1. CURVED GRAM-NEGATIVE Chapter 20
BACILLI

2. OBJECTIVES
 Describe the gram stain morphology of Vibrio species.
 Discuss the clinical significance of Vibrio cholerae.
 List the selective media for the isolation of Vibrio species and
describe the different colony morphologies observed on the
media.
 Discuss the mode of transmission of Vibrio cholerae.
 Identify Vibrio cholerae based on biochemical reactions.
 Explain the role of the vibrio toxin the pathogenesis of cholera
infections.
 Differentiate the species of Vibrio species discussed in this
unit based on colony morphology on Thiosulfate Citrate Bile
Salts Sucrose (TCBS) and clinical symptoms of patient.

3. VIBRIO SPECIES

4. GENERAL CHARACTERISTICS
 More than 30 recognized species
 Approximately 12 species have been associated with human
disease
 Persists in the environment:
 Grows in saltwater, freshwater
 Causes human disease because it can adapt to colonize intestinal tract
if ingested

5. PROGRESSION OF INFECTION

6. MICROSCOPIC CHARACTERISTICS
 Gram-negative, curved rod
 Motile  single polar flagellum
 “Darting” motility
 “Shooting star” motility

7. GENERAL BIOCHEMICAL
CHARACTERISTICS
 Most are:
 Indole positive
 Urea negative
 Nitrate positive
 Most are oxidase positive
 Posses the vibriostatic compound O/129

8. MEDIA AND GROWTH
 Media
 Grow quite readily on various routine media
 Use of Thiosulfate-Citrate-Bile Salts-sucrose agar (TCBS) has
enhanced recovery
 Alkaline peptone water - enrichment broth
 Conditions for growth
 Facultative anaerobes
 Cultures should be incubated at 35°C in air or CO 2

9. COLONY MORPHOLOGY
 On TCBS agar:
 Sucrose fermenters are yellow
 V. cholera and V. alginolyticus
 Non-sucrose fermenters are green
 V. parahaemolyticus
 On BAP and chocolate agar:
 Most are iridescent with a greenish hue

10. VIBRIO CHOLERAE

11. EPIDEMIOLOGY
 Mostly found in aquatic environments
 Transmitted:
 Fecal-oral route
 Contact with contaminated water
 Ingestion of contaminated shellfish or other seafood

12. VIRULENCE FACTORS
Production of Cholera toxin or choleragen
 Provokes an accumulation of cyclic AMP in the cell
membrane which causes mucosal cells to
hypersecrete electrolytes and water into the lumen of
the GI tract which results in watery diarrhea and fluid
loss = rice -water stools
 Such a rapid fluid and electrolyte loss leads to severe
dehydration, hypovolemic shock, metabolic acidosis
and death in just a matter of hours

13. CLINICAL SIGNIFICANCE
 Etiologic agent of cholera
 Cholera is mostly seen in Asia and South America
 Causes both intestinal and extraintestinal infections
 Infections are acquired by ingesting contaminated food or
water
 Characteristic “rice-water” stools

14. EPIDEMIOLOGY
Contaminated water enters stomach
Bacterial cells adhere to gastric and intestinal
mucosal epithelial cells
Enterotoxin is produced
 Increase in cyclic AMP - prevents the re-absorption of
Na+ ions and the excretion of sodium bicarbonate and
potassium
 Causes water to leave the epithelial cells into
intestinal lumen causing "rice water" stools
It may lead to death in matter of hours

15. SPECIMEN MANAGEMENT
 Stool specimens are preferred - must be transported in Cary -
Blair medium
 Rectal swabs acceptable

16. MEDIA FOR ISOLATION
 Will grow on BAP, Choc, and MAC (non -lactose fermenter on
MAC)
 Alkaline peptone water broth for enrichment
 Thiosulfate Citrate Bile Salts Sucrose (TCBS) - selective and
differential media (yellow or green colonies)
 Yellow - ferment sucrose
 Green - do not ferment sucrose

17. COLONY MORPHOLOGY
Vibrio cholerae - BAP

18. COLONY MORPHOLOGY
Vibrio cholerae - MAC

19. COLONY MORPHOLOGY
Vibrio cholerae - TCBS

20. BIOCHEMICAL CHARACTERISTICS
 Oxidase positive
 Sucrose positive (yellow on TCBS)
 Lactose negative (clear on MAC)
 Lysine positive
 Ornithine positive
 Arginine negative
 String test positive – 0.5% deoxycholate

21. BIOCHEMICAL CHARACTERISTICS
 String test
 Principle:
 Addition of 0.5% sodium deoxycholate causes most Vibrio spp. to lyse and
release DNA, which can be pulled up into a string with a inoculating loop

22. TREATMENT
 Therapy - fluid and electrolyte replacement
 Antimicrobial therapy will shorten the course of the disease
and may decrease bacterial excretion
 Tetracycline is the drug of choice

23. VIBRIO ALGINOLYTICUS

24. CLINICAL SIGNIFICANCE
 Least pathogenic for humans and least isolated
 Wound, ears, or eyes after trauma and sea water exposure

25. MEDIA AND GROWTH
 Strict halophile = requires added NaCl for optimal growth
 Yellow colonies on TCBS

26. COLONY MORPHOLOGY
Vibrio alginolyticus - BAP

27. COLONY MORPHOLOGY
Vibrio alginolyticus - TCBS

28. BIOCHEMICAL CHARACTERISTICS
 Sucrose positive

29. VIBRIO
PARAHAEMOLYTICUS

30. CLINICAL SIGNIFICANCE
 Acute gastroenteritis associated with the ingestion of raw
contaminated seafood, particularly oysters
 Usually mild but can be fatal
 Endemic in Japan; #1 cause of “summer diarrhea”
 Usually self-limiting disease
 Rare extraintestinal pathogen

31. COLONY MORPHOLOGY
Vibrio parahemolyticus - BAP

32. COLONY MORPHOLOGY
Vibrio parahemolyticus - TCBS

33. BIOCHEMICAL CHARACTERISTICS
 Sucrose negative
 Beta-hemolytic on blood agar

34. VIBRIO VULNIFICUS

35. CLINICAL SIGNIFICANCE
 Second most pathogenic
 Causes wound infections:
 Wound infections – after exposure to marine animals or marine
environment
 Wound infections may lead to septicemia
 Primary septicemia – 24 hours after ingestion of raw oysters

36. COLONY MORPHOLOGY

37. COLONY MORPHOLOGY
Vibrio vulnificus - green or yellow on TCBS Agar

38. BIOCHEMICAL CHARACTERISTICS
 Sucrose variable

39. CAMPYLOBACTER SPECIES
& HELICOBACTER PYLORI

40. OBJECTIVES
 Discuss the clinical significance of Campylobacter and
Helicobacter pylori.
 Summarize specimen collection and transport procedures for
Campylobacter and Helicobacter.
 Select appropriate culture media and incubation conditions
for Campylobacter cultures.
 Compare the methods for obtaining microaerobic conditions.
 Describe the microscopic morphology of Campylobacter and
Helicobacter.
 Differentiate species of Campylobacter based on biochemical
identification.
 Explain the principle and procedure of the CLO test and breath
tests for the detection of Helicobacter.

41. CAMPYLOBACTER SPECIES

42. CLINICAL SIGNIFICANCE
 Gastroenteritis - Campylobacter spp. is one of the leading
causes of bacterial diarrhea worldwide
 Septicemia

43. EPIDEMIOLOGY
 Transmitted via contaminated food, milk, or water

44. SPECIMEN MANAGEMENT
 Stool and rectal swabs - if specimen cannot be cultured within
4 hours of collection, place in Cary -Blair transport medium
 Blood

45. GROWTH CONDITIONS
 Optimal temperature for growth is 42 ̊C
 Microaerophilic (5-10% oxygen)
 Capnophilic (8-10% CO 2 )
 Needs at least 48-72 hours for growth

46. MICROSCOPIC CHARACTERISTICS
Curved gram negative rods
Comma shapes, "S" shapes and gull wing
forms are seen
Organisms may occur in short or occasionally
long chains
Oxidase positive
Non-sporeforming
Cells have a single polar unsheathed flagellum
at one or both ends = “darting motility”

47. MEDIA FOR ISOLATION
 Media
 Campy BAP (most commonly used)
 Butzler Medium
 Skirrow medium
 Campy-Thio for enrichment
 Examine at 24, 48 and 72 hours

48. COLONY MORPHOLOGY
 Colonies smooth
 Convex
 Translucent
 May be flat and watery with irregular edges
 They may be pinpoint to spreading over large areas of the
plate

49. COLONY MORPHOLOGY

50. BIOCHEMICAL CHARACTERISTICS
 Catalase positive
 Oxidase positive
 Darting motility

51. BIOCHEMICAL CHARACTERISTICS
 C. coli
 Hippurate negative
 Resistant to cephalothin
 Susceptible to nalidixic acid
 C. jejuni
 Hippurate positive
 Resistant to cephalothin
 Susceptible to nalidxic acid

52. TREATMENT
 Antimicrobic therapy is not recommended in most cases of
diarrhea
 Supportive care and fluid replacement
 When needed:
 Erythromycin is the drug of choice
 Systemic infections are treated with Gentamycin

53. HELICOBACTER PYLORI

54. CLINICAL SIGNIFICANCE
 Found only on mucus-secreting epithelial cells of the stomach
 Causative agent of active chronic gastritis (type B gastritis) =
Peptic ulcer disease

55. MICROSCOPIC MORPHOLOGY
 Gram negative
 Small curved (gull wing and U shapes)
 Slightly plump bacilli

56. SPECIMENS
 Stool cultures are not often done for detection of Helicobacter
pylori
 Specimens/test of choice include:
 Invasive (tissue biopsy)
 CLO rapid urease test
 Non-invasive
 Urea breath test
 Serological detection of IgG

57. BIOCHEMICAL CHARACTERISTICS
 Oxidase positive
 Catalase positive
 Urease positive

58. WHAT’S NEXT?
 Review Mahon, chapter 20
 Complete AUMoodle Review