Microbiology

Gram-negative Rods:
Microaerophiles

Campylobacter and Helicobacter

Chaisit Niyasom, Ph.D. (medical microbiology)

Medical Microbiology
Medical Microbiology
 Campylobacter
• Phylum: Proteobacteria
• Class : Epsilonproteobacteria
• Order : Campylobacterales
• Family : Campylobacteraceae
• Genus : Campylobacter
 General Characteristics
 Gram-negative
 Helical (spiral or curved) morphology; Tend to be
pleomorphic
 Characteristics that facilitate penetration and
colonization of mucosal environments (e.g.,
motile by polar flagella; corkscrew shape)
 Microaerophilic atmospheric requirements
 Become coccoid when exposed to oxygen or
upon prolonged culture
 Neither ferment nor oxidize carbohydrates
 History of Campylobacter
 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
Morphology & Physiology of Campylobacter
 Small, thin (0.2 - 0.5 um X 0.5 - 5.0 um), helical (spiral or
curved) cells with “Gull-winged” appearance
 Distinctive rapid darting motility
• Long sheathed polar flagellum at one (polar) or both
(bipolar) ends of the cell
• Motility slows quickly in wet mount preparation
 Microaerophilic & capnophilic 5%O2, 10%CO2, 85%N2
 Thermophilic (42-43C) (except C. fetus)
• Body temperature of natural avian reservoir

 May become nonculturable in nature

Campylobacter Species Associated
with Human Disease
Guillain-Barre Syndrome (GBS)
กิลแลง-บาเร

 Low incidence potential sequelae

 Reactive, self-limited, autoimmune disease
 Campylobacter jejuni most frequent antecedent
pathogen
 Immune response to specific O-antigens cross-reacts
with ganglioside surface components of peripheral
nerves (molecular or antigenic mimicry)
• Acute inflammatory demyelinating neuropathy (85%
of cases) from cross reaction with Schwann-cells or
myelin
• Acute axonal forms of GBS (15% of cases) from
molecular mimicry of axonal membrane
กลุ่มอาการที่เกิดจาก การอักเสบเฉียบพลันของเส้น
ประสาทหลายเส้นพร้อมกัน
- ก่อให้เกิดอาการกล้ามเนื้ออ่อนแรงเฉียบพลัน
หรืออาจเป็ นอัมพาต
- อาจต้องใช้เครื่องช่วยหายใจสาเหตุจากกล้าม
อัตราเกิดประมาณ 1-3 รายต่อประชากร 100,000 คนต่อ
Epidemiology of Campylobacteriosis

 Zoonotic infections in many animals particularly avian

(bird) reservoirs
 Spontaneous abortions in cattle, sheep, and swine, but
generally asymptomatic carriage in animal reservoir

 Humans acquire via ingestion of contaminated food

(particularly poultry), unpasteurized milk, or improperly
treated water

 Infectious dose is reduced by foods that neutralize gastric

acidity, e.g., milk. Fecal-oral transmission also occurs
Epidemiology of Campylobacteriosis

 Contaminated poultry accounts for more than half of

the camylobacteriosis cases in developed countries but
different epidemiological picture in developing countries

 In U.S. and developed countries: Peak incidence in

children below one year of age and young adults (15-24
years old)

 In developing countries where campylobacters are

hyperendemic: Symptomatic disease occurs in young
children and persistent, asymptomatic carriage in adults
Epidemiology of Campylobacteriosis

 Globally, C. jejuni subsp. jejuni accounts for more

than 80% of all Campylobacter enteritis

 C. coli accounts for only 2-5% of the total cases in the

U.S.; C. coli accounts for a higher percentage of cases
in developing countries
 Pathogenesis & Immunity
 Infectious dose and host immunity determine
whether gastroenteric disease develops
• Some people infected with as few as 500 organisms
while others need >106 CFU
 Pathogenesis not fully characterized
• No good animal model
• Damage (ulcerated, edematous and bloody) to the
mucosal surfaces of the jejunum, ileum, colon
• Inflammatory process consistent with invasion of the
organisms into the intestinal tissue; M-cell (Peyer’s
patches) uptake and presentation of antigen to
underlying lymphatic system
 Non-motile & adhesin-lacking strains are
avirulent
 Putative Virulence Factors
Cellular components:
Endotoxin
Flagellum: Motility
Adhesins: Mediate attachment to mucosa
Invasins
GBS is associated with C. jejuni serogroup
O19
S-layer protein “microcapsule” in C. fetus:

Extracellular components:
Enterotoxins
Cytopathic toxins
 Laboratory Identification
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
Skirrow’s medium (vancomycin, polymyxin B,
trimethoprime)
 Filtration: pass through 0.45 μm filters

Campylobacter Selective
(Skirrow) Agar
Grey spreading colonies
 Laboratory Identification

• 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 oC
 Hippurate hydrolysis (C. jejuni is positive)
 Susceptibility to nalidixic acid & cephalothin
 Principle of Hippurate hydrolysis test

• detect the ability of bacteria to hydrolyse hippurate into glycine and

benzoic acid by action of hippuricase enzyme present in bacteria.

• Previously, it was tested using ferric chloride as indicator to detect

benzoic acid which took 48 hours to produce result

• but now a rapid test (2 hours compared to 48 hours) is used in which

an oxidizing agent ninhydrin is used as an indicator. Ninhydrin reacts
with glycine to form a deep blue or purple color.
Laboratory Identification (cont.)
Treatment, Prevention & Control
 Gastroenteritis:
• Self-limiting; Replace fluids and electrolytes
• Antibiotic treatment can shorten the excretion period;
Erythromycin is drug of choice for severe or complicated
enteritis & bacteremia; Fluroquinolones are highly active (e.g.,
ciprofloxacin was becoming drug of choice) but fluoroquinolone
resistance has developed rapidly since the mid-1980s apparently
related to unrestricted use and the use of enrofloxacin in poultry
• Azithromycin was effective in recent human clinical trials
• Control should be directed at domestic animal reservoirs and
interrupting transmission to humans
 Guillain-Barre Syndrome (GBS)
• Favorable prognosis with optimal supportive care
• Intensive-care unit for 33% of cases
Helicobacter
 Helicobacter
• Phylum: Proteobacteria
• Class: Epsilonproteobacteria
• Order: Campylobacterales
• Family: Helicobacteraceae
• Genus: Helicobacter
History & Taxonomy of Helicobacter
 Family not yet named (17 species by rRNA sequencing)
 First observed in 1983 as Campylobacter-like organisms
(formerly Campylobacter pyloridis) in the stomachs of
patients with type B gastritis
 Nomenclature of Helicobacter was first established in
1989, but only three species are currently considered to
be human pathogens
Important Human Pathogens:
 Helicobacter pylori (human; no animal reservoir)
 H. cinaedi (male homosexuals; rodents)
 H. fenneliae (male homosexuals; rodents)
 General Characteristics of Helicobacter
 Helicobacter pylori is major human pathogen
associated with gastric antral epithelium in patients with
active chronic gastritis
 Stomach of many animal species also colonized
 Urease (gastric strains only), mucinase, and
catalase positive highly motile microorganisms
 Other Helicobacters:
H. cinnaedi and H. fenneliae
• Colonize human intestinal tract
• Isolated from homosexual men with proctitis,
proctocolitis, enteritis, and bacteremia and
are often transmitted through sexual practices
Procto = anus + rectum
 Morphology & Physiology of
Helicobacter
 Gram-negative; Helical (spiral or curved)
(0.5-1.0 um X 2.5-5.0 um);
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. fennellae & H. cinaedi

Helicobacter Species Associated
with Human Disease
Epidemiology of Helicobacter Infections
 Family Clusters
 Orally transmitted person-to-person (?)

Worldwide:
 ~ 20% below the age of 40 years are infected
 50% above the age of 60 years are infected
 H. pylori is uncommon in young children
Epidemiology of Helicobacter Infections (cont.)

Developed Countries:
 United States: 30% of total population infected
• Of those, ~1% per year develop duodenal ulcer
• ~1/3 eventually have peptic ulcer disease(PUD)
 70% gastric ulcer cases colonized with H. pylori
 Low socioeconomic status predicts H. pylori infection
Developing Countries:
 Hyperendemic
 About 10% acquisition rate per year for children between 2
and 8 years of age
 Most adults infected but no disease
• Protective immunity from multiple childhood infections
Pathogenesis of Helicobacter Infections
 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
H. pylori
Virulence Factors of Helicobacter
Virulence Factors of Helicobacter
 Multiple polar, sheathed flagella
• Corkscrew motility enables penetration into viscous
environment (mucus)
 Adhesins: Hemagglutinins; Sialic acid binding
adhesin; Lewis blood group adhesin
 Mucinase: Degrades gastric mucus; Localized
tissue damage
 Urease converts urea (abundant in saliva and
gastric juices) into bicarbonate (to CO 2) and
ammonia
• Neutralize the local acid environment
• Localized tissue damage
 Acid-inhibitory protein
 Urea Hydrolysis

Urease
C=O(NH2)2 + H+ + 2H2O  HCO3- + 2 (NH4+)
(Urea) (Bicarbonate) (Ammonium)

And then…

HCO3-  CO2 + OH-

Virulence Factors of Helicobacter (cont.)

Tissue damage:
 Vacuolating cytotoxin: Epithelial cell damage
 Invasin(s)(??): Poorly defined (e.g., hemolysins;
phospholipases; alcohol dehydrogenase)

Protection from phagocytosis & intracellular killing:

 Superoxide dismutase
 Catalase
 Laboratory Identification

 Recovered from or detected in endoscopic

antral gastric biopsy material (antrum)
 Many different transport media
 Culture media containing whole or lysed blood
 Microaerophilic
 Grow well at 37oC, but not at 25 nor 42oC
 Like Campylobacter, does not use
carbohydrates, neither fermentatively nor oxidatively
 Treatment, Prevention & Control

Triple Chemotherapy (synergism):

 Proton pump inhibitor (ลดกรด)
(e.g., omeprazole = Prilosec(R))
 One or more antibiotics
(e.g., clarithromycin; amoxicillin; metronidazole)
 Bismuth compound (เคลือบแผล)

Inadequate treatment results in recurrence of symptoms