5. Vibrio cholerae,Treponema pallidum, B.burgdoferi,genus Campilobacter and Helicobacter

Laboratory Diagnosis of Infectious Diseases

• Focus on pathogens such as Vibrio cholerae, Treponema pallidum, Borrelia species, Campylobacter, and Helicobacter.

• Presenting information is from Assist. Prof. Denitsa Tsaneva-Damyanova, MD.

Genus Vibrio

• More than 100 species of Gram-negative, curved rods.

Vibrio cholerae

• Characteristics:

  • Gram-negative, comma-shaped bacterium, approximately 2-3 mm long.

  • Facultative anaerobe with polar flagellum, crucial for motility.

  • Displays polymorphism: cocci, S-shaped, spirals.

  • Non-spore forming and lacks capsules, limiting infection spread to the intestine.

Classification

• Key pathogenic species:

  1. Vibrio cholerae - Causes cholera.

  2. Vibrio parahaemolyticus - Causes bacterial gastroenteritis.

  3. Vibrio vulnificus - Causes septicemia and wound infections.

• All strains have lipopolysaccharides (endotoxin, core polysaccharide, O polysaccharide).

• 200 serogroups of V. cholerae, including O1 and O139, known for cholera toxin production and cholera epidemics.

Serogroup Classification

• V. cholerae O1 agglutinates with specific antiserum, subdivided into:

  • Serotypes: Inaba, Ogawa, Hikoshima.

  • Biotypes: Classical and El Tor, distinguished by phenotypic/morphologic properties.

• Non-O1 group includes other toxigenic isolates, significant for isolating cholera cases.

Physiology

• Vibrio species thrive in simple media at temperatures between 14°C to 40°C.

• Sodium chloride (NaCl) is essential for growth; V. cholerae can grow without added salt, while halophilic species depend on it.

Culture Properties

• Strictly aerobic and prefers alkaline media (pH 8.5-9.2).

• Susceptible to stomach acids; must collect samples early in the disease.

• Optimal growth conditions involve temperatures 16-42°C in alkaline peptone water.

• Grows on common media like blood agar, MacConkey agar, and selective media (e.g., TCBS agar).

Biochemical Activity

• Positive for gelatin hydrolysis, H₂S formation, indole production, and nitrate reduction (Cholerarot reaction).

• Ferments glucose, maltose, mannitol, and manose to acid without gas; oxidase-positive.

Determinants of Pathogenicity

• Secretes enterotoxin choleragen, which comprises A (active) and B (binding) regions.

• Genes for cholera toxin carried by CTXφ bacteriophage, integrating into V. cholerae genome.

Cholera Pathogenesis

• Sequence of ingestion leads to severe secretory diarrhea:

  1. Adherence/Colonization: V. cholerae attaches to intestinal microvilli and multiplies.

  2. Cholera Toxin Secretion: Released near GM₁ ganglioside receptors.

  3. cAMP Accumulation: Activates adenyl cyclase, leading to hypersecretion of fluids.

  • High fluid loss (up to 1 liter/hour) in severe cases.

Epidemiology

• Responsible for major cholera pandemics, with significant risks in developing countries.

• Found in marine environments, primarily in shellfish; fecal-oral transmission common via contaminated water and food.

• Rarely spreads person-to-person due to high infectious dose.

Clinical Disease

• Range from asymptomatic to severe diarrhea, with untreated mortality around 60%.

• Survival leads to spontaneous recovery and local immunity development.

Laboratory Diagnosis

• Best samples: feces, vomit, bile; stained via Gram or Fuchsin.

• Cultures on specific media (TCBS, alkaline peptone broth) and agglutination tests.

Treatment and Prevention

• Rapid fluid replacement essential; antibiotics include tetracycline, chloramphenicol, and cephalosporins.

• Traditional oral vaccines provide limited short-lived immunity; new genetically engineered vaccines under development.

Order Spirochaetales

• Grouped by common morphologic properties, includes Treponema, Borrelia, and Leptospira - important human pathogens.

Genus Treponema

• Long, thin, corkscrew-shaped organisms (0.1-0.3 μm wide).

• Contain axial fibrils for movement; Gram-negative but do not stain easily.

• Visualized via dark-field or fluorescent dyes.

Physiology of Treponema pallidum

• Fastidious; does not grow in cell-free cultures, limited growth in rabbit epithelial cells.

Pathogenesis and Immunity

• Travels quickly from the site of infection to lymph nodes, then to bloodstream.

• Adheres to endothelial cell surface proteins, promotes infiltration and tissue damage through immune response.

Epidemiology of Treponema pallidum

• Humans are the only natural host; transmitted through direct contact and transplacentally.

• Occurs worldwide without seasonal incidence.

Clinical Disease Stages

• Primary Syphilis: Chancre develops 3-4 weeks post-infection, healing spontaneously after 14 days.

• Secondary Syphilis: Symptoms appear 3-8 weeks post-chancre; systemic symptoms and skin rashes develop.

• Tertiary Syphilis: Long-term complications (gummas, neurological issues) emerge years after initial infection; known as the "great imitator".

Congenital Syphilis

• T. pallidum can cross the placental barrier after 16 weeks gestation, leading to severe outcomes for the fetus.

Laboratory Diagnosis for Treponema pallidum

• Samples include secretions from lesions or lymph nodes; cannot be cultured in vitro.

• Nucleic acid tests available in research labs only.

Treatment and Prevention for Treponema pallidum

• Penicillin is the drug of choice; alternatives include doxycycline or azithromycin.

• No vaccine available due to limited surface proteins for effective immunization.

Genus Borrelia

• Comprises over 19 pathogenic species; notable diseases include Lyme disease (B. burgdorferi) and relapsing fever (B. recurrentis).

Characteristics of Borrelia

• Gram-negative, larger spirochetes (0.2-0.5 μm wide).

• Complex motility due to periplasmic flagella and twisting movement.

Borrelia burgdorferi

• Microaerophilic with slow growth (12-18 hours doubling time); cultivated in specialized media (e.g., BSK medium).

Pathogenicity of Borrelia burgdorferi

• Contains toxic LPS and outer surface proteins critical for transmission.

• Rarely isolated after disease onset, suggesting immune response significantly contributes to its pathogenicity.

Epidemiology of Borrelia

• Lyme disease risk factors include exposure in endemic tick areas; seasonal patterns seen.

• Relapsing fever transmitted via human lice or rodent reservoirs.

Lyme Disease Stages

• Stage 1: Early localized infection with erythema migrans skin lesions.

• Stage 2: Neurological or cardiac symptoms due to dissemination.

• Stage 3: Untreated leads to chronic arthritis or skin manifestations.

Relapsing Fever

• Characterized by fever and chills; symptoms recur after intervals of afebril periods.

Laboratory Diagnosis for Borrelia

• Microscopic examination of blood during febrile periods; culture sensitivity is low.

Treatment for Borrelia

• Tetracyclines and penicillins effective for relapsing fever.

• Lyme disease managed with oral amoxicillin, doxycycline.

Genus Campylobacter

• Gram-negative, comma-shaped, microaerophilic rods responsible for human infections (C. jejuni, C. fetus, C. coli).

Culture Properties of Campylobacter

• Thermophilic and microaerophilic, requiring specific growth conditions; non-fermentative.

Virulence and Epidemiology of Campylobacter

• Zoonotic infections common; virulence factors poorly defined; transmission through contaminated food and water.

Clinical Disease and Treatment for Campylobacter

• Gastroenteritis and systemic infections (especially C. fetus) are primary concerns; treated with erythromycin or azithromycin.

Genus Helicobacter

• Curved Gram-negative rods; H. pylori is significant for human pathology.

Classification of Helicobacter

• Distinction based on gastric colonization (H. pylori) and intestinal colonization.

H. pylori Physiology

• Microaerophilic with specific growth requirements; known for urease production.

Pathogenicity of H. pylori

• Multiple virulence factors promote gastric colonization and inflammation, contributing to ulcers and cancer risk.

Epidemiology of Helicobacter

• Common in lower socioeconomic categories; primarily fecal-oral transmission.

Clinical Diseases Caused by Helicobacter

• Lead to chronic gastritis and peptic ulcers, with potential progression to gastric cancer.

Laboratory Diagnosis for Helicobacter

• Microscopic examination, culture on enriched media; various tests for urease activity.

Treatment for Helicobacter

• Combination therapies (PPIs, macrolides, beta-lactams) necessary for effective management.