1. Lab 10 BACTERIOLOGY OF THE GASTROINTESTINAL TRACT Lab Manual (p.67-82)

2. THE GASTROINTESTIONAL TRACT

3. THE GASTROINTESTINAL TRACT HOST DEFENSE MECHANISMS Anatomical characteristics/mechanisms to prevent/fight infection: High acidity of stomach prevents most microbes from surviving, making it almost sterile. Normal microbiota – a variety of microorganisms that compete with each other for space and nutrients, aid in digestion, immunity and preventing infection from pathogens. –Enterobacteriaceae (enteric Gram-negative rods), Staphylococcus spp., Streptococcus spp., Enterococcus spp. and various anaerobic bacteria

4. ENTEROBACTERIACEAE (ENTERIC Gram-negative RODS) Enterobacteriaceae are a family of Gram- negative rods that include both harmless and pathogenic bacteria: –Characteristics: Gram-negative rods Facultative anaerobic Ferment glucose Oxidase negative If motile, peritrichious flagella

5. ENTEROBACTERIACEAE (ENTERIC Gram-negative RODS) Enterobacteriaceae are commonly found as 1.) Normal flora or 2.) Pathogenic in the gastrointestinal tract (termed “enterics”) Non-pathogenic in the gastrointestinal tract: –E.coli (exception: Shiga toxin-producing E. coli) –Klebsiella spp. –Proteus spp. –Citrobacter spp. Pathogenic in the gastrointestinal tract: –Salmonella sp. –Shigella sp.

6. INFECTIONS OF THE GASTROINTESTINAL TRACT Cholera Diarrhea or Gastroenteritis Colitis

7. THE GASTROINTESTINAL TRACT Specimens & Diagnosis Fresh stool or stool in preservative (Cary- Blair transport) –Routine Pathogens detected: Salmonella, Shigella, Campylobacter, and Shiga toxin-producing bacteria –Toxigenic Clostridium difficile (CDIFF) –Special bacteria requests: Yersinia enterocolitica, Aeromonas spp., Vibrio spp.

8. THE GASTROINTESTINAL TRACT Specimens & Diagnosis Parasites (requires special preservative [EcoFix]): –Ova and Parasite screening: Giardia spp. and Cryptosporidium spp. –Ova and Parasites extended testing: Other than above, including all protozoa, eggs and worms in stool. Viruses –Not routinely tested

9. BIOCHEMICAL TESTS

10. BIOCHEMICAL TESTS SIM (Sulfide, Indole and Motility MRVP (Methyl Red and Voges Proskauer) Citrate TSI (Triple Sugar Iron) Agar

11. SIM TEST This is a multipurpose medium that can evaluate 3 different physiological characteristics in one tube The SIM (Sulfur Indole Motility) test is used to determine if the bacteria: –(S): to determine the production of sulfide –(I): to determine the presence of an enzyme tryptophanase, which is responsible for producing the end product indole –(M): to determine if the bacterium is motile

12. METHYL RED / VOGES PROSKAUER (MRVP) TEST 2 Biochemical tests performed in the same media (MRVP broth) that contains glucose MR – Methyl red VP – Voges-Proskauer Both of these tests are performed on MR-VP Medium (contains glucose)

13. METHYL RED TEST The MR TEST determines if a bacterium is capable of mixed acid fermentation Mixed acid fermentation results when glucose is fermented to produce acids, which lowers the pH of the media The isolate is inoculated to a MRVP broth and incubated overnight Methyl Red is added to the tube to detect whether or not large amounts of acid has been produced

14. VOGES-PROSKAUER TEST The VP TEST determines if a bacterium ferment sugars to produce the neutral alcohol end products acetoin and butanediol The isolate is inoculated to a MRVP broth and incubated overnight Barritt’s reagents are added to the tube to detect whether the neutral products have been produced

15. CITRATE TEST Citrate agar contains citrate (carbon source), ammonium phosphate (nitrogen source), and bromthymol blue (pH indicator) –Used to determine if a bacterium can transport citrate into their cell to utilize citrate as a source of energy –Nitrogen (from a source such as ammonium - NH 4 + ) is also a requirement for growing bacteria Utilization of citrate and nitrogen release alkaline by-products which raise the pH that is detected by a color change in the citrate agar slant

16. CITRATE TEST

17. TSI (Triple Sugar Iron agar) Triple-Sugar Iron agar is a differential medium used to identify Gram- negative enteric bacteria based on their ability to ferment selected carbohydrates and produce hydrogen sulfide (a metabolic by-product) Many biochemical tests are performed simultaneously in a single tube

18. TSI (Triple Sugar Iron agar) Each TSI slant contains: 1 part glucose and another carbohydrate/sugar 10 parts glucose 10 parts lactose 10 parts sucrose) Phenol Red (pH indicator) Two sources of sulfur (used to detect the production of sulfur) Cysteine and thiosulfate An iron salt Peptone

19. TSI (Triple Sugar Iron agar) The bacterium is inoculated and incubated overnight at 35C Glucose utilized within 8 – 12hrs Butt remains acidic after 18 –24 hrs Slant reverts to alkaline (red) due to oxidation of the fermentation products under aerobic conditions on the slant results in: CO 2 and H 2 0 and the oxidation of peptones in the medium to alkaline amines Slant reverts back to yellow This means that the fermentation of lactose and/or sucrose has occurred, yielding acidic products Slant stays red No fermentation of lactose and/or sucrose

20. TSI (Triple Sugar Iron agar) Production of gas Positive for gas (CO 2 or H 2 ) - Cracks or bubbles in the agar Negative for gas – No cracks or bubbles in the agar Black precipitant in the butt Desulfurization of sulfur-containing amino acids Indicates the production of ferrous sulfide and H 2 S gas

21. BIOCHEMICAL TESTS SET UP

22. SETUP: SIM TEST

23. SETUP: MR-VP TEST

24. SETUP: CITRATE TEST

25. SETUP: TSI SLANT Loose cap on all tubes!

26. BIOCHEMICAL TESTS RESULTS

27. RESULTS: SIM TEST

28. RESULTS: MR-VP TEST

29. METHYL RED TEST –POSITIVE MR TEST: Large amounts of acid = medium remains RED (pH below 4.4) –NEGATIVE MR TEST: Neutral products produced = medium turns YELLOW (pH above 6.2)

30. VOGES-PROSKAUER TEST – POSITIVE TEST: Production of RED color – NEGATIVE TEST: No RED color

31. RESULTS:CITRATE TEST

32. IMViC ( Indole, Methyl Red, Voges-Proskaeur, Citrate) The IMViC tests to identify enteric Gram-negative rods to the Genus or even Species level

33. RESULTS: TSI SLANT 1.Slant (Lactose and/or sucrose) Acid = yellow Alkaline = red 2.Butt (Glucose) Acid = yellow Alkaline = red 3.Gas production Positive = bubbles or cracks in the media Negative = intact agar with no air bubbles 4.Hydrogen sulfide production Positive = black to dark charcoal gray insoluble precipitate Negative=no black color detected