The mechanism of multiple antibiotic resistance in six isolates of Salmonella enterica serovar Typhimurium recovered from a patient treated with ciprofloxacin was studied to investigate the role of efflux in the resistance phenotype. Compared to the patient's pretherapy isolate (L3), five of six isolates accumulated less ciprofloxacin, three of six isolates accumulated less chloramphenicol, and all six accumulated less tetracycline. The accumulation of one or more antibiotics was increased by carbonyl cyanide m-chlorophenylhydrazone to concentrations similar to those accumulated by L3 for all isolates except one, in which accumulation of all three agents remained approximately half that of L3. All isolates had the published wild-type sequences of marO and marR. No increased expression of marA, tolC, or soxS was observed by Northern blotting; however, three isolates showed increased expression of acrB, which was confirmed by quantitative competitive reverse transcription-PCR. However, there were no mutations within acrR or the promoter region of acrAB in any of the isolates.

The quinolone resistance-determining regions (QRDRs) of the gyrA genes of quinolone-resistant clinical and veterinary salmonella isolates were sequenced. Substitutions analogous to a substitution of a Ser to a Phe at position 83 (Ser83-->Phe) and Asp87-->Gly or Tyr in Escherichia coli were found, as was a single novel mutation outside of the QRDR resulting in Ala119-->Glu. The data suggest that gyrA mutations are associated with quinolone resistance in veterinary and clinical salmonella isolates and that the limits of the QRDR may require revision.

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 fluoroquinoloneresistant isolates, whether they were C. jejuni or C. coli, had a mutation in gyrA that resulted in the substitution Thr-863Ile. The other gyrA mutations detected were Thr-863Ala (n ‫؍‬ 17) and Asp-903Asn (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. Twentyfive 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-863Ile. Resistant isolates were isolated from the feces of some flocks up to the point of slaughter, which may have consequences for public health.

Two patients (patients A and B) infected with Salmonella typhimurium failed ciprofloxacin therapy, and the posttherapy isolates had reduced susceptibilities to quinolones; 6 of 11 isolates from patient B were also cross-resistant to chemically unrelated agents. No transferable resistance, chloramphenicol-acetylating enzymes, or 13-lactamases were detected. For 13 of 14 isolates, the concentrations of ciprofloxacin that inhibited DNA synthesis by 50%o were similar to the MICs, suggesting a mutation in gyrA. Insertion of pNJR3-2 (gyrA) in the posttherapy isolate from patient A and 5 of 11 of the posttherapy isolates from patient B resulted in lower quinolone MICs, also suggesting that resistance was due to a mutation in gyrA.

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 ␤-lactamase was typically associated with resistance to ampicillin, amoxicillin (amoxicilline), penicillin, and ticarcillin. Regardless of ␤-lactamase production, all isolates were resistant to piperacillin (MICs > 256 g/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 ␤-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 ␤-lactamase, CjBla2, with a pI of 9.2 and molecular mass of 32.4 kDa. Mass spectrometry confirmed that the most prevalent ␤-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 ␤-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.