MICROBIOLOGY LECTURE 12 – Other Spirochaetales Notes from Lecture USTMED ’07 Sec C - AsM Order Spirochaetales (2 Families, 7 Genera) • Treponema • Borrelia • Leptospira TREPONEMA
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T. T. T. T.
pallidum ssp. Pallidum pallidum ssp. Endemicron pallidum spp. Pertenue carateum
- Syphilis - Bejel - Yaws - Pinta
Structure • spirochete 0.1 X 5 – 15 um, coiled • too thin to be seen by light microscopy even with grams or giemsa stain • motile forms – darkfield illumination • appreciated thru staining with specific antibodies labeled with flourescent dyes
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Diffuse chronic inflammation devastating destruction of virtually any organ (arthritis, dementia, blindness Gummas-granulomatous lesions
D. Congenital In-utero can lead to fetal death organ malformations, or latent infections (desquamation maculopapular rash body destruction, cardiovascular syphilis common in untreated infants Laboratory Diagnosis Diagnostic Test Microscopy Culture Serology
Physiology
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Grown in cell cultures (rabbit epithelium) Doubling time is 30 hours Maintained only for a few generations Can utilize glucose oxidatively
Natural syphilis is not found in any other species
Virulence Factors • Outer membrane proteins – adherence to surface of host’s cells • Hyaluronidase – perivascular infiltration • Fibronectin coating from host cells antiphagocytic Pathogenesis Tissue destruction and lesions are primarily the result of the patient’s immune response to infection Epidemiology 1. Incidence • worldwide; no seasonal incidence • true incidence underestimated • in US, next only to Neisseria and Chlamydia • high incidence among blacks and Hispanic population
Method or Examination * Darkfield * Direct fluorescent antibody staining Not used Nontreponemal tests VDRL RPR Treponemal tests FTA-ABS MHA-TP Darkfield examination. Illumination for darkfield microscopy is obtained when light rays strike the objection in the field at an oblique angle so that no light rays pass directly from the condenser to the objective of the microscope, but only the light rays that are reflected from the object will enter. A treponema is seen in the center field, distinguished by its corkscrew, spiral shape
Direct fluorescent antibody for Treponema pallidum (DFA-TP). A tissue impression smear from a brain biopsy specimen shows fluorescent treponemes. In the DFA-TP test, a monoclonal antibody labeled with fluorescein isothiocyanate specific for T. pallidum binds to the treponemes in either smears or tissue sections prepared from paraffin-embedded tissue blocks. Because a monoclonal antibody is used in the DFA-TP test, motility of the organism is not required for identification. VDRL TEST
2. Transmission • Man is the only natural host • Sexually transmitted • Congenital transmission thru infected birth canal • Transfusion with contaminated blood 3. Who are at risk
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People with multiple sexual partners Newborns of infected mothers Patient with AIDS increased risk for neurosyphilis
Clinical Syndromes A. Primary Syphilis • Chancre (at site of inoculation) painless, indurated ulcer, with regional lymphadenopathy
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Reacive Weakly Reactive
Non-reactive RPR TEST
Represents local focus for spirochete multiplication Heals spontaneously after 2 months
B. Secondary Syphilis • Disseminated disease • Flu-like syndrome (fever, sorethroat, myalgia • Generalized mucocutaneous rash (highly infectious) • Resolve spontaneously C. Late Syphilis, Tertiary
Non-treponemal Tests • Measures reagin antibodies developed against lipids released from damaged cells and also on surface cells of treponemes
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Antigen used is cardiolipin – derived from beef heart
VDRL – Venereal Disease Research Laboratory RPR – Rapid Plasma Reagin for suspected neurosyphilis – only VDRL should be used can be used to monitor therapy of patients specificity = 98% Treponemal Tests • Specific antibody tests – to confirm positive VDRL and RPR • They are positive even before non-treponemal tests become positive • Remain positive even when the non-treponemal tests are negative FTA-ABS – Flourescent Treponemal Antibody Absorption MHA – TP – Microhemagglutination Test for T. pallidum WESTERN BLOT ASSAY they remain generally positive for life a negative test is unreliable for AIDS patients Medical Conditions Associated with False-positive Treponemal and Non-treponemal Serology Tests
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persist for years and result in hypopigmented lesions and scarring (Mexico, Central and South America) spread by direct contact with infected lesions
Diagnosis Microscopy Darkfield microscopy for Spirochetes Treatment Penicillin Tetracyclines Chloramphenicol BORRELIA
Relapsing fever (Borrelia recurrentis) epidemic or louseborne endemic or tick born
Lyme disease – tick borne disease protean manifestations with initial skin lesion erythema migrans
1. Non treponemal Tests • Viral infection • Collagen-vascular disease • Acute or chronic illness • Pregnancy • Recent immunization • Heroin addiction • Leprosy • Malaria
Structure • Gram negative bacilli resembling spirochetes • Weakly staining, 0.2 – 0.5 X 3 – 30 um • Stains well with Aniline dyes (Wright and Giemsa) • With periplasmic flagella
2. Treponemal Tests • Pyoderma • Skin neoplasm • Acne vulgaris • Mycoses • Crural ulceration • Rheumatoid arthritis • Psoriasis • Systemic lupus erythematosus • Pregnancy • Drug addiction • Herpes genitalis
Pathogenesis and Immunity • Able to spread in the blood stream and the clinical manifestations of relapsing fever are in part a response to the release of endotoxins • Organism can undergo antigenic variation hence those residing in internal tissues can alter their serotype specific outer envelop proteins by gene rearrangement and emerge as antigenically novel organism
Physiology • Microaerophilic with complex nutritional requirements • Recovery in culture difficult • Generation time – 18 hours
Treatment, Prevention and Control • Penicillin drug of choice • alternatives – tetracyclines, erythromycin, chloramphenicol • Safe sex techniques • Adequate treatment of partners with documented infections Other Treponemes 1. T. pallidum ssp. Endemicum (bejel) or endemic syphilis • Initial oral lesions • Secondary lesions are oral papules and mucosal patches • Late manifestation of gummas of skin, bones and nasopharynx • (Africa, Asia and Australia)
Crowded unsanitary conditions required for maintenance of disease.
2. T. pertenue – Yaws
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Granulomatous disease with early skin lesions and late destructive lesions of the skin, lymph nodes and bones (South America, Central Africa and SEA) Spread by direct contact with infected skin lesions
3. T. carateum (Pinta) • Start as small pruritus papules, enlarge and
Epidemiology of Borrelia Infections Clinical Syndromes 1. RELAPSING FEVER
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1 week incubation period abrupt onset of shaking cells, fever, myalgia, headache Splenomegaly and hepatomegaly frequent corresponds to the bacteremia phase Relief after 3-7 days. Recurrence of manifestation after 1 week, with milder symptoms 2-3 relapses, but as many as 13 More severe with epidemic than endemic Mortality less than 5 in endemic; 4-40% in epidemic
Physiology • obligate aerobes, motile (two peripasmic flagella) • Utilize alcohol and fatty acids • Grown on enriched media – rabbit or bovine serum albumin • Fletchers, EMTH, Tween 80 – albumin 6-16 hrs incubation Pathogenesis and Immunity • Subclinical infection • Mild flu-like illness
• 2. LYME DISEASE • Incubation period – 3 –30 days
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Severe systemc disease with renal, hepatic failure, extensive vasculitis myocarditis
One or more skin lesion at bite site (erythema migrans Malaria, headache fever, lymphadenopathies ( 4 weeks)
chills,
myalgia,
Late manifestations (80% of untreated cases) one week to 2 years later – 10 – 15% of cases o Phase 1 neurologic and cardiac o Phase 2 arthritis and antralgia
Laboratory Diagnosis
Epidemiology • Dogs, cattle rodents and wild animals are reservoir • Transmission thru break in skin after exposure to urine contamination wadding in streams, fields, flood waters occupational exposure – farmers, meat-handlers, veterinarians worldwide distribution more common in warm months Clinical Syndrome
A. Lyme Disease • Isolation of Borrelia burgdorferi • Demonstration of diagnostic levels of IgM or IgG (ELISA, Western Blot) B. burgdorferi is rarely seen in clinical specimens or blood Serologic test should not be performed in the absence of appropriate history and clinical disease
Stages of Icteric and Anicteric Leptospirosis
B. Borrelia Recurrentis • Microscopy – Giemsa or Wrights stain of blood during febrile periods (70%) o increased sensitivity with mouse inoculation test • Serologic test not useful because of antigenic phase variation • • •
Borreiia sp.
Indirect immunofluorescent test for Antibodies to Borrelia burgdorferi.
Prevention and Control • Relapsing fever; tetracycline (drug of choice except for pregnant women and young children), chloramphenicol
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Lyme disease; doxycline, amoxicillin, erythromycin for early manifestations, ceftriaxone, penicillin, doxycycline, amoxicillin for late manifestations Avoid ticks and their natural habitats, wear protective clothing, and use insect repellants Control of rodents (epidemic relapsing fever) Delousing sprays and improved hygiene important to control of epidemic louse-borne disease
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Organism can be demonstrated in the blood and CSF early in disease and later in urine Clearance of organisms occurs when humoral immunity develops Clinical manifestations may be related to immunologic reactions with the organisms meningitis develop after organism have been removed from CSF immune complexes have been detected in renal lesions 10% mortality with icteric form of generalized disease Surviving patients with hepatic and renal damage usually attains full recovery Congenital leptospirosis can also occur
Diagnostic Tests for Leptospirosis
LEPTOSPIRA
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Leptospira interrogans – pathogenic for many wild domestic animals and humans Leptospira biflexa – free living saphrophyte
Structure • Thin coiled bacilli 0.1 X 6-20 um with a hook at one both ends LEPTOSPIRA INTERROGANS
Icterohemorrhagiae, Weil’s disease, pomona, Swineherd’s disease, Autumnalis, Fort Bragg fever or pre-tibial eruptions
Mode of Control • Antimicrobials (PCN, Tetracycline, Doxycyline • Prophylaxis – Doxycycline • Vaccination of livestock and pets • Control of rodents CAMPYLOBACTER
Structure • Gram negative bacillus, 0.3 – 0.6 um diameter • Comma shaped, motile with polar flagella Physiology • Grows best at 5 – 7% O2, 5 – 10% CO2 • Oxidase and Catalase (+) Pathogenesis and Immunity Development of disease is associated with: • Infectious dose of organisms • Level of specific immunity of host Low gastric acids Antibodies (higher among people in endemic areas – less severe Hypogamma globulinemia associated with prolonged severe infection • Development of destruction of mucosal surface crypt abscess and infiltration into the lamina propria. Enteroxins, cytopathic toxins and endotoxic activity detected but precise roles, poorly defined • Campylobacter fetushas the propensity to go systemic especially among immunocompromised patients. It is covered with S protein that prevents complement-mediated killing Epidemiology of Campylobacter Most common cause of bacterial gastroenteritis in US Transmission is associated with contaminated food (especially poultry), milk and water Asymptomatic carriage in animals is an important reservoid for human disease Fecal-oral transmission as wells as person to person is also possible It has world wide distribution but more common in warm months Clinical Syndrome Acute enteritis with diarrhea, malaria, fever and abdominal pain, blood stools may be present Disease self-limiting Laboratory Diagnosis 1. Microscopy small curved bacilli, pairs resemble wings of sea gull – cannot be easily seen on gram stain Darting motility by darkfield a phase contrast microscopy 2. Culture Microaerophilic selective media containing blood or charcoal and antibiotic slow growers (48-72 hours incubation). Identification based on biochemical reactions Campylobacter sp.
infection low during childhood but increase to 50% in older adults early infections among low socio-economic classes and developing nations identified in 70 – 100% of patients with gastritis no animal reservoir identified infection via food or water not demonstrated ubiquatous and worldwide no seasonal incidence
Laboratory Diagnosis Detected by histological examination of gastric biopsis Warthin-starry silver stain is most sensitive; also seen by hemolytic, eosin and gram stain Urease test most rapid test, directly on clinical specimen Culture may be done but not very sensitive due to extraneous factors Serology cannot discriminate between past and current infection. Titers do not correlate with severity of disease or response to therapy Control • Antibiotics combined with bismuth nitroimidazole and either Amoxycillin or Tetracycline • Prevention is difficult because organism is ubiquitous LEGIONELLA PNEUMOPHILA
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ubiquitous aquatic saprophyte, that causes human respiratory infections
Structure • 0.3 – 0.9 X 2-5 um long pleomorphic in cultures, appreciated by Dieterle silver stain in tissues, motile Physiology • nutritionally fastidious, growth enhanced • by iron solutions and L-cysteine • Non-fermentative, catalase positive, • liquify gelatin, do not reduce nitrate nor hydrolyze urea Epidemiology • worldwide distribution • organism commonly present in natural bodies of water, air conditioning power and water systems, survives in moist environments at relatively high temperature • most infections occur in late summer and fall
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Elderly and decreased cellular immunity and people with compromised pulmonary function are at risk Transmission by inhalation of aerosols
Clinical Syndromes Pontiac fever – flu-like illness, self-limited Legionnaire’s Disease 2-10 days incubation, with abrupt onset of fever, chills, dry cough, primary manifestations of pneumonia, with common multiple organ involvement, mortality of 15 – 20% in compromised patients Laboratory Diagnosis Microscopy
Mode of Control Antibiotics – Erythromycin, Tetracyclines, Aminoglycoside, Clindamycin Proper food prepration, Pasteurization of milk, potable water HELICOBACTER PYLORI
Potential
Associated with gastritis, recently implicated in gastric and duodenal ulcers and gastric cancer Sequence analysis of 16S rRNA – hence their reclassification; previously classified as Campylobacter Virulence Factors urease production (protection from gastric acids Motility mucinase activity adherence factors – anchors bacteria at the intracellular junction of enteric cells heat-labile cytotoxin, hemolysin, lipopolysaccharide
Epidemiology
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direct flourescent antibody test (DFA) – sensitivity is low because antibody present are species-specific, organism is relatively small and predominantly intracellular
Culture – BCYE • small colonies with ground glass appearance Antigenic Detection and Serology • limited usefulness – due to limited sensitivity Legionella pneumophilia on buffered charcoal-yeast extract (BCYE) agar. BCYE agar is a selective medium for the recovery of Legionella spp. This buffered medium is the agar of choice for the isolation of Legionella spp. because it contains the requirements for optimal growth of microorganism: Lcystein, iron salts and a pH of 6.9. Antibiotics are added to inhibit the growth of other bacteria.
Growth appears in 2 to 3 days and the colonies are circular, glistening, entire and measure up to 4 mm as shown here.
Mode of Control • Antibiotics Erythromycin, Flouroquinolones • Hyperchlorination of water supply
Rifampicin
Classification and Clinical Features of Periodontitis
and
SPIRILUM MINOR
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agent of rat-bite fever gram negative bacillus, polar flagella has not been cultured in vitro found in nasopharynx of rats and small rodents produce ulcrations at bite site with lymphadenopathy and recurrent febrile episodes in untreated cases diagnosed by darkfield examination of blood ulcer exudates or lymph node aspirates or by intraperitoneal inoculation of rats
TREATMENT
Penicillin Tetracycline
ORAL MICROBIOLOGY
Most diseases of the oral cavity are the result of bacterial colonization and infection. The predominant etiologic agents are colonized microorganisms and their producets à an adherent mass or the teeth called dental plaque.
Microbial Profile for Periodontitis
Bacterial Plaque
total bacterial count – 108 – 1011 per gram removed from teeth by mechanical means factors enhancing rate of colonization inadequate oral hygiene diet rich in fermentable carbohydrate malocclusions and malposed teeth reduced oral immune factors impaired saliva flow rough teeth surfaces early plaque development are formed by streptococci and gram positive bacilli plaque that extends underneath the gingiva where there is less O2 tensin will grow facultative and strict anaerobic bacteria – mainly bacilli and filamentous bacteria the mixed complex colonies have > 200 sp. of bacteria the types of bacterial vary among individuals, with age of dental plaque area of the oral cavity where it was collected
Clinical Syndromes 1. Dental Caries • soft, leather like consistency of teeth
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demineralization and decalcification accelerated by acids hydrolysis of enamel phosphoprotein by phosphoprotein phosphatase oral bacteria penetrate the enamel, mostly streptococci (streptococcus mutans)
2. Periodontal Disease Gingivitis reversible plaque is the primary etiologic agent organisms involved mostly gram + cocci, bacilli and filamentous forms
Trench mouth or Vincent infection (ANUG – Acute Necrotizing Ulceration Gingival stomatitis) very painful, malodorous bacteria involved are spirochetes, vibrios and filamentous forms Periodontitis inflammation extends beyond soft tissues into the alveolar bone ligament and cementum with loss of gingival attachment irreversible dental disease generally associated with age of onset and types of bacteria present in the pockets surrounding the teeth
Laboratory Diagnosis Made primarily by clinical means There are no laboratory diagnostic tests specifically for oral bacteria associated with gingivitis Treatment, Prevention and Control 1. Dental Caries
mechanical removal of carious lesion PCN, Erythromycin
2. Gingivitis
improved oral hygiene
3. Periodentitis measures as for control of gingivitis surgical approaches if necessary systemic antibiotics in the presence of abscess Palliative therapy for ANUG Antiplaque mouth rinse like Chlorhexidine Dietary considerations Reduction of acids from fermentable carbohydrate
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