pVir and bloody diarrhea in Campylobacter jejuni enteritis

Emerging Infectious Diseases, June, 2005 by Dobryan M. Tracz, Monika Keelan, Jasmine Ahmed-Bentley, Amera Gibreel, Kinga Kowalewska-Grochowska, Diane E. Taylor

The plasmid pVir may play a role in the virulence of Campylobacter jejuni, a leading cause of bacterial gastroenteritis. The pVir plasmid was identified in 17% of 104 C. jejuni clinical isolates studied and was significantly associated with the occurrence of blood in patient stool, a marker of invasive infection. The pVir plasmid was not associated with greater occurrence of diarrhea, fever, pain, vomiting, or need for patient hospitalization. Isolates containing pVir were also associated with the presence of a tetracycline-resistance plasmid, but pVir did not transfer with tetracycline-resistance plasmids to recipient strains of C. jejuni. The association of pVir and bloody stool suggests that pVir may be clinically relevant in C. jejuni infections.

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Campylobacter jejuni is a major foodborne pathogen and a leading cause of bacterial gastroenteritis (1,2). Infection with C. jejuni can result in a wide array of clinical symptoms, including diarrhea, fever, abdominal pain, and vomiting, as well as bloody stool with severe invasive infection (3). Various virulence factors, which allow for adherence, colonization, and invasion of the intestinal epithelium, have been proposed to contribute to the pathogenesis of C. jejuni (4). Potential virulence components include flagella (5,6), invasion proteins (7), and toxins (8,9). Although the genome of C. jejuni has been sequenced (10), its mechanisms of pathogenicity remain poorly understood (11).

A number of bacterial enteric pathogens contain plasmids that contribute to pathogenesis, including Shigella sp. (12), Salmonella sp. (13), and enteropathogenic Escherichia coli (14). No evidence was seen for the involvement of plasmids in the virulence of C. jejuni until Bacon et al. (15) identified plasmid pVir in strain 81-176. pVir is an [approximately equal to] 37.5-kb plasmid that contains components of a type IV secretion system (T4SS) (15,16) known to be important for the virulence of a number of major bacterial pathogens (17). Bacon et al. (15) suggested that the pVir plasmid is important in vitro for both adherence and invasion of intestinal epithelial cells in culture. In a survey of fresh clinical isolates of C. jejuni from Thailand, 10% (n = 58) contained pVir (15).

C. jejuni gastroenteritis is primarily self-limiting and is usually treated by supportive therapy (fluid and electrolyte replacement) (3). Erythromycin is the drug of choice for treating severe clinical infections with C. jejuni (18), and fortunately the prevalence of erythromycin resistance has remained low (19). Worldwide, tetracycline resistance in C. jejuni is high: 56% in Canada (20) and up to 95% in Thailand (21). In Alberta, Canada, only 8.6% of human clinical isolates were tetracycline-resistant in 1981 (22). In C. jejuni, tetracycline resistance is primarily mediated by plasmids that carry the tet(O) gene (23,24). The Tet(O) protein binds to the bacterial ribosome and displaces tetracycline (25,26). The aim of this study was to investigate the prevalence of the pVir plasmid in C. jejuni isolated from clinical specimens in Alberta and the relationship of pVir to the clinical expression of the disease in gastroenteritis.

Materials and Methods

Source of C. jejuni Isolates

We obtained a random sample of 104 human isolates of C. jejuni (fresh and frozen at -70[degrees]C in double-strength skim milk) cultured from stool samples from 1999 to 2002 at the University of Alberta Hospital and Provincial Laboratory for Public Health (Microbiology) in Edmonton, Alberta, Canada, for this study. All stool specimens were routinely cultured for enteric pathogens tested according to standard laboratory protocols. Most C. jejuni isolates were from northern Alberta and represented [approximately equal to] 10% of C. jejuni infections in Alberta reported annually to Health Canada. The Health Research Ethics Board (Biomedical Panel) of the University of Alberta approved the protocol for access to human isolates and collection of patient information for this study.

Collection of Clinical Data

Clinical data were collected by letter or phone from family physicians' offices and emergency medical records and included information on age, sex, place of residence, travel within the last month, coexisting conditions, symptoms (diarrhea, bloody diarrhea, pain, fever, vomiting) and their duration (interval from symptom onset to initial evaluation), hospitalization (if needed), antimicrobial therapy, and complications. Fever was defined as a complaint of fever stated by the patient or a documented temperature of >38[degrees]C. Blood in the stool was observed by the patient and reported to the attending physician. The investigators conducting the chart review and laboratory analyses (pVir screening, antimicrobial susceptibility testing) were blinded to each other's data.

Culture of C. jejuni Isolates

Isolates of C. jejuni were spread on brain heart infusion agar (Difco-Becton-Dickinson, Sparks, MD, USA), supplemented with 0.4% yeast extract (Difco), and incubated at 37[degrees]C in microaerobic conditions (5% C[O.sub.2], 10% [H.sub.2], 85% [N.sub.2]) for 48 h. Isolates were not passaged more than once. All isolates were stored frozen in brain heart infusion broth (Difco) with 20% glycerol at -80[degrees]C.

Antimicrobial Susceptibility and Plasmid Isolation

C. jejuni susceptibility was tested by using the disk diffusion method; the following antimicrobial disks (Oxoid, Nepean, Ontario, Canada) were included: tetracycline (30 [micro]g), kanamycin (30 [micro]g), erythromycin (15 [micro]g), chloramphenicol (30 [micro]g), and nalidixic acid (30 [micro]g). All nalidixic acid-resistant isolates were further tested for resistance to ciprofloxacin (1 [micro]g). Susceptibility testing to all antimicrobial agents was carried out on Mueller-Hinton agar plates that were spread with a 0.5 McFarland standard suspension of C. jejuni in phosphate-buffered saline (Sigma, St. Louis, MO, USA) and incubated for 48 h at 37[degrees]C under microaerobic conditions. Zones of inhibition were measured as described by Gaudreau and Gilbert (27). Plasmid isolations were performed by using a Qiagen Midi or Mini Plasmid Kit (Qiagen, Mississauga, Ontario, Canada), by alkaline lysis (28) or by a method used for Helicobacter pylori (29). Tetracycline resistance detected by disk diffusion was confirmed by Etest (Oxoid) and conventional agar dilution.