L29 - Medical Microbiology Laboratory (Aeromonas, Helicobacter and Campylobacter spp.) by Hussein A. Abid

1. Medical Microbiology Laboratory
Gram Negative Rods (Bacilli)
(Aeromonas, Helicobacter and
Campylobacter spp.)
Hussein A. Abid
Medical Laboratory Scientist
Member at American Society of Microbiology
Chairman of Iraqi Medical Laboratory Association
Teacher at Middle Technical University

2. TAXONOMY
Scientific name Rank
• Aeromonadaceae Family
• Aeromonas Genus
• A. hydrophila
• A. caviae
• A. veronii
Species
(medically important spp.)
2

3. Aeromonas spp.
 Gram-negative rod-shaped bacteria that morphologically
resemble members of the family Enterobacteriaceae
 Facultative anaerobic
 Most of the 14 described species have been associated
with human diseases
 The organisms are ubiquitous in fresh and brackish water
 Acquired by ingestion of or exposure to contaminated
water or food
 Motile species have single polar flagellum (nonmotile
species apparently not associated with human disease)
3

4. 4
Aeromonas spp.
 Associated with gastrointestinal disease:
 Chronic diarrhea in adults
 Self-limited acute, severe disease in children resembling
shigellosis with blood and leukocytes in the stool
 3% carriage rate
 Wound infections:
 Opportunistic systemic disease in immunocompromised
 Putative virulence factors include: endotoxin;
hemolysins; eneterotoxin; proteases; siderophores;
adhesins

5. TAXONOMY
Scientific name Rank
• Helicobacteraceae Family
• Helicobacter Genus
• H. pylori
• H. cinaedi
• H. fenneliae
Species
(medically important spp.)
5
Important human pathogen with their reservoirs:
• H. pylori (human; no animal reservoir)
• H. cinaedi (male homosexuals; rodents)
• H. fenneliae (male homosexuals; rodents)

6. Helicobacter spp.
 First observed in 1983 as
Campylobacter-like organisms
(formerly Campylobacter pyloridis)
in the stomachs of patients with
type B gastritis.
6
 Nomenclature of Helicobacter was first established in
1989.
 Only three species are currently considered to be human
pathogens

7. Helicobacter spp.
 Helicobacter pylori is major human pathogen associated
with gastritis; peptic ulcer disease and neoplasia
 Stomach of many animal species also colonized
 Urease (gastric strains only), mucinase, and catalase
positive, highly motile microorganisms
 Other Helicobacters: H. cinaedi and H. fenneliae
 Colonize human intestinal tract
 Isolated from homosexual men with proctitis (inflammation
of rectum and anus), proctocolitis, enteritis, and bacteremia
and are often transmitted through sexual practices
7

8. Helicobacter spp.
 Gram-negative; helical (spiral or curved); blunted/
rounded ends in gastric biopsy specimens; cells become
rod-like and coccoid on prolonged culture
 Produce urease, mucinase, and catalase
 H. pylori tuft (lophotrichous) of 4-6 sheathed flagella
(30um X 2.5nm) attached at one pole
 Single polar flagellum on H. fenneliae & H. cinaedi
 Smooth cell wall with unusual fatty acids
8

9. PATHOGENESIS (H. pylori)
 Colonize mucosal lining of stomach & duodenum in
man & animals
 Adherent to gastric surface epithelium or pit epithelial
cells deep within the mucosal crypts adjacent to gastric
mucosal cells
 Mucosa protects the stomach wall from its own gastric
milleu of digestive enzymes and hydrochloric acid
 Mucosa also protects Helicobacter from immune
response
 Most gastric adenocarcinomas and lymphomas are
concurrent with or preceded by an infection with 9

10. LAB IDENTIFICATION (H. pylori)
 Recovered from or detected in endoscopic antral gastric
biopsy material; multiple biopsies are taken
 Many different transport media
 Culture media containing whole or lysed blood
 Microaerophilic
 Grow well at 37 o
C, but not at 25 nor 42 o
C
 Like Campylobacter, does not use carbohydrates, neither
fermentatively nor oxidative.ely
10

11. H. pylori
Clinical specimens:
 Biopsy for histopathological examination
 Stool for antigen detecting (by ELISA)
 Blood for serological testing
11

12. H. pylori (microscopy)
 Gram-negative bacilli, curve or spiral shape
12

13. H. pylori (culture)
 Blood agar, chocolate agar and Columbia blood agar,
small, convex, translucent, non hemolytic colonies.
13
Blood agar Columbia blood agar

14. 14
BIOCHEMICAL TESTS
1. Catalase: positive (+ve)
2. Oxidase: positive (+ve)
3. Mucinase: positive (+ve)
4. Motility: motile
5. Hippurate hydrolysis: negative (-ve)
6. Urease: positive (+ve), within 2 minutes

15. 15
BIOCHEMICAL TESTS
+VE -VE
Motility testing

16. 16

17. 17

18. 18
UREA BREATH TEST

19. 19
SEROLOGICAL TESTS

20. 20
TREATMENT
Triple Chemotherapy (synergism):
 Proton pump inhibitor (e.g., omeprazole = Prilosec(R))
 One or more antibiotics (e.g., clarithromycin;
amoxicillin; metronidazole)
 Bismuth compound
Inadequate treatment results in recurrence of symptoms

21. TAXONOMY
Scientific name Rank
• Campylobacteraceae Family
• Campylobacter Genus
• C. jejuni
• C. coli
• C. fetus
Species
(medically important spp.)
21

22. Campylobacter spp.
 First isolated as Vibrio fetus in 1909 from spontaneous
abortions in livestock
 Campylobacter enteritis was not recognized until the mid-
1970s when selective isolation media were developed for
culturing campylobacters from human feces
 Most common form of acute infectious diarrhea in
developed countries; higher incidence than Salmonella &
Shigella combined
 In the U.S. > 2 million cases annually, an annual
incidence close to the 1.1% observed in the United
Kingdom; Estimated 200-700 deaths 22

23. Campylobacter spp.
 Small, thin, helical (spiral or curved) cells with typical gram-
negative cell wall; “Gull-winged” appearance
o Tendency to form coccoid & elongated forms on prolonged
culture or when exposed to O2
 Distinctive rapid darting motility:
o Long sheathed polar flagellum at one (polar) or both
(bipolar) ends of the cell
o Motility slows quickly in wet mount preparation
 Microaerophilic & capnophilic 5% O2,10% CO2, 85% N2
 Thermophilic (42-43 ºC) (except C. fetus)
 May become non-culturable in nature
 Oxidase: positive 23

24. PATHOGENESIS (Campylobacter spp.)
 Campylobacteriosis is an infection by Campylobacter
 The common routes of transmission are fecal-oral, ingestion of contaminated food or
water, and the eating of raw meat.
 It produces an inflammatory, sometimes bloody, diarrhea, periodontitis or dysentery
syndrome, mostly including cramps, fever and pain. The infection is usually self-
limiting and in most cases, symptomatic treatment by liquid and electrolyte
replacement is enough in human infections. The use of antibiotics, on the other
hand, is controversial. Symptoms typically last for five to seven days.
 The sites of tissue injury include the jejunum, the ileum, and the colon. Most strains
of C jejuni produce a toxin (cytolethal distending toxin) that hinders the cells from
dividing and activating the immune system. This helps the bacteria to evade the
immune system and survive for a limited time in the cells. A cholera-like enterotoxin
was once thought to be also made, but this appears not to be the case.
 In some cases, a Campylobacter infection can be the underlying cause of Guillain–
Barré syndrome. Gastrointestinal perforation is a rare complication of ileal infection.
 Infectious dose and host immunity determine whether gastroenteric disease
develops (Some people infected with as few as 500 organisms while others need
>106
CFU) 24

25. LAB IDENTIFICATION (Campylobacter spp.)
Specimen collection and processing:
 Feces refrigerated & examined within few hours
 Rectal swabs in semisolid transport medium
 Blood drawn for C. fetus
 Care to avoid oxygen exposure
 Selective isolation by filtration of stool specimen
 Enrichment broth & selective media
 A selective blood agar medium (Skirrow's medium) can
be used. Greater selectivity can be gained with an
infusion of a cocktail of antibiotics: vancomycin,
polymixin-B, trimethoprim and actidione, (Preston's agar).
25

26. LAB IDENTIFICATION (Campylobacter spp.)
Microscopy:
 Gull-wing appearance in gram stain
 Darting motility in fresh stool (rarely done in clinical lab)
 Fecal leukocytes are commonly present
Identification:
 Growth at 25, 37, or 42-43 o
C
 Hippurate hydrolysis (C. jejuni is positive)
 Susceptibility to Nalidixic acid & Cephalothin
26

27. 27
Campylobacter (microscopy)
 Gram negative (-ve), curved rods

28. 28
Campylobacter (culture)
 Butzler’s agar (blood agar + antibiotics): color white-
gray, mucoid-slight, microaerophilic O2 5% & CO2 10%,
growth at 37 °C & 42 °C, no growth at 25°C. Non
hemolytic, droplet like colonies, transparent to gray-white.

29. 29
Campylobacter (culture)
 Blood agar plate (BAP):
On Blood agar: C. jejuni and C. coli produce non-
hemolytic spreading, droplet-like colonies

30. 30
BIOCHEMICAL TESTS
1. Catalase: positive (+ve)
2. Oxidase: positive (+ve)
3. Motility: motile
4. Hippurate hydrolysis: positive for C. jejuni, negative
for C. coli
5. Urease: negative (-ve)

31. 31
BIOCHEMICAL TESTS
Hippurate hydrolysis testing
+ve -ve

32. 32
BIOCHEMICAL TESTS
Campylobacters are motile therefore dispersed growth is
observed in motility medium