corneal ulceration →pannus formation (invasion of vessels into the cornea,) →blindness Endemic in the Middle East, North Africa, and India (dry and sandy regions); predominantly in children. Leading global causes of blindness (500 million infected, 7 to 9 million blinded). Transmission: eye-to-eye by droplet, hands, contaminated clothing, flies.

Urogenital infections Venereal infections caused by serotypes of D to K. The most common sexually transmitted bacterial disease in U.S. 2.8 million new cases annually, largely in males (50 million worldwide). In women: 80% asymptomatic; bartholinitis, cervicitis, pelvic inflammatory disease, which can lead to sterility and ectopic pregnancy. In men: 25% asymptomatic; nongonococcal urethritis (NGU)

Nongonococcal Gonorrhea urethritis 1. Mild 1. Severe 2. Slow and prolonged 2. Acute 3. Dysuria is mild 3. Severe dysuria 4. Urethral discharge is 4. Purulent clear or white, thin discharge and mucoid

Nongonococcal Urethritis (NGU) Urethritis caused by pathogens other than gonococcus C. trachomatis (35-50% of cases) Ureaplasma urealyticum (10-30% of cases) Mycoplasma hominis Gardnerella vaginalis Trichomonas vaginalis Candida albicans Herpesvirus hominis (?) Cytomegalovirus (?)

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. Auto-inoculation, oral-genital contact

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

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

Lymphogranuloma venereum (LGV) A chronic sexually transmitted disease caused by C. trachomatis L1, L2, L2a, L3. More common in men, with male homosexuals being the major reservoir. Small, painless lesions at site of infection (genitalia). Fever, headache, myalgia. Swelling of regional lymph nodes (inguinal nodes), painful buboes , rupture. Proctitis is common in women. Resolve spontaneously or progress to ulceration or genital elephantiasis .

Lab diagnosis Symptomatic infections are easier to diagnosis than asymptomatic infections 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 strain to detect inclusions; the most specific methods for diagnosis.

Iodine-stained Chlamydia trachomatis inclusion bodies (arrows)

Chlamydial urethritis (elementary bodies in direct smear of urethral cell, fluorescein antibody stain)

Lab diagnosis Nucleic acid amplification tests (NAATs) – test of choice for lab diagnosis of C. trachomatis infection Serologic tests – limited value for adult urogenital infections; good for LGV. CF test or EIAs: genus-specific LPS, fourfold increase or >1:256 MIF test: species- and serovar-specific antigen (MOMPs)

2. Chlamydophilia pneumoniae Was first isolated from the conjunctiva of a child in Taiwan - TWAR stain. An important cause of bronchitis, pneumonia and sinusitis. Infection is common, especially in adults and transmitted person-to-person by respiratory secretions.

Clinical disease Most infections are asymptomatic or mild - persistent cough. Can’t be differentiated with other atypical pneumonia - M. pneumoniae, Legionella pneumophila, and respiratory viruses. Detected in atherosclerotic lesions in blood vessels. However, the role in the development of atherosclerosis is not clear. (Koch’s postulate)

Lab diagnosis Diagnosis is difficult Do not grow in cell lines NAATs are OK for use. Complement fixation test (not specific) or MIF test (specific)

3. Chlamydophilia psittaci Caused Psittacosis (parrot fever). The natural reservoir is any species of birds (Ornithosis) Veterinarians, zookeepers, pet shop workers, employees of poultry industry.

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

C. psittaci has three forms of infection Asymptomatic infection Transient flu-like illness: high fever, headache, chills, myalgia Serious pneumonia: non-productive cough, rales, CNS involvement is common, carditis, hepatomegaly, splenomegaly

Diagnosis and treatment for C. psittaci Diagnosis: complement fixation test with group antigen, fourfold rise in specific antibody Treatment: tetracyclines or macrolides Treat birds with chlortetracycline HCl for 45 days.

C. trachomatis C. pneumoniae C. psittaci Disease mild and chronic Disease severe Glycogen in inclusions Glycogen absent Inclusions can be stained No staining with iodine with iodine Susceptible to sulfonamides Sulfonamide resistant  

Rickettsia and Orientia Ehrlichia, Anaplasma, Coxiella (Historically classified in Rickettsiaceae)

Order Rickettsiales Family Rickettsiaceae. Genena Rickettsia Order Rickettsiales Family Rickettsiaceae Genena Rickettsia Orientia Family Anaplasmataceae Genena Ehrlichia Anaplasma Neorickettsia Wolbachia

Rickettsia and Orientia

General characteristics G(-) bacilli, obligate intracellular parasites. Were thought to be virus: small (0.3x1m), 800 genes. True bacteria: DNA+RNA, binary fission, sensitive to antibiotics, use host cell ATP.

Maintain in animal and arthropod reservoirs. Transmitted to humans by arthropod vectors (ticks, mites, lice, fleas), and maintained in arthropod hosts by transovarian transmission. Humans are accidental hosts: acquired by arthropod bite or contact of arthropod excreta with abraded skin. The distribution of rickettsial diseases is determined by the distribution of the arthropod host/vector.

Pathogenesis Rickettsia (also Ehrlichia) is unstable and die quickly outside host cells. Coxiella highly resistant to desiccation, remain viable in environment for months to years. No toxins, no immunopathology Rickettsia replicate in endothelial cells, cause cell damage and blood leakage, skin rash, microthrombi, focal ischemia, hemorrhage. Hypovolemia, hypoproteinemia, reduced perfusion, organ failure.

Important Rickettsial Diseases Spotted fever group R. rickettsii RMSF (>90%) R. akari Rickettsialpox (100%) Typhus group R. prowazekii Epidemic typhus (40-80%) R. typhi Murine typhus (50%) Scrub typhus group O. tsutsugamushi Scrub typhus (<50%) (Parentheses: % of rash)

Spotted fever Have a restricted geographic distribution; Rocky mountain spotted fever (RMSF) is the prototype of the group, caused by R. rickettsii. Organisms are maintained in hard ticks (wood tick and dog tick) by transovarian transmission. Transmitted to humans by ticks (need 24-48h to establish infection). High fever, chills, headache, skin rash (>90%, extremities to trunk) GI symptoms, respiratory failure, encephalitis, renal failure.

Diagnosis is urgent, because the prognosis depends on the duration of illness. (identify key clinical signs – rash) Culture: 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 Molecular diagnosis: PCR, not species-specific

Prevention/Control: Tetracyclines (e.g., doxycline) No vaccine Prevent tick bites (can survive for as long as 4 years without feeding)

Epidemic (louse-borne) typhus R. prowazekii transmits from man to man by human head and body lice. Humans are the primary reservoir (lice die 2 to 3 wk after infection). Epidemics occur among people living in crowded, unsanitary condition - war, famine, or natural disaster. High fever, severe headache, chills, followed by a generalized skin rash; complications: myocarditis and CNS involvement.

O

Brill-Zinsser Disease A recrudescent form of epidemic typhus arising years after the initial attack.

Diagnosis: T/P/C: MIF test Tetracyclines, Chloramphenicol Louse-control Formaldehyde-inactivated vaccine

Endemic (murine) typhus R. typhi transmits to man from rodents reservoir hosts by rat flea. Endemic all over the world, primarily in warm, humid areas. Fever, severe headache, chills, skin rash (50%) on chest and abdomen

o

Diagnosis: T/P/C: IFA test Tetracyclines, doxycycline, Chloramphenicol Pest control No vaccine

Scrub typhus A rickettsial disease caused by Orientia tsutsugamushi Transmitted to humans by red mites (chiggers) Organisms are maintained in mites by transovarian transmission. Endemic in eastern Asia, Australia, and Japan. Fever, severe headache, skin rash (<50%), spread centrifugally to extremities. Generalized lymphadenopathy, splenomegaly, CNS complication, heart failure

O

T/P/C: Prompt treatment with tetracyclines, doxycycline, chloramphenicol Avoid exposure to chiggers No vaccine

Ehrlichia, Anaplasma, Coxiella

Ehrlichia and Anaplasma Intracellular bacteria that lodge in phagosomes of mononuclear and granulocytic phagocytes, but not RBC. multiply in phagosomes = morulae Grow cycle: three stages - elementary body, initial body, morula

Ehrlichia inclusions (peripheral blood smear, Wright-Giemsa)

Clinical disease Human monocytic ehrlichiosis E. chaffeensis : infect blood monocytes and mononuclear phagocytes in tissues and organs Vector - Lone Star tick Reservoir - white-tailed deer, domestic dogs

Canine granulocytic ehrlichiosis E. ewingii Vector - Lone Star tick Reservoir -white-tailed deer, domestic dogs. Human anaplasmosis Anaplasma phagocytophilium : infect bone marrow myeloid cell (i.e., neutrophils) Vector - Ixodes ticks Reservoir - small mammals

Clinical disease Fever, headache, malaise, leukopenia, thrombocytopenia Skin rash (10 to 40%) 50% patients require hospitalization, 1 to 3% mortality

T/P/C: Diagnosis is urgent. Prompt treatment with doxycycline No vaccine Avoid tick-infected areas

Coxiella burnetii Biologically and genomically distinct from Rickettsia; more closely related to Legionella and Francisella. Obligate intracellular pathogen Multiply in phagolysosome

Epidemiology Can infect mammals, birds, and ticks Primary reservoirs: farm animals, cats, dogs, rabbits Ticks are vector for disease in animals but not in humans

Epidemiology High concentrations of bacteria are present in placenta of infected livestock. Spores are able to survive in nature under dry environmental conditions for months. Transmit to man by the respiratory route from contaminated soil, not from arthropod vector. Those handling pregnant or lactating cows or sheep, drinking unpasteurized milk, or working in slaughter-houses are at highest risk.

Pathogenesis Target tissue is the lung, proliferate in phagolysosomes of infected cells, then disseminate to other organs Undergo antigenic variation (cell wall LPS): phase I antigen: LPS with a complex carbohydrate, can block antibody binding; infectious form phase II antigen: modified LPS, expose surface proteins to antibody, less infectious form Formation of immune complex: cause of signs and symptoms

Q fever Most infections are mild or asymptomatic Acute disease: Pneumonia - high fever, severe headache, chill, myalgias, resemble “atypical pneumonia” Granulomatous hepatitis, hepatosplenomegaly, Chronic disease: subacute endocarditis with long incubation period and poor prognosis

Diagnosis Serologic tests (IFA, ELISA, CF) Acute Q fever: IgM and IgG are developed against phase II antigen. Chronic Q fever: antibodies against both phase I and II antigens are elicited. (phase I antigen: weak antigenic)

T/P/C: Doxycycline for prolonged period Vaccine is available (single dose with no booster immunization for uninfected people)