2015-10-27 Systematic Bacteriology for Pharmacy Students Spirochetes & other Spiral Bacteria Instructor: Mohsen Amin General Characteristics • Long, slender, helically coiled, motile • Endoflagella (axial filaments) • A series of cytoplasmic tubules • Three human pathogens 1. Treponema 2. Borrelia 3. Leptospira Treponema • T. pallidum subsp. pallidum causes syphilis • T. pallidum subsp. pertenue causes yaws • T. pallidum subsp. endemicum causes endemic syphilis (bejel) • T. carateum causes pinta 1 2015-10-27 Treponema pallidum • Morphology: regular spiral coils, 0.2 µm x 5-15 µm • Culture: has never been cultured. Non pathogenic strains (Reiter) can be cultured • Growth characteristics: microaerophilic Reiter strain grows on defined medium • Drying and 42°C kills the spirochete rapidly • Penicillin is treponemicidal • Genome is highly conserved, and does not have transposable elements Fontana tribondeau staining (silver nitrate) Electron micrograph of T. pallidum 2 2015-10-27 Antigenic structure • Membrane proteins • Endoflagella • Hyaluronidase Pathogenesis A. Acquired syphilis: limited to the human host through sexual contact • Skin or mucous membrane lesions • Primary lesions: In 2-10 weeks after infection, a papule develops and then an ulcer (hard chancre) • Infiltration of lymphocytes and plasma cells • Secondary lesions: red maculopapular rash anywhere on the body Pathogenesis • Both primary and secondary are rich in spirochetes and subside spontaneously • Tertiary lesions: development of granulomatous lesions (gummas) in skin, bones, and liver; degenerative changes in the CNS (neurosyphilis); or cardiovascular lesions • In tertiary lesions, treponemes are very rare 3 2015-10-27 Pathogenesis B. Congenital syphilis: A pregnant syphlitic woman can transmit T. pallidum C. Experimental disease: rabbits can be experimentally infected Diagnostic tests • Specimens: tissue fluid and blood • Dark-field examination • Immunofluorescence • Serologic tests Serologic tests for syphilis • The tests use either nontreponemal or treponemal antigens 1. Nontreponemal antigens: cardiolipin + lecithin + cholesterol. The Ag reacts with syphlitic “reagin” Ab (mix of IgM and IgG) – Venereal Disease Research Lab (VDRL) – Unheated serum reagin (USR) – Rapid plasma reagin (RPR) – Toluidine red unheated serum test (TRUST) 4 2015-10-27 Serologic tests for syphilis 2. Treponemal antibody tests: measure antibodies against T. pallidum antigens – T. pallidum-particle agglutination (TP-PA) – T. pallidum hemagglutination (TPHA) – ELISA – Fluorescent treponemal antibody absorbed (FTA-ABS) Immunity • If early syphilis is treated adequately the individual again becomes fully susceptible Treatment • Penicillin 0.003 U/ml • Latent syphilis: penicillin G IM, 3 times at weekly intervals 5 2015-10-27 Progress of syphilis Reported cases of syphilis Diseases related to syphilis • Bejel: T. pallidum subsp. endemicum • Yaws: T. pallidum subsp. pertenue • Pinta: T. carateum 6 2015-10-27 Nodules on the elbow resulting Treponema pallidum pertenue Borrelia spp. • Epidemic relapsing fever caused by B. recurentis, transmitted by the human body louse • Endemic relapsing fever caused by borreliae, transmitted by ticks Borrelia recurentis • Morphology: irregular spirals 0.3 µl x 10-30 µl • Smears: Giemsa’s stain or Wright’s stain • Culture: fluid media containing blood, serum, or tissue, but it rapidly loses pathogenicity 7 2015-10-27 Borrelia in a peripheral blood smear Antigenic variation • The relapsing course of the disease is due to the multiplication of antigenic variation Pathogenesis • Incubation period: 3-10 days • Sudden onset with chills and fever which lasts for 3-5 days 8 2015-10-27 Treatment • Tetracyclines, erythromycin, and penicillin Epidemiology • Main reservoir: rodents which serve sources of infection for ticks of the genus Ornithodoros • Borrelia is transmitted transovarially from generation to generation • The tick-borne disease is not epidemic • Sever epidemics may occur in louse- infected populations Ornithodoros 9 2015-10-27 Borrelia burgdorferi & lyme disease • First recognized in the US in 1975 following a mysterious outbreak of juvenile rheumatoid arthritis near the community of Lyme, Connecticut • 4% of population had rheumatoid arthritis • In 1982, the cause of Lyme disease was discovered by Willy Burgdorfer Lyme disease • Patients often live near wooded areas • Onset of illness during summer and early fall, and not contagious • Symptoms: Bull’s eye rash (erythema migrans) occurs in 60-80% of patients Lyme disease Other initial symptoms: • Fatigue, chills & fever, headache, muscle & joint pain, swollen lymph nodes Long term symptoms: • Arthritis, numbness, pain, Bell’s palsy, meningitis Puzzled, researchers looked at several possible causes, such as contact with microbes in water or air 10 2015-10-27 Lyme disease Hypothesis: Disease is transmitted through water or air If disease is transmitted through water or air, then it should be possible to find the microbial cause in the air or water Observation & Experimentation • Analyzed air and water for known microbes • Results were negative! Lyme disease New Data • Victim saved tick he was bitten by Deer tick: Ixodes Hypothesis/Prediction: This tick carries disease agent Lyme disease Observation & Experimentation • Isolated spirochete (bacteria) in tick • Tested blood of victims for spirochete: Results were positive 11 2015-10-27 Lyme disease Conclusion Published in the June 18, 1982, edition of Science Lyme disease is caused by the spirochete Borrelia burgdorferi Lyme incidence map, 2007 Antigenic structure • A large number of lipoproteins, including outer surface proteins OspA-F • Differential expression of these proteins helps B. burgdorferi live in the very different tick and mammalian hosts 12 2015-10-27 Diagnostic tests • Specimens: Blood, CSF or joint fluid • Smears: B. burgdorferi in tissue sections can be identified using Ab • Culture: not performed because it takes 6-8 weeks to complete and lacks sensitivity • PCR • Serology: EIA and IFA Treatment • Doxycycline or amoxicillin for 14-21 days Leptospira interrogans & leptospirosis • Leptospirosis is a zoonosis of worldwide • Morphology: tightly coiled, thin, flexible spirochetes 0.2 µm x 5-15 µm; one end is often bent, forming a hook • Culture: aerobic condition at 28-38°C in semisolid medium with 0.1% agar and 5- fluorouracil • Leptospira oxidizes long FAs 13 2015-10-27 Leptospira interrogans Antigenic structure • The outer envelope contains large amounts of LPS that is variable from one strain to another Pathogenesis • From water, entering the body through breaks, cuts and abrasions • Ingestion is less important • Incubation period: 1-2 weeks • Fever and bacteremia • Spread to kidney and liver producing necrosis and hemorrhage • “Aseptic meningitis” with intense headache and stiff neck 14 2015-10-27 Diagnostic tests • Specimens: blood, CSF, or tissue • Dark-field microscopy • Serology: 4-fold rise of leptospiral Ab Treatment • Doxycycline, ampicillin or amoxicillin Epidemiology and control 15 2015-10-27 Other spirochetal diseases • Spirillum minor: one form of rat-bite fever (sodoku) • Local lesion, regional gland swelling, skin rashes, and fever • Spirochetes of the normal mouth and mucous membrane: Borrelia buccalis, Treponema denticola 16.