1. Laboratory aspects of Bacterial GIT infection
Dr. Mohammad shakeeb , MD
specialist in clinical pathology /Microbiology and immunology

2. Introduction
Bacterial agents are a common cause of infectious diarrhea.
Mechanisms of infections range from toxin production to mucosal invasion.
The microbiologist has an important role in isolating these organisms to lead to diagnosis.

3. Collection and Transport of fecal specimen
Stool also is preferred for detection of bacteria responsible for infectious diarrhea but a rectal swab specimen is an acceptable alternative.
Stool specimens should be collected in a clean container with a tight lid.
specimen should not be contaminated with urine, barium, or toilet paper.
Specimen should be transported promptly (within 2 hours) to the laboratory and processed as soon as possible.

4. Specimen Processing and culture media
Processing stool or rectal swab specimens for detection of bacteria is based on the organism or group of organisms expected to be present.
Specimens received for “routine” bacterial culture should be processed to allow recovery of Shigella, Salmonella, and Campylobacter jejuni/coli by plating to appropriate differential, inhibitory and noninhibitory media.
Also ,examination of shiga-like toxin producing E. coli (STEC) and Aeromonas species might be considered.
Consideration of Yersinia enterocolitica, Vibrio cholerae, or other Vibrio species, or Plesiomonas shigelloides depends on prevelance of gastroenteritis caused by these microorganisms

5. To detect STEC, the stool specimen is inoculated onto sorbitol MacConkey agar.
containing 1% D-sorbitol instead of lactose
a medium that differentiates isolates of STEC, which do not ferment sorbitol, from almost all other E. coli, which are sorbitol-positive.

7. When isolation of Y. enterocolitica is requested, Cefsulodin-irgasan- novobiocin(CIN) agar is inoculated and incubated at room temperature.
It is selective and differential media for the recovery of Yersinia enterocolitica.
Colonies will appear as bull’s-eyes with red centers and transparent borders.

9. xylose-lysine deoxycholate (XLD) agar and Hektoen enteric (HE) agar are more selective differential media that are especially useful in selecting for Salmonella spp. and Shigella spp. in heavily contaminated specimens such as stool.

10. Salmonella enterica on Hektoen enteric agar
Salmonella enterica on Hektoen enteric agar. Note the black center of the transparent colonies, indicating H2S production in the absence of carbohydrate fermentation.

11. Green colonies of shigella spp on HE agar

12. Colonies of Salmonella enteritidis (lactose-negative) growing on XLD

13. Colorless Colonies of Shigella (lactose-negative) growing on XLD

14. Proteus vulgaris on Hektoen enteric agar
Proteus vulgaris on Hektoen enteric agar. Note the yellow-orange colonies, indicating the fermentation of at least one of the carbohydrates present in the medium.

16. campylobacter jejuni
Campylobacter spp. are small (0.5–8 µm long × 0.2–0.5 µm wide).
motile,
non–spore-forming,
curved (comma-shaped), (seagull wing shaped) or S-shaped gram- negative bacilli .
grow optimally in an atmosphere containing 5%–10% oxygen and, therefore, are considered to be microaerophilic.
A single stool specimen is generally adequate to detect Campylobacter spp .

17. Campylobacter jejuni Gram stain: Note the comma-shaped appearance of the bacilli.

19. Several media can be used for the selective isolation of Campylobacter spp.
In general, Campylobacter spp. produce gray, flat, irregular, spready colonies, which may become round, convex, and glistening.
Incubation of the plates at 42° C increases selectivity for C. jejuni.
A typical Gram stain appearance and a positive oxidase reaction from a colony growing on selective media at 42° C can be reported as Campylobacter spp.
An enzyme immunoassay (EIA) is available for direct detection of Campylobacter jejuni and Campylobacter coli antigens in stool specimens.

21. Helicobacter Helicobacter spp. are spiral-shaped or curved.
gram-negative , non–spore forming bacilli.
measuring 0.3–1.0 μm wide and 1.5–10 μm long.
They are motile by multiple bipolar or monopolar flagella.
Microaerobic.
Typically, nonculture methods have been utilized to diagnose H. pylori infection.
urea breath test

22. noninvasive test that detects urease activity of H
noninvasive test that detects urease activity of H. pylori by measuring 14C- and 13C-labeled CO2 in the patient’s expelled air after ingestion of labeled urea.
Serologic assays are also widely used in symptomatic patients to detect antibodies against H. pylori.
nonspecific test
for epidemiologic or surveillance tool.
In some cases, biopsies of the affected tissues are obtained and are examined histologically.

23. A commercially available EIA for detection of H
A commercially available EIA for detection of H. pylori antigen in stool is a noninvasive alternative for diagnosis of H. pylori infection.

25. Vibrio Vibrio spp. are facultatively anaerobic. Oxidase-positive.
Short, curved, or straight gram-negative bacilli.
Usually motile by means of polar flagella.
They ferment carbohydrates .
Reduce nitrates to nitrites.
With the exception of V. cholera and V. mimicus, growth of this group of organisms requires media containing NaCl.

27. Selective media containing sucrose, such as thiosulfate citrate bile salts medium, are very useful for culturing stool specimens for Vibrio spp.
V. cholerae ferment the sucrose and appear as yellow colonies on this medium.
An enrichment medium, such as alkaline peptone water, may be used prior to subculture to solid medium to enhance recovery of Vibrio spp. from stool.

29. The string test can be used to separate Vibrio spp. from Aeromonas spp
The string test can be used to separate Vibrio spp. from Aeromonas spp. and P. shigelloides.
In this test, organisms are emulsified in 0.5% sodium deoxycholate, which lyses the vibrio cells, but not those of Aeromonas spp. and P. shigelloides.
With cell lysis there is release of DNA, which can then be pulled up into a string using an inoculating loop.

30. String test used to separate Vibrio spp. (positive) from Aeromonas spp
String test used to separate Vibrio spp. (positive) from Aeromonas spp. and P. shigelloides (negative).

31. Biochemical tests useful for identification of salmonella and shigella

32. Citrate Utilization Test Urea Hydrolysis
Kligler Iron agar (KIA):determine bacterial ability for
Sugar fermentation of lactose & glucose
Production of gas
Hydrogen sulfide (H2S) production
Citrate Utilization Test
Urea Hydrolysis
SIM Medium (H2S, Indole, Motility) Test

33. KIA Principle
Fermentation of glucose (1 part) (ONLY) → entire medium becomes acidic (yellow) in 8-12 hours.
1- Butt (anaerobic conditions) remains acidic (yellow) even after the recommended hour incubation period.
2- Slant (aerobic) reverts to alkaline (red) because of aerobic oxidative deamination of peptones present in the medium into alkaline amines.
Fermentation of lactose sucrose (10 parts) (in addition to glucose) → slant reverts to acid (yellow) because larger organic acids formed will neutralize the alkaline amines, provided the reaction is read in hours.
Reactions should NOT be read beyond 24 hours of incubation, because aerobic oxidation of fermentation products from lactose does proceed and slant will eventually revert to alkaline (red).

35. KIA Key of Results

36. KIA

38. Citrate Utilization Test
Use: to determine bacterial ability to use citrate as the sole source of carbon.
Culture medium: Simmons citrate agar; contains sodium citrate, inorganic ammonium salts (sole source of nitrogen), & pH indicator bromthymol blue (neutral; green & alkaline; blue).
Principle: citrate use → ammonia production → alkaline pH.
Method:
1- Use a needle to lightly touch tip of single 16- to 24-hour-old colony & inoculate.
2- Incubate at 35°C.
3- Observe for development of blue colour.

39. Citrate Utilization Test

40. Urea Hydrolysis
Use: to determine bacterial ability to hydrolyze urea (by urease enzyme) into CO2 & ammonia which alkalinizes the medium.
Culture medium: Christensen’s urea agar or Stuart's urea broth: both contain urea, & phenol red indicator.
Method:
1- Streak agar surface with portion of well-isolated colony or inoculate urea broth with 1-2 drops from overnight enrichment broth.
2- Leave cap on loosely & incubate at 35°C.

41. Urea Hydrolysis Results:
1- Positive: enzyme present, ammonia produced, high pH (bright pink colour).
2- Negative: enzyme absent, NO colour change (yellow orange).
Important urease-positive bacteria:
Proteus sp.
Helicobacter sp.

42. SIM Medium (H2S, Indole, Motility) Test
Hydrogen sulfide (H2S): cystine desulfanase enzyme → sodium thiosulfate reduction → H2S (+ ferrous salts indicator; ferrous sulphate or ferrous ammonium sulphate) → ferrous sulfide black precipitate.
Indole: Positive; tryptophanase enzyme degrades tryptophan → indole + Kovac’s reagent → red ring on top of medium. Negative; enzyme absent, indole NOT produced → clear yellow ring (NO colour change).
Motility in semisolid agar: Positive (motile); fuzzy growth feathering away from stab line creating cloudy appearance & Negative (nonmotile); growth strictly along stab line.

43. SIM Medium (H2S, Indole, Motility) Test

44. SIM Medium (H2S, Indole, Motility) Test

45. Salmonella Do not ferment lactose Negative for indole
Produce hydrogen sulfide(salmonella paratyphi A is exception)
Negative for urease
Positive for citrate

46. Salmonella
Commercially available polyvalent antisera designated A, B, C1, C2, D, E, and Vi are commonly used to preliminarily group Salmonella spp.
The antisera A through E contain antibodies against somatic (“O”) antigens
the Vi antiserum is prepared against the capsular (“K”) antigen of S. typhi
Typing is performed using a slide agglutination test.
S. typhi is positive with Vi and group D.

47. shigella Do not ferment lactose Non-motile
They do not produce gas from glucose(exception is shigella flexneri)
Do not hydrolyze urea
Do not produce hydrogen sulfide
Citrate negative

48. shigella
Preliminary serologic grouping of Shigella spp. Is also performed using commercially available polyvalent somatic (“O”) antisera designated A, B, C, and D.