CLBACT - Spirochetes and Atypical Bacteria

Spirochetes

• Large, helically curved, spiral or cork-screw-shaped gram-negative bacteria
• Motile by axial filaments (endoflagella)
• Demonstrated using special staining and microscopy techniques

Axial filaments

Also called endoflagella (periplasmic flagella); spirochetes are motile by axial filaments.

Treponema

Slender, tightly coiled; 6–10 axial filaments, 1 insertion disk; direct contact or venereal transmission depending on species.

Borrelia

Thicker, loosely coiled; 30–40 axial filaments, 2 insertion disks; arthropod-borne.

Leptospira

Tightly coiled with hooked ends ("question mark"); 2 axial filaments, 3–5 insertion disks; zoonotic.

T. pallidum subsp. pallidum

Causative agent of syphilis; cannot be cultured in the laboratory.

Syphilis transmission

Sexual contact, transplacental passage (congenital), blood transfusion.

Primary syphilis

Chancre at site of infection, regional lymphadenopathy.

Secondary syphilis

Disseminated rash, generalized lymphadenopathy.

Latent syphilis

Recurrence of secondary syphilis symptoms in up to 25% of individuals; 72% no further complications, 28% progress to tertiary.

Tertiary syphilis

Gumma, cardiovascular syphilis, late neurological complications.

Syphilis direct detection

Darkfield microscopy or DFA-TP of exudate from a chancre / moist lesion (motile corkscrew organisms); PCR; silver stain of tissue.

Non-treponemal tests

VDRL and RPR; detect reagin; used for screening + therapy monitoring.

Treponemal tests

FTA-ABS, TP-PA, MHA-TP; detect antibody to T. pallidum antigens; confirmatory; reactive for life.

Traditional testing

Quantitative RPR; + → TP-PA or other specific treponemal test (+ = syphilis; − = differential diagnosis, syphilis unlikely); RPR − → no further testing.

Reverse testing

EIA; + → quantitative RPR (+ = syphilis); RPR − → TP-PA or other specific tests (+ = syphilis; − = syphilis unlikely).

T. pallidum subsp. pertenue

Yaws (frambesia): direct contact; elevated granulomatous nodules and disseminated raspberry-like lesions.

T. pallidum subsp. endemicum

Bejel (endemic syphilis): direct contact or sharing of utensils; skin papules that progress to gummas.

T. pallidum subsp. carateum

Pinta: direct contact; scaling painless skin papules, then an erythematous rash that becomes hypopigmented.

B. burgdorferi sensu lato

Agent of Lyme disease; most common tick-borne disease in the US.

Lyme disease vector

Deer ticks Ixodes scapularis and I. pacificus.

Erythema migrans

Bull’s eye rash at the bite site: Stage 1 (early localized) Lyme disease.

Lyme disease Stage 2

Early disseminated: hematogenous spread to organs and tissues including the bones, heart, and CNS.

Lyme disease Stage 3

Late persistent infection, months to years after the bite; chronic arthritis and a diffuse skin rash.

Western blot (Lyme)

Most accurate method for antibody detection in Lyme disease; others: silver staining, PCR, IFA, ELISA.

Relapsing fever

Caused by other Borrelia species: abrupt fever, headache and myalgia, an afebrile period as antibodies form, then another febrile period.

Borrelia recurrentis

Louse-borne or epidemic relapsing fever; vector Pediculus humanus (body louse).

Borrelia hermsii

Tick-borne relapsing fever (with B. parkeri, B. duttonii); vector Ornithodoros (soft ticks).

Borrelia miyamotoi

Hard tick-borne relapsing fever; vector Ixodes (hard ticks).

Relapsing fever diagnosis

Darkfield microscopy, Giemsa-stained blood films.

Leptospira interrogans

Over 200 serovars; causative agent of leptospirosis; direct contact with urine of animals carrying the organism or urine-contaminated soil or water.

Anicteric leptospirosis

Self-limiting illness in two phases: leptospiremic (septicemic) phase, then immune phase.

Leptospiremic phase

Septicemic phase: non-specific, flu-like symptoms (high fever and headache).

Immune phase

Appearance of IgM antibodies; hallmark is aseptic meningitis.

Weil's disease

Icteric leptospirosis: severe form caused by L. interrogans serovar Icterohaemorrhagiae; liver, kidney, or vascular dysfunction.

Leptospirosis diagnosis

Darkfield microscopy or silver staining; culture of blood or urine in EMJH or Fletcher’s medium; microscopic agglutination test; PCR.

Leptospira biflexa

Non-pathogenic species found in water and soil; over 60 serovars.

Typical bacteria

Obligate intracellular: No · Peptidoglycan: Yes · Growth on non-living media: Yes

Chlamydiae

Obligate intracellular: Yes · Peptidoglycan: No · Growth on non-living media: No

Rickettsiae

Obligate intracellular: Yes · Peptidoglycan: Yes · Growth on non-living media: No

Mycoplasmas

Obligate intracellular: No · Peptidoglycan: No · Growth on non-living media: Yes

Chlamydia

Obligate intracellular parasites; like gram-negative bacilli but the lipopolysaccharide has little endotoxic activity.

Reticulate body (RB)

Intracellular, replicative form of Chlamydia; divides by binary fission.

Elementary body (EB)

Extracellular, metabolically inert, infective form of Chlamydia.

Chlamydia life cycle

Phagocytosis of EB (0 hour) → reticulate bodies (8 hours) → multiplication (24 hours) → reorganization into elementary bodies (30 hours) → release (35 to 40 hours).

C. trachomatis (differentiation)

15 serovars (A–K, L1–L3); round EB; round, vacuolar inclusion; glycogen +; sulfonamide sensitive.

C. pneumoniae (differentiation)

1 serovar (TWAR); pear-shaped EB; round, dense inclusion; glycogen −; sulfonamide resistant.

C. psittaci (differentiation)

≥ 4 serovars; round EB; variable shape, dense inclusion; glycogen −; sulfonamide resistant.

Serovars A, B, Ba, C

Endemic trachoma that leads to blindness; hand to eye from fomites; specimen – conjunctival scrapings.

Serovars D–K

Oculogenital (genital tract) infections: urethritis, cervicitis, PID, conjunctivitis; can lead to sterility and ectopic pregnancy.

Serovars L1, L2, L2a, L3

Lymphogranuloma venereum (LGV); transmitted through sexual contact.

Frei test

Susceptibility (skin) test for lymphogranuloma venereum.

C. trachomatis cytology

Demonstration of inclusion bodies in Giemsa-stained smears.

C. trachomatis culture

Cell lines: cycloheximide-treated McCoy cells; also HeLa-229.

C. trachomatis serology

Detection of antibody to chlamydial LPS (CF) or MOMP (MIF test).

TWAR

Former name of the C. pneumoniae strain: Taiwan acute respiratory (isolates TW-183 and AR-39).

Chlamydia pneumoniae

Person to person by aerosolized droplets; mild respiratory infections; risk factor for atherosclerosis (coronary artery disease).

C. pneumoniae culture

HEp-2 or HL cells; diagnosis often by fluorescent-labeled C. pneumoniae antibodies.

Chlamydia psittaci

Agent of psittacosis (ornithosis, "parrot fever") in birds; acquired by inhalation of aerosols or fomites; can progress to pneumonia.

C. psittaci detection

Preferred means: serology and PCR; occupational hazard to farmers, pet shop employees and bird owners.

Mycoplasma

• Smallest free-living bacteria
• Lack cell wall: pleomorphic, resistant to cell-wall antibiotics (penicillins, β-lactams)
• Require cholesterol for growth

Mycoplasma media

Enriched artificial media which generally contain animal serum (sterols) and yeast extract.

Dienes stain

Directly stains the very small Mycoplasma colonies, visualized using a stereomicroscope (dissecting).

Eaton agent

Former name of Mycoplasma pneumoniae (also PPLO).

Mycoplasma pneumoniae

Tracheobronchitis and primary atypical ("walking") pneumonia; direct respiratory contact; usually diagnosed by serology (cold agglutinin test, ELISA, IFA).

M. pneumoniae identification

Overlaying SP4 agar culture with guinea pig red blood cells in 1% agar: hemadsorption after 20–30 min at RT, β-hemolysis after 24 h.

Mycoplasma hominis

UGT infections (prostatitis, PID, amnionitis, NGU, APN); only species that grows on SBA and CA: pinpoint colonies with "fried egg" appearance.

Ureaplasma urealyticum

NGU and other UGT infections; smaller, granular dark brown colonies on A8 agar due to urease production; T-strain Mycoplasma.

Mycoplasma genitalium

NGU in men; cervicitis and endometritis in females.

Mycoplasma fermentans

Opportunistic infections in immunocompromised patients.

Mycoplasma biochemicals

Glucose / Arginine / Urease
M. pneumoniae, M. genitalium: + / − / −
M. hominis: − / + / −
U. urealyticum: − / − / +
M. fermentans: + / + / −

Mycoplasma specimens

Body fluids, tissues, swabs (Dacron or polyester on aluminum or plastic shafts); transport within 1 h on ice or at 4°C.

Mycoplasma specimen storage

4 °C for up to 48 h; freeze at −70 °C if longer.

Mycoplasma cultivation media

SP-4 agar, PPLO agar or broth, Modified New York City medium, A7 or A8 medium, Shepard’s broth.

Rickettsiae and Ehrlichiae

Fastidious obligate intracellular, arthropod-borne; triad of fever, headache, and skin rashes.

Weil-Felix test

Serologic test for rickettsial infections (with IFA, EIA); also immunohistology and PCR.

Morulae

Typical inclusions in WBCs (monocytes and granulocytes) in ehrlichiosis; Giemsa-stained blood smear or buffy coat.

Rickettsia prowazekii

Epidemic (louse-borne) typhus, Brill-Zinsser disease; vector Pediculus humanus.

Rickettsia typhi

Endemic (murine) typhus; vector Xenopsylla cheopis.

Rickettsia rickettsii

Rocky Mountain spotted fever; vectors Dermacentor variabilis, D. andersoni.

Rickettsia conorii

Boutonneuse fever (Mediterranean or Israeli spotted fever, Indian tick typhus); vector Rhipicephalus sanguineus.

Rickettsia sibirica

North Asian (Siberian) tick typhus; vector Dermacentor spp.

Rickettsia akari

Rickettsialpox (TRG); vector Liponyssoides sanguineus.

Rickettsia australis

Queensland tick typhus (TRG); vectors Ixodes holocyclus, I. tasmani.

Rickettsia felis

Flea-borne (cat flea) spotted fever (TRG); vector Ctenocephalides felis.

Orientia tsutsugamushi

Scrub typhus; vector Leptotrombidium deliense.

Ehrlichia chaffeensis

Human monocytic ehrlichiosis; vector Amblyomma americanum.

Ehrlichia ewingii

Human granulocytic ehrlichiosis; vector Amblyomma americanum.

Anaplasma phagocytophilum

Human granulocytic anaplasmosis; vectors Ixodes scapularis, I. pacificus.

Coxiella burnetii

Causative agent of Q fever; smaller than Rickettsia, spore-like life cycle, survives extracellularly; no skin rash; inhaled aerosols from infected animals.

Q fever diagnosis

Serologic techniques: IFA, complement fixation, and EIA.

Ureaplasma colonies (correction)

Handout: "T-strain Mycoplasma (tiny, spherical fried egg colonies)".
Correct: tiny, granular, dark brown colonies on A8 agar; the "fried egg" colony belongs to M. hominis.

Coxiella spread (correction)

Handout: "is highly contagious".
Correct: highly infectious (a handful of inhaled organisms causes Q fever), but almost never spreads from person to person.