Isolation and identification of Campylobacter bacteria.pptx

Microbiology, campylobacter

1. Isolation and identification of Campylobacter bacteria Presented by: Muhammad Hussain Taqi BS Applied Microbiology 7 th

2. What is Campylobacter ? Genus of Gram-negative, spiral-shaped bacteria. Major cause of bacterial gastroenteritis worldwide. Common species: Campylobacter jejuni and Campylobacter coli . Significance of Isolation and Identification: Responsible for foodborne illnesses. Typically transmitted through contaminated food (especially poultry), water, or unpasteurized milk. Proper diagnosis crucial for effective management and control of outbreaks. Introduction

3. General Characteristics of Campylobacter Morphology: Gram-negative. Motile, spiral, and comma-shaped rods. Single polar flagellum, providing "corkscrew" motility. Oxygen Requirement: Microaerophilic (requires low oxygen levels to grow, around 5% O₂). Growth Conditions: Prefer temperatures around 42°C (thermophilic). Sensitive to environmental conditions like oxygen, desiccation, and cold temperatures.

4. Specimen Collection and Transportation Specimen Types: Commonly stool samples. Other samples: Blood, water, food, and environmental surfaces. Transportation: Transport media: Cary-Blair or alkaline peptone water. Store specimens at 4°C if immediate processing is not possible (do not freeze).

5. Primary Isolation Procedure Media Used: Selective media: Skirrow’s agar. Campy-CVA agar. Blood-free media (e.g., charcoal cefoperazone deoxycholate agar). Contains selective antibiotics (e.g., cefoperazone , vancomycin) to suppress competing flora. Incubation Conditions: Incubate at 42°C for 48 hours in a microaerophilic atmosphere (5% O₂, 10% CO₂, 85% N₂). Special incubators or gas-generating sachets used to create these conditions.

6. Colony Morphology and Microscopy Colony Appearance: Small, grayish to colorless, moist, flat or slightly raised colonies. Microscopic Examination: Use a Gram stain: Gram-negative rods. Characteristic spiral or "gull-wing" shapes. Motility: Wet mount preparation under phase-contrast microscopy shows typical darting motility.

7. Biochemical Identification Tests 1. Oxidase Test Campylobacter species are oxidase-positive. Procedure: Place colonies on filter paper with oxidase reagent (tetramethyl-p-phenylenediamine); positive result shows a dark purple color within 10 seconds. 2. Catalase Test Positive for catalase enzyme (especially C. jejuni ). Procedure: Add hydrogen peroxide to a bacterial colony; bubbles indicate a positive result. 3. Hippurate Hydrolysis Test Differentiates C. jejuni ( hippurate -positive) from other species. Procedure: Inoculate a tube of sodium hippurate broth; add ninhydrin after incubation. Purple color indicates a positive result.

8. Biochemical Identification Tests (continued) 4. Nitrate Reduction Test Most Campylobacter species reduce nitrate to nitrite. Procedure: Inoculate nitrate broth with the isolate and check for nitrite presence by adding sulfanilic acid and α-naphthylamine. 5. Growth in 3.5% NaCl Helps in differentiating certain species of Campylobacter . Procedure: Inoculate the medium with 3.5% NaCl and check for growth. 6. Urease Test Campylobacter species are urease-negative. Procedure: Inoculate urea broth; no color change indicates a negative result.

9. Molecular Identification (Optional for Higher Specificity) PCR (Polymerase Chain Reaction): Procedure: Extract DNA from bacterial colonies. Amplify specific genes (e.g., 16S rRNA, flaA gene) for Campylobacter species identification. Use gel electrophoresis to analyze PCR product. Advantages of PCR: High sensitivity and specificity. Allows identification at the species level

10. Antimicrobial Susceptibility Testing Purpose: Determines the susceptibility of Campylobacter isolates to antibiotics. Methods: Disk diffusion test. E-test (for determining minimum inhibitory concentration). Commonly Tested Antibiotics: Macrolides (e.g., erythromycin). Fluoroquinolones (e.g., ciprofloxacin). Tetracyclines (e.g., doxycycline).

11. Reporting and Interpretation Colony Identification: Based on morphology, motility, and biochemical tests (oxidase, catalase, hippurate , etc.). Susceptibility Reporting: Determine resistance profiles for potential clinical treatment. Report as per CLSI (Clinical and Laboratory Standards Institute) guidelines.

12. Quality Control Controls for Testing: Positive control: Known Campylobacter strain (e.g., C. jejuni ). Negative control: Escherichia coli or Salmonella spp. Importance of QC: Ensures accuracy and reliability of results.

13. Common Errors and Challenges Challenges in Isolation: Overgrowth of competing flora. Failure to achieve appropriate microaerophilic conditions. False-Negative Results: Inadequate transport and handling. Use of inappropriate media.

14. Conclusion Summary: Campylobacter is a significant pathogen requiring careful isolation and identification. Use of selective media and microaerophilic conditions is essential for successful isolation. Biochemical tests such as oxidase, catalase, and hippurate hydrolysis are key for identification. Clinical Importance: Accurate identification assists in the treatment and management of gastroenteritis and helps control the spread of infection.

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