Quinolone-resistant Campylobacter infections in Denmark: risk factors and clinical consequences

by Jorgen Engberg, Jakob Neimann, Eva Moller Nielsen, Frank Moller Aarestrup, Vivian Fussing

We integrated data on quinolone and macrolide susceptibility patterns with epidemiologic and typing data from Campylobacter jejuni and C. coli infections in two Danish counties. The mean duration of illness was longer for 86 patients with quinolone-resistant C. jejuni infections (median 13.2 days) than for 381 patients with quinolone-sensitive C. jejuni infections (median 10.3 days, p = 0.001). Foreign travel, eating fresh poultry other than chicken and turkey, and swimming were associated with increased risk for quinolone-resistant C. jejuni infection. Eating fresh chicken (of presumably Danish origin) was associated with a decreased risk. Typing data showed an association between strains from retail food products and broiler chickens and quinolone-sensitive domestically acquired C. jejuni infections. An association between treatment with a fluoroquinolone before stool-specimen collection and having a quinolone-resistant C. jejuni infection was not observed.

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Campylobacter is a leading cause of bacterial gastroenteritis in industrialized and developing countries worldwide (l). Most Campylobacter infections need not be treated with antimicrobial agents. However, in a subset of patients Campylobacter may cause severe complications and increased risk for death and therefore requires treatment. A recent Danish study has shown that patients with Campylobacter infections have higher acute- and long-term death rates than controls after coexisting conditions were taken into account (2). The drag of choice is a macrolide (e.g., erythromycin or a newer agent) for treatment of enteric Campylobacter infections after the microbiologic diagnosis. However, for the empiric treatment of adults with suspected bacterial gastroenteritis, the drug of choice typically includes a fluoroquinolone (e.g., ciprofloxacin) because of their activity against almost all enteric bacterial pathogens. Antimicrobial drug resistance in Campylobacter infections, in particular to quinolones, has increased dramatically in many countries during the 1990s as reviewed by Engberg et al. (3). According to a recent published report by World Health Organization (4), the sources of antimicrobial drug-resistant Campylobacter strains and the clinical impact of such strains need to be determined.

We conducted a 1-year prospective study to address the prevalence of macrolide and quinolone resistance in human Campylobacter isolates. Human isolates were compared with isolates from retail food products and broiler chickens. A systematic approach integrating standardized epidemiologic, antimicrobial susceptibility, and typing data was used. We also conducted a case-comparison study to identify risk factors associated with acquiring quinolone-resistant C. jejuni infections.

Materials and Methods

Surveillance and Susceptibility Testing of Campylobacter Isolates

The study included all culture-positive Campylobacter infections from May 1, 2001, through June 10, 2002, from two counties with a catchment area of approximately 1.1 million persons (approximately one fifth of the Danish population). The county of Copenhagen, a metropolitan residential area, has a population of 619,000, and the county of Funen, an island with both urban and rural areas, has a population of 472,000. Because of inconsistencies in the patient enrolment from the county of Funen during May and June 2002, patients from this county who were infected after April 31,2002, were excluded. Epidemiologic data were captured on self-completed standardized patient questionnaires forwarded by the Danish Zoonosis Centre. Patients were interviewed about clinical symptoms, travel history, and exposures to food, water, and animals in the 7 days before illness onset. Completed questionnaires were returned to the Danish Zoonosis Centre and linked with microbiologic data.

All isolates included in the study were tested for resistance to nalidixic acid and erythromycin. All human isolates from the county of Copenhagen and isolates obtained from retail food products and broiler chickens were screened by a disk-diffusion test using Oxoid disks on 5% blood agar plates. On the basis of zone sizes, this method grouped the isolates in two well-separated populations of susceptible and resistant isolates with both antimicrobial drugs. The few isolates that fell between these populations were retested by using the standardized tablet diffusion and E-test procedures described previously (5), with the modifications that resistance to nalidixic acid was defined as an MIC >64 mg/L for the MIC method and a zone size [less than or equal to] 27 mm for the tablet method. All human isolates from the county of Funen were tested by the standardized tablet diffusion test with both antimicrobial drugs. Finally, all isolates found to be resistant and sensitive to nalidixic acid from out case-comparison study were retested by both the standardized tablet diffusion and E-test procedure.

Case-Comparison Study

In the second half of the study period (from December 1, 2001, to June 10, 2002), characteristics of patients with quinoione-resistant and quinolone-sensitive C. jejuni infections were compared. Each patient with a resistant isolate was matched with two randomly selected patients with sensitive isolates. Patients were matched on date of specimen collection.

Patients answered, either by phone or by mail, a short additional questionnaire, which included questions about use of fluoroquinolones the month before onset of illness, use of fluoroquinolones after onset of illness but before specimen collection, use of antimicrobial drugs after specimen collection, and other clinical information. When patients could not answer questions about exposure to fluoroquinolones before fecal sampling, the information was gathered from their healthcare providers.

Food and Animal Isolates

As part of a national surveillance program, food samples from retail outlet stores were analyzed for Campylobacter at the regional food safety authorities, according to accredited methods of the Nordic Committee on Food Analysis (6). The samples were taken from whole poultry and different cuts of poultry (frozen and fresh), including chicken and turkey. Samples of pork and beef products were also analyzed. Imported as well as domestic food products were sampled.

As part of a national surveillance program for Campylobacter in broiler chickens, chickens were sampled at slaughter and analyzed for Campylobacter. In this study, isolates from broiler chicken farms located in Funen County were included (one isolate per flock). Copenhagen County does not have any broiler chicken farms.

Serotyping and Molecular Subtyping of Campylobacter Isolates

One isolate from the primary isolation on modified charcoal cefoperazone deoxycholate agar (mCCDA) from each patient, as well as one isolate from each retail food sample and broiler chicken fecal sample were characterized at Statens Serum Institut and the Danish Veterinary Institute. Speciation, serotyping, and RiboPrinting (automated ribotyping) were undertaken as previously described (7,8), with the following modifications for the RiboPrinting method: 1-[micro]L eye needle was filled with bacterial culture and dissolved in 100 [micro]L sample buffer. Ten microliters of 10 g/L lysozyme was added, and the solution was left at 37[degrees]C for 10 min. From this solution, 30 [micro]L was transferred to a sample carrier for heat treatment. The RiboPrinter was run according to the SEC protocol at 37[degrees]C for 2 h.

Statistical Analysis

Conditional logistic regression was applied to calculate a matched odds ratio for the exposure variables. Variables, which reached a significance level of [less than or equal to] 0.15 in the univariate analysis of the case comparison study, were selected for the multiple logistic regression analysis. Stepwise conditional logistic regression with a backward elimination procedure was conducted to obtain a reduced model. Variables with a p value [less than or equal to] 0.05 were kept in the model. All excluded variables were retested in the final model. The statistical software SAS Release v.8.00 (SAS Institute Inc., Cary, NC) and Epi Info version 6.04d (Centers for Disease Control and Prevention, Atlanta, GA) were used to analyze the data.

Results

Surveillance and Resistance

Of 975 culture-confirmed Campylobacter infections in the study, 177 (18.2%) were infected with a quinolone-resistant isolate, whereas 3 (0.3%) isolates were erythromycin-resistant. Linked microbiologic and epidemiologic data were obtained from 678 (69.5%) patients. In total, 152 (22.4%) patients had been outside Denmark within 1 week before illness, whereas 526 (77.6%) were domestically acquired infections. The three erythromycin-resistant isolates were all C. coli, two of them were also quinolone-resistant, and these were both isolated from travelers returning to Denmark from Spain and Portugal, respectively.