1. Diagnostic microbiology lecture: 7 CAMPYLOBACTER Abed ElKader Elottol MSc. Microbiology 2010 1

2. GENERAL CHARACTERISTICS OF CAMPYLOBACTER Small, delicate, spirally or curved. Gram-negative bacteria Oxidase positive Fastidious Microaerophilic Motile by polar flagella 2

3. SPECIES 1. Camplylobacter fetus subsp. fetus Non pathogenic 2. Campylobacter fetus subsp. intestinalis 3. Campylobacter fetus subsp. jejuni 4. Campylobacter pylori or Helicobacter pylori (Peptic ulcer) 3

4. Sources of Campylobacters: GIT and reproductive organs of: Chicken Sheeps Cattles Dogs Cats 4

5. 5 Transmission routes Direct occupational Contact Farmers,Butchers, Poultry processors Direct domestic Contact Dogs,Cats Children to children Indirect transmission Untreated water Raw milk Undercooked meat

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7. DISEASES 1.meningitis and meiningoencephalitis in infants: Mortality rate is 50% despite intensive treatment. Specimens include blood, CSF and stool Mostly caused by C. intestinalis. 2. Bacteremia: In children: caused by both C. intestinalis and jejuni. Specimen is blood. 3. Disseminated infections in adults: Usually in debilitated patients with one or more of the following conditions: 7

8. 1. Cardiovascular disease. 3. Endocrinological disorders 4. Chronic alcoholism 5. Malignancies Causative organism: Mostly C. intestinalis = Specimen: Blood, pericardium, pleura, joints and stool. II. Enteric Infections: Species of the genus campylobacter, are one of the major etiologic agents of bacterial enteritis. Campylobacter enteritis is accompanied by fever, headache, muscular pain, nausea and pain. 24 hours following this acute phase, diarrhea develops which may be bloody, mucoid and watery. 8

9. Isolation of Campylobacter from Stool 1. Filtration Technique: 1. One gram of stool specimen is suspended in 20 ml saline 2. Agitate vigorously (on Vortex for 10-15 seconds). 3. Centrifuge at 650-800 rpm for 10 minutes. 4. Take four to five milliliters into a syringe 5. Pass the liquid through two 25 μm filter chamber: =The upper non-sterile chamber is fitted with an 8.0 and 1.2 μm Millipore membrane filter. = The lower steam-sterilized chamber contains a 0.65 um membrane. 6. 2-4 drops of the filtrate are spread onto chocolate agar and incubated. 7. Both C. jejuni and intestinalis grow well on this medium and are very Characteristics. 9

10. Incubation conditions 1.Microaerophilic environment: 5% O2 10% CO2 85% N2 2. To increase the size of C. jejuni, incubate at 42 oC. 10

11. Selective media The introduction of selective media made the isolation of Campylobacter species from a stool sample possible: EXAMPLE: Blood Agar + Antibiotic solution. 11

12. Skirrow agar: Campylobacter Skirrow Agar is a selective solid medium used for the cultivation and isolation of Campylobacter jejuni from fecal specimens. Vancomycin, Polymyxin B, Trimethoprim Colonies of C. jejuni will appear on Campylobacter Skirrow agar as small, mucoid, flat or slightly raised, non-hemolytic translucent and gray. 12

13. Butzler solution Bacitracin Cyclohexamide Colistine B sulfate Cephazolin sodium Novobiocin Campy-BAP it contain Vancomycin Trimethoprim 13

14. Advantages of filtration technique over the selective media 1. All non-campylobacter organisms are retained by the filters. 2. Both C. intestinalis and jejuni will grow on Chocolate agar unlike the selective media which allows only one of the two. 3. You do not need to worry about the decay of antibiotics as in the selective media. 14

15. SEROLOGICAL TESTS: Serological tests are not suitable for routine investigation due to the lack of standardized antigen suspensions and reference sera. ANTIBIOTIC SENSITIVITY: Erythromycin is the drug of choice. Chloramphenicol, aminoglycosides, carbenicillin, clindamycin and tetracycline are also effective. 15

16. HELICOBACTER PYLORI 16

17. Helicobacter pylori Previous name : Campylobacter pylori First isolated in 1983 from human stomach Gram-negative bacilli Curved, spiral or seagull-shaped Motile with multiple polar flagella 17

18. CULTURAL CHARACTERISTICS Grow best at 42-43 oC Microaerophile (7% oxygen) Strongly urease positive (different from Campylobacter) Grow on enriched & selective media Modified Skirrow’s agar (Blood agar with antibiotics) 18

19. HABITAT Human gastric mucosa (world-wide distribution) Approx. 50% of adults >60 years are infected TRANSMISSION (person to person) Oral-oral Fecal-oral There are clusters of infection in families DISEASES BY H. PYLORI Gastritis Gastric & peptic ulcer Gastric cancer Most individuals tolerate the presence of H. pylori for decades 19

20. CLINICAL FEATURES Incubation period : few days Nausea, flatulence & bad breath Recurrent epigastric pain & dyspepsia Epigastric burning sensation Bleeding from ulcer No dissemination 20

21. LAB IDENTIFICATION Specimen : gastric biopsy Direct gram-stained smear of crushed biopsy Direct urease test Place a piece of biopsy in urea broth- red color change in few min to 2 hrs Culture : On Skirrow’s medium ° Oxidase +ve, Urease +ve, catalase +v Serodiagnosis ° ELISA to detect IgG (not established to differentiate active vs past infection ) 21

22. H. pylori Stool Antigen (HpSA) test Urea Breath Test Patient ingests radio-labeled 14C urea If H. pylori infection present : Urease produced by the organism, hydrolyses urea to NH3 and radio-labeled -HCO3 that is exhaled as CO2 which is detected by spectrometer Has good sensitivity & specificity. 22

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24. TREATMENT Triple therapy Metronidazole + Clarythromycin or amoxycillin + Omeprazole Vaccine development : under tria l 24

25. END OF LECTURE 25