BSCI 424 — PATHOGENIC MICROBIOLOGY — Fall 2000

Campylobacter Summary

History:

 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

Taxonomy:

 Campylobacter, Helicobacter, Wolinella, Arcobacter and Flexispira belong to a single phylogenetic group that is distinct from other Gram-negative bacteria and based on 16S rRNA sequencing, DNA hybridization, genus-specific probes, cell wall protein and lipid characterization, serological and biochemical analyses

 Campylobacter and Helicobacter (formerly Campylobacter) are the most clinically important members of the rRNA superfamily

 Recently defined family Campylobacteriaceae contains the genera Campylobacter and Arcobacter

 Helicobacter, Wolinella, and Flexispira now included in a phylogenetically distinct family, as yet unnamed

 Campylobacter (Family Campylobacteriaceae): 15 species, 6 subspecies by rRNA sequencing; 12 of 15 species associated with human disease

 Campylobacter jejuni (poultry; humans)

  C. jejuni subsp. jejuni (major human enteric pathogen worldwide)

  C. jejuni subsp. doylei (gastroenteritis, gastritis, septicemia)

  C. jejuni subsp. fetus

 Campylobacter coli (porcine; humans)

 Campylobacter fetus (cattle; sheep; occasionally humans)

  C. fetus subsp. fetus (major veterinary pathogen)

 C. fetus subsp. venerealis (Infrequent septicemia)

 Many Minor Pathogens:

  Campylobacter lari (avian; humans: gastroenteritis, diarrhea, septicemia): can be confused with C. jejuni on primary isolation but rarely associated with human enteritis)

  Campylobacter concisus (Oral, gingival crevices in humans)

  Campylobacter hyointestinalis (watery or bloody diarrhea, vomiting)

  Campylobacter upsaliensis (acute watery diarrhea, septicemia, abscesses)

Morphology & Physiology:

General Characteristics Common to Superfamily:

  Pleomorphic helical (spiral or curved) Gram-negative microaerophilic (see WebLinked Campylobacter image)

  Characteristics that facilitate penetration and colonization of mucosal environments (e.g., motile by polar flagella; corkscrew shape)

  Become coccoid when exposed to oxygen or upon prolonged culture

  Neither ferment nor oxidize carbohydrates; low DNA G+C base ratio

Campylobacters:

  Cellular Structure:

 Microscopically, helically-shaped cells have "gull-winged" appearance

 Characteristic rapid darting motility by means of long sheathed polar flagellum at one (polar) or both (bipolar) ends of the cell that may be up to several times the length of the cell (see WebLinked image); motility slows quickly in wet mount microscopy preparation due to entangling of flagella and aerotoxicity (oxygen sensitivity)

Cells tend to form coccoid and elongated forms on prolonged culture or upon exposure to oxygen; may enter a nonculturable form in nature

 Immunodominant antigens include:

  Lipopolysaccharide (LPS)

  Flagellum

  Porin: peptidoglycan-associated major outer membrane protein (MOMP):

  Adhesins/binding proteins (PEBs)

 C. fetus covered with capsule-like surface protein arrayed in crystalline lattices (S-protein or S-layer)

  Culture: various commercial media available

Microaerophilic and capnophilic; Optimal growth with reduced oxygen (5-7%) and increased CO₂ (10%)

  Thermophilic species (C. jejuni, C. coli) grow better at 42-43^oC (natural body temperature of avian species) than 37^oC and this can be used for selective isolation from stool specimens

  Metabolism:

 Produce oxidase and catalase; despite these enzymes, still unusually sensitive to superoxides and free radicals, as well as, physical and chemical agents

 Do not utilize carbohydrates

Clinical Syndromes:

  Gastroenteritis (campylobacteriosis):

 Primarily caused by thermophilic enteropathogenic C. jejuni and C. coli

 Inflammatory bacterial diarrhea; Damage to mucosal surfaces of jejunum; ileum; colon

 Most commonly: acute enteritis with diarrhea, malaise, fever, and abdominal pain; nausea but not vomiting

 Incubation period of 1-7 days with acute onset with abdominal cramps and diarrhea

 About 1/3 of gastroenteritis patients suffer prodromal fever, headache, dizziness, myalgia, and other non-specific flu-like complaints 12-24 h before intestinal symptoms

 Symptoms range from few loose stools to profuse prostrating water diarrhea with ten or more bowel movements per day during peak in affected patients, stools may contain mucus or gross blood

 Generally self-limiting, symptoms may last for a week or longer

 Range of clinical manifestations can include: colitis; acute abdominal pain; bacteremia (<1% of patients)

 Important late onset complications following gastroenteritis include: Guillain-Barre Syndrome; Reiter’s syndrome; reactive arthritis; chronic infection in immunodeficient patients

  Guillain-Barre syndrome (GBS): Low incidence potential sequela

 Reactive, self-limited, autoimmune disease

 Campylobacter jejuni most frequent antecedent pathogen; Infection induces humoral and cellular immune responses

 Immune response to specific heat stable O-antigens (O-side chain of LPS) cross-react 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

  Septicemia (occasionally leading to meningitis); spontaneous abortion: C. jejuni and C. coli, but primarily caused by C. fetus

  Infrequently: Proctitis; septic arthritis; enterocolitis; acute appedicitis or "pseudoappendicitis"; cholecystitis; hepatitis; pancreatitis; peritonitis; cystitis; prostatitis; bacteremia

  C. fetus subsp. fetus most commonly associated with systemic infections

 Presents initially as gastroenteritis with propensity to spread from gastrointestinal tract to bloodstream and systemically to distal foci, particularly in debilitated and immunocompromised patients, e.g., those with liver disease, diabetes mellitus, chronic alcoholism, or malignancies

 Bacteremia; Septic thrombophlebitis; Arthritis; Septic abortion; Meningitis

  Other Species: Uncommonly associated with gastroenteritis or systemic infections

Epidemiology:

  Campylobacteriosis is most common form of acute infectious diarrhea in developed countries; higher incidence than both the gastroenteric pathogens Salmonella and Shigella combined

 In the U.S., more than 2 million cases may occur annually, an annual incidence close to the 1.1% observed in the United Kingdom; estimated 200-700 deaths

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

 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

  Zoonotic infections in many animals particularly avian reservoirs (bird) species and shedding into the environment; 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

 Contaminated poultry accounts for more than half of the camylobacteriosis cases in developed countries but different epidemiological picture in developing countries

  Sporadic infections in humans far outnumber those affected in point-source epidemics; sporadic cases peak in the summer in temperate climates with a secondary peak in the late fall seen in the U.S.

  Infectious dose (some people infected with as few as 500 organisms while others need >10⁶) and host immunity appear to be major factors in determining whether gastroenteric disease develops

  Hypochlorhydric or achlorhydric patients are at higher risk (lower infectious dose)

Pathogenesis & Immunity:

  Pathogenesis of campylobacter gastroenteritis not fully characterized

  Non-motile strains and those lacking adhesin are avirulent

  Damage (edematous and bloody) to the mucosal surfaces of the jejunum, ileum, and colon

 Histologically, mucosa is ulcerated, with crypt abscesses in the epithelial glands and infiltration of the lamina propria with neutrophils, mononuclear cells, and eosinophils

 Inflammatory process is consistent with invasion of the organisms into the intestinal tissue

 M-cell (Peyer’s patches) uptake and presentation of antigen to underlying lymphatic system

  

 Cellular components:

  Endotoxin

  Flagellum

  Adhesins (e.g., PEBs): mediate attachment to mucosa

  Invasins

  GBS is highly associated with C. jejuni serogroup O19 (LPS)

  S-layer protein "microcapsule" in C. fetus:

Antiphagocytic

Prevents complement-mediated killing in serum (inhibition of C3b binding to bacteria protects against bactericidal effect)

Removal of S-layer renders C. fetus avirulent in animal model

 Extracellular components:

  Enterotoxins

  Cytopathic toxins (cytotoxin)

  Immune Response: Inflammatory response consistent with invasive disease

 Following exposure, specific serum and secretory antibodies develop that provide strain-specific immunity and protection from disease caused by the homologous strain, although recolonization may occur

 High level of endemic disease results in the development of specific serum and secretory antibodies and less severe disease

 Hypogammaglobulinemia results in prolonged, severe disease by C. jejuni

 For Campylobacter fetus:

  In vitro studies have shown that C. fetus is resistant to complement- and antibody-mediated serum killing (due to S-layer), but C. jejuni is killed rapidly

 S-layer (S-protein) on surface of C. fetus inhibits C3b binding and subsequent complement-mediated phagocytosis and killing 

Laboratory Identification:

  Specimen Collection and Processing:

 Feces in plain container, refrigerated, examined within few hours

 Rectal swabs in semisolid transport medium (e.g., Cary-Blair transport medium; Wang’s medium)

 Care to avoid oxygen exposure

  Microscopy

 Gull-wing appearane in gram stain

 Darting motility in wet mount preparation under phase-contrast or dark field illumination; direct detection of darting organisms is possible in fresh stool (rarely done in clinical lab)

 Fecal leukocytes are commonly present and can be readily visualized with methylene blue staining of wet or dried fecal smears

  Culture

 Diarrheic stools should be routinely cultured for Campylobacter in most clinical laboratories

 Selective media: (e.g., Butzler's agar; Skirrow's agar; Campy-BAP)

  Slow-growing pinpoint colonies

  Isolation requires growth in microaerophilic atmosphere (5-7% oxygen, 5-10% carbon dioxide, balance nitrogen); For C.jejuni supsp. jejuni (5% O₂, 10% CO₂, 85% N₂)

  For thermophilic species (C. jejuni; C. coli) incubation at 42-43.5C for 24-72

  For nonthermophilic species (C. fetus; C. hyointestinalis) 37oC is optimal

 Enrichment broth:

  Campy-thioglycollate enrichment (refrigerated from 8 h to overnight then sub-cultured to selective agar)

  Useful for environmental and food samples where organisms may be stressed or in low numbers

 Selective isolation of Campylobacter by filtration of stool specimen:

  Small size of campylobacters compared to other enteric bacteria can be utilized to selectively isolate; Campylobacter cells can pass through 0.45 μm, 0.65 μm or 0.8 μm pore-size filter membranes, while most other enteric microorganisms are retained

  Filtrate can then be grown out on nonselective agar

  Filtration method has a lower sensitivity than direct culture onto selective media

 For C. fetus from blood or other body fluids: nonselective media

  Characterization on the basis of biotyping, serotyping, phage typing, molecular typing can be used but are not routinely available:

 Hippurate hydrolysis (C. jejuni is positive)

 Indoxyl acetate hydrolysis (C. fetus is negative

 Growth temperature (C. fetus not thermophilic; will grow at 25C)

 Susceptibility to:

  Nalidixic acid (C. jejuni; C. coli sensitive): naladixic acid is a quinolone (see below about resistance to the floroquinolones)

  Cephalothin (C. fetus sensitive)

PCR and DNA probes are on the horizon

  

 Lior serotyping scheme: Heat-labile flagellar and capsular antigens; Slide agglutination assay

 Penner serotyping scheme: Heat-stable O-polysaccharide antigen; Indirect hemagglutination assay

  

 Correlates of protection ("markers" that predict protection of host from disease) have not been established

 Latex particle agglutination kits are available commercially but expensive

 Serodiagnosis may be important in the immunological sequelae reactive arthritis or Guillain-Barre syndrome

Treatment, Prevention & Control:

  Gastroenteritis:

  Generally self-limiting; Replacement of fluids and electrolytes as needed

  Antibiotic treatment may not shorten duration of disease symptoms but can shorten the excretion period of the organisms

  Proper food handling, preparation and storage

  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 patients; Require intubation; assisted ventilation

  Infusions of IgG or plasma exchange may shorten the course of disease (Immunomodulation)

  Antimicrobial Susceptibility

  Erythromycin continues to be the drug of choice for severe or complicated enteritis (500mg b.i.d. for 5 days); Continue for 4 weeks with bacteremia; Azithromycin was shown to be effective in recent human clinical trials

  Consistently sensitive to macrolides, aminoglycosides, and nitrofurans (furazolidone activity limited to gut); Tetracycline and chloramphenicol are also active; Inherently resistant to trimethoprim and most cephalosporins

  Fluroquinolones were 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; In the Netherlands, the rise in resistance among poultry strains has been closely paralleled by the rise in resistance among human clinical strains