Amoxicillin therapy of poultry flocks: effect upon the selection of amoxicillin-resistant commensal Campylobacter spp.

BACKGROUND: The aim of this study was to investigate the effect of amoxicillin therapy of poultry flocks upon the persistence of commensal Campylobacter spp. and the incidence of antibiotic resistance. METHODS: Four poultry flocks naturally colonized with Campylobacter were treated with amoxicillin and monitored before, during and up to 4 weeks post-treatment. The numbers of Campylobacter were determined and the isolates speciated and typed by flaA short variable region (SVR) sequence analysis and PFGE. The susceptibility of the isolates to antibiotics, presence of the Cj0299 gene encoding a beta-lactamase and beta-lactamase production (nitrocefin hydrolysis) were also determined. RESULTS: Amoxicillin-resistant Campylobacter were isolated from Flock 1 before and during treatment, but Campylobacter were not detected afterwards. Flock 2 was colonized by amoxicillin-susceptible strains throughout sampling. No amoxicillin-resistant isolates arose during or after treatment. Flock 3 contained amoxicillin-susceptible and -resistant types pre-treatment. Resistant isolates were detected during treatment, while antibiotic-susceptible isolates re-emerged at 3 weeks post-treatment. All Campylobacter isolates from Flock 4 were amoxicillin resistant, irrespective of sampling time. All but one of the 82 amoxicillin-resistant (MICs 16 to >128 mg/L) Campylobacter jejuni and Campylobacter coli tested for the presence of Cj0299 carried the gene and all of these produced beta-lactamase. Co-amoxiclav remained active against amoxicillin-resistant isolates. CONCLUSIONS: Amoxicillin therapy had little effect on the numbers of amoxicillin-resistant commensal Campylobacter except for one flock where amoxicillin-resistant Campylobacter temporarily dominated. Amoxicillin therapy did not select amoxicillin-resistant isolates from a previous susceptible strain. Co-amoxiclav remained active against amoxicillin-resistant isolates.

Beta-lactamase-mediated beta-lactam resistance in Campylobacter species: prevalence of Cj0299 (bla OXA-61) and evidence for a novel beta-Lactamase in C. jejuni.

Fifty-two percent of 1,288 poultry isolates of campylobacters were ampicillin resistant, and resistance was more common among Campylobacter coli isolates (67.4%) than among Campylobacter jejuni isolates (47.5%). Production of beta-lactamase was typically associated with resistance to ampicillin, amoxicillin (amoxicilline), penicillin, and ticarcillin. Regardless of beta-lactamase production, all isolates were resistant to piperacillin (MICs >or= 256 microg/ml), and most were resistant to carbenicillin, cloxacillin, and cephalosporins. Of all ampicillin-resistant campylobacters tested, 91% (347/380) carried the bla(OXA-61) gene, and 77% (136/175) of those tested with nitrocefin produced a beta-lactamase, presumably OXA-61. The isoelectric point (pI) of OXA-61 was 8.7, and the molecular mass was 31.0 kDa. Insertional inactivation of bla(OXA-61) in C. jejuni NCTC 11168 and two ampicillin-resistant isolates resulted in increased susceptibility to ampicillin, co-amoxiclav (amoxicillin and clavulanic acid), penicillin, carbenicillin, oxacillin, and piperacillin, but the effects on MICs of cephalosporins and imipenem were negligible. Some C. jejuni isolates that lacked bla(OXA-61) produced a beta-lactamase, CjBla2, with a pI of 9.2 and molecular mass of 32.4 kDa. Mass spectrometry confirmed that the most prevalent beta-lactamase was the product of bla(OXA-61), but CjBla2 was not identified. OXA-61 is prevalent among Campylobacter spp. of veterinary origin and is similar to the beta-lactamase previously reported in human isolates. Production of OXA-61 was associated with resistance to penams but not cephalosporins. Co-amoxiclav remained active against all isolates tested.

Prevalence and subtypes of ciprofloxacin-resistant Campylobacter spp. in commercial poultry flocks before, during, and after treatment with fluoroquinolones.

Five commercial broiler chicken flocks were treated with either difloxacin or enrofloxacin for a clinically relevant infection, as instructed by a veterinarian. Campylobacters were isolated from individual fecal samples and from samples associated with the broiler environment before, during, and after treatment. Ciprofloxacin-resistant Campylobacter jejuni and/or C. coli strains were detected pretreatment in four flocks, but they constituted a very small proportion of the campylobacters present. When the broilers were treated with a fluoroquinolone, a rapid increase in the proportion of ciprofloxacin-resistant campylobacters was observed. During treatment nearly 100% of campylobacters were resistant, and in some flocks a high proportion of resistant strains persisted for up to 4 weeks after treatment. Prior to treatment a variety of campylobacter subtypes were present. During and after treatment considerable changes in both species and subtype prevalence were observed, but no single fluoroquinolone-resistant clone became dominant. Instead, resistant C. coli strains or a mixture of resistant C. coli and C. jejuni strains became dominant, whereas susceptible C. jejuni strains had usually been dominant prior to treatment. The resistant subtypes which emerged and became dominant were not always the same as those detected pretreatment. The persistence of resistant strains for up to 4 weeks posttreatment has important implications for any strategy designed to avoid the introduction of such strains into the food chain.

Incidence and mechanism of ciprofloxacin resistance in Campylobacter spp. isolated from commercial poultry flocks in the United Kingdom before, during, and after fluoroquinolone treatment.

Five commercial broiler flocks were treated with a fluoroquinolone for a clinically relevant infection. Fresh feces from individual chickens and environmental samples were cultured for campylobacters before, during, and weekly posttreatment until slaughter. Both Campylobacter jejuni and C. coli were isolated during all treatment phases. An increased proportion of quinolone-resistant strains was seen during treatment, and these strains persisted posttreatment. One quinolone-resistant isolate of each species, each serotype, and each phage type from each sample at all treatment phases was examined for its phenotype and mechanism of resistance. Two resistant phenotypes were isolated: Nal(r) Cip(r) and Nal(r) Cip(s). The majority (269 of 290) of fluoroquinolone-resistant isolates, whether they were C. jejuni or C. coli, had a mutation in gyrA that resulted in the substitution Thr-86-->Ile. The other gyrA mutations detected were Thr-86-->Ala (n = 17) and Asp-90-->Asn (n = 10). The genotypic variation, based on the silent mutations in gyrA identified by the denaturing high-performance liquid chromatography pattern and DNA sequencing, was used to supplement typing data and provided evidence for both the spread of preexisting resistant strains and the selection of spontaneous resistant mutants in treated flocks. Multidrug resistance was significantly (P < 0.01) associated with resistance to ciprofloxacin. Twenty-five percent (73 of 290) of ciprofloxacin-resistant isolates but only 13% (24 of 179) of susceptible isolates were resistant to three or more unrelated antimicrobial agents. In conclusion, quinolone-resistant campylobacters were isolated from commercial chicken flocks in high numbers following therapy with a veterinary fluoroquinolone. Most ciprofloxacin-resistant isolates had the GyrA substitution Thr-86-->Ile. Resistant isolates were isolated from the feces of some flocks up to the point of slaughter, which may have consequences for public health.

Selection of moxifloxacin-resistant Staphylococcus aureus compared with five other fluoroquinolones.

Fluoroquinolone-resistant mutants were selected from Staphylococcus aureus NCTC 8532 (F77), and two GrlA mutants of F77 (F193 and F194) with moxifloxacin, sparfloxacin, ofloxacin, grepafloxacin, levofloxacin and trovafloxacin. For mutants selected from F77, moxifloxacin, grepafloxacin and sparfloxacin selected preferentially for mutations in gyrA (Glu-88-->Lys). Ofloxacin and trovafloxacin selected most commonly for mutations in grlA, conferring substitutions for Ser-80. Three mutants of F77 were shown to have substitutions in both GrlA (Phe-80) and GyrA (Lys-88). Of the mutants selected from F193 (GrlA Phe-80), restriction fragment length polymorphism analysis of gyrA showed that 76/94 had a mutation at codon 84; those analysed in detail all had the substitution Ser-->Leu. Two mutants selected with grepafloxacin contained the substitution Lys-88. One mutant selected with trovafloxacin contained a novel mutation in gyrA substituting Gly-82-->Cys. Of the mutants selected from F194 (GrlA Tyr-80), 6/8 had a mutation in gyrA codon 84; of which three contained Leu. The MICs of most agents for mutants selected from F193 and F194 were similar, irrespective of the mutation selected. No mutants had any changes in grlB, and only one had a mutation in gyrB giving rise to the novel substitution Asp-437-->His. The mutations arising in first-step mutants were influenced by the fluoroquinolone used for selection. The phenotypes and genotypes of second-step mutants, derived from mutants with existing mutations in grlA, were similar, regardless of the selecting antibiotic.

Fluoroquinolone resistance in Campylobacter species from man and animals: detection of mutations in topoisomerase genes.

Consecutive isolates of quinolone-resistant campylobacter isolated over a 5 year period (1990-1995) from the faeces of patients with enteritis in Plymouth, UK, were examined for the epidemiology of mutations in gyrA (n = 127). In addition, clinical isolates and poultry isolates from Germany, The Netherlands and other regions of the UK collected before 1995 were examined for mutations in the quinolone resistance-determining region of gyrA by single-stranded conformational polymorphism analysis and direct sequencing of a 270 bp fragment of PCR-generated DNA. The majority of isolates (173/208) carried a mutation at codon 86 in gyrA resulting in substitution of Ile for Thr; all of these were resistant to ciprofloxacin (MIC > or = 2 mg/L). One isolate of Campylobacter jejuni had a mutation at Asp-90, and another had a double mutation at Thr-86 and Pro-104. Only two resistant isolates showed no mutation in gyrA. A novel gyrA sequence was amplified from two Campylobacter lari and one C. jejuni, which exhibited a valine at codon 86. Only 8/192 isolates had changes in gyrB; all were shown to relate to silent mutations in gyrB and presumably reflect natural polymorphisms in the gene.