Campylobacter coli isolates from Finnish farrowing farms using aminopenicillins: high prevalence of bla(OXA-61) and ß-lactamase production, but low MIC values.

Antimicrobial treatment of animals may select resistance in Campylobacter to antimicrobial agents belonging to several classes of compounds. We investigated the effect of widely used aminopenicillin therapy on the minimum inhibitory concentration (MIC) levels in porcine Campylobacter coli isolates and investigated the presence of a ß-lactamase gene and ß-lactamase production. Epidemiological cut-off values (ECOFFs) were applied to detect decreased susceptibility. Fifty-three isolates were obtained from aminopenicillin-treated (ampicillin or amoxicillin) sows and piglets during and up to 3 weeks post-treatment. All isolates had ampicillin MICs below the ECOFF (≤ 8 µg/mL). An additional 63 isolates were sampled before treatment or from other untreated sows and piglets. Of these isolates, four had ampicillin MICs above the ECOFF. All ciprofloxacin MICs were below the ECOFF (≤ 1 µg/mL), except for three isolates from untreated sows and four isolates after aminopenicillin therapy. One isolate originating from an untreated sow had an erythromycin MIC above the ECOFF (> 16 µg/mL). None of the isolates had MICs above the ECOFFs for two or three studied antimicrobials simultaneously. Of the 116 C. coli isolates, 90 (77.6%) isolates carried the bla(OXA-61) ß-lactamase gene, and 63 (70.0%) of those produced ß-lactamase. The isolates producing ß-lactamase had higher ampicillin MICs than those without the bla(OXA-61) gene and production of ß-lactamase. Proportion of the bla(OXA-61)-positive C. coli isolates was similar among untreated animals or during and after the treatment. In conclusion, C. coli isolates did not acquire high ampicillin MICs even though aminopenicillins were administered at therapeutic levels for several days. The bla(OXA-61) gene and production of ß-lactamase increased ampicillin MICs in C. coli, but the values remained mainly under the ECOFF. We also demonstrated that aminopenicillin therapy did not select simultaneously resistance to the major antimicrobials used in human therapy against campylobacteriosis (i.e., erythromycin and ciprofloxacin).

Establishing streptomycin epidemiological cut-off values for Salmonella and Escherichia coli.

This study was conducted to elucidate the accuracy of the current streptomycin epidemiological cut-off value (ECOFF) for Escherichia coli and Salmonella spp. A total of 236 Salmonella enterica and 208 E. coli isolates exhibiting MICs between 4 and 32 mg/L were selected from 12 countries. Isolates were investigated by polymerase chain reaction for aadA, strA, and strB streptomycin resistance genes. Out of 236 Salmonella isolates, 32 (13.5%) yielded amplicons for aadA (n = 23), strA (n = 9), and strB (n = 11). None of the 60 Salmonella isolates exhibiting MIC 4 mg/L harbored resistance genes. Of the Salmonella isolates exhibiting MICs 8 mg/L, 16 mg/L, and 32 mg/L, 1.6%, 15%, and 39%, respectively, tested positive for one or more genes. For most monitoring programs, the streptomycin ECOFF for Salmonella is wild type (WT) ≤32 or ≤16 mg/L. A cut-off value of WT ≤32 mg/L would have misclassified 13.5% of the strains as belonging to the WT population, since this proportion of strains harbored resistance genes and exhibited MICs ≤32 mg/L. Out of 208 E. coli strains, 80 (38.5%) tested positive for aadA (n = 69), strA (n = 18), and strB (n = 31). Of the E. coli isolates exhibiting MICs of 4 mg/L, 8 mg/L, 16 mg/L, and 32 mg/L, 3.6%, 17.6%, 53%, and 82.3%, respectively, harbored any of the three genes. Based on the European Committee on Antimicrobial Susceptibility Testing guidelines (ECOFF ≤16 mg/L), 25% of the E. coli strains presenting MIC ≤16 mg/L would have been incorrectly categorized as belonging to the WT population. The authors recommend an ECOFF value of WT ≤16 mg/L for Salmonella and WT ≤8 mg/L for E. coli.

Mutations in the rpsL gene are involved in streptomycin resistance in Campylobacter coli.

To characterize the mechanisms of streptomycin (STR) resistance in Campylobacter coli, we chose 17 isolates that were resistant to STR, erythromycin (ERY), or both, and the putative STR resistance target genes rpsL, rrs, and gidB were analyzed for mutations. The presence of the aadE gene encoding aminoglycoside 6-adenylyltransferase was also evaluated. To reveal putative connection between ERY and STR resistance mechanisms, 13 C. coli isolates initially susceptible to STR and ERY were exposed to STR, and resistant variants were characterized. We also assessed the development of ERY resistance with a similar method. Finally, the effect of the putative CmeABC efflux pump inhibitor phenyl-arginine-beta-naphthylamine on STR resistance was tested. Our studies showed an association between mutations in the rpsL gene and STR resistance in C. coli. Further, mutations obtained in vitro were more diverse than those occurring in vivo. However, we observed no resistance associated mutations in the other genes studied, and selection with STR did not result in variants resistant to ERY and vice versa. None of the isolates harbored the aadE gene, and no differences in STR minimum inhibitory concentration levels were detected in the presence or absence of phenyl-arginine-beta-naphthylamine. In conclusion, we found that STR resistance was associated with mutations in the rpsL gene, but no obvious association between STR and ERY resistance mechanisms was found in C. coli.

The temporal, PFGE and resistance pattern associations suggest that poultry products are only a minor source of human infections in western Finland.

In order to compare human and retail poultry meat thermophilic Campylobacter isolates originating in a regional area in Western Finland, minimum inhibitory concentration (MICs) for six antimicrobials (96 isolates) and pulsed-field gel electrophoresis (PFGE) (102 isolates) were analysed. Campylobacter spp. were detected in 10.5% out of 305 fresh poultry products studied; 29 (90.5%) isolates were identified as Campylobacter jejuni. Among the 70 human isolates, 66 (94.3%) isolates were identified as C. jejuni. Only one C. jejuni domestic poultry isolate showed resistance (ampicillin), whereas domestic human C. jejuni isolates were more commonly resistant to ciprofloxacin, nalidixic acid, ampicillin and tetracycline. The resistance in foreign human isolates was significantly more common than among domestic isolates. PFGE analysis with KpnI restriction enzyme resulted in 59 different PFGE types among the poultry and human isolates. Three types were detected first in poultry meat and thereafter during the following month in domestic human samples, whereas the other conjoint types were detected only after many months. This study suggests that poultry products play only a minor role in human campylobacteriosis in the study area and that the resistance found in domestic human isolates is not likely related to retail poultry meat products.

Prevalence of Campylobacter spp. in cattle in Finland and antimicrobial susceptibilities of bovine Campylobacter jejuni strains.

The study investigated the prevalence of Campylobacter spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (n=952) and carcass surface samples (n=948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples. Campylobacter jejuni was isolated from 19.5%, Campylobacter coli from 2.2%, and presumptive Campylobacter hyointestinalis from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (n=730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal C. jejuni isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of C. jejuni by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass C. jejuni isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187 C. jejuni isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.