Sir: Extensive use of antimicrobials in human and animal treatment, in the latter as growth promoters, has resulted in a considerable increase in resistance to quinolones among Salmonella1. The aim of this study was to identify the mechanisms of resistance to quinolones in Salmonella enterica serovar Infantis clinical isolates in the city of Corrientes (Argentina). For this purpose, six isolates of Salmonella infantis, in all probability linked to a foodborne community outbreak, were included in this study. All isolates were recovered from stool specimens over a two-day period and were cultured, identified, and serotyped by standard methods2. Antimicrobial susceptibility was tested with disk diffusion assay, according to CLSI (formerly NCCLS)3 criteria, against nalidixic acid 30 μg, ciprofloxacin 5 μg, and other antimicrobials with therapeutic or epidemiological purposes. To assess the activity of the AcrAB-like efflux pump, in addition to antimicrobial diffusion disk susceptibility, minimal inhibitory concentrations (MICs) of nalidixic acid and ciprofloxacin were performed following the CLSI guidelines3 by agar dilution test in the presence and absence of Phe-Arg-β-naphthylamide (Sigma, St. Louis, USA) at a concentration of 20 mg/L4 The quinolone-resistant determining regions (QRDR) of the gyrA gene were amplified by PCR and sequenced and analyzed using a method previously described by Boucheron et al5.
Clonal relationships between the isolates were assessed by ERIC-PCR according to the method described by Beyer et al6 using two parts of the primer ERIC27. PCR products were separated on 2% agarose gels, stained with ethidium bromide, visualized and photographed on an UV transilluminator.
Among the six strains studied, two were nalidixic acid-sensitive (NALs) with MICs of 4 and 8 μg/mL, respectively, and four were nalidixic acid-resistant (NALr), with MICs ≥256μg/ mL. These strains presented decreased susceptibility against ciprofloxacin (MIC = 2 μg/mL). The zone diameters for both antimicrobials increased and the MICs decreased two-fold against nalidixic acid and one-fold against ciprofloxacin when the strains were tested in the presence of AcrAB inhibitor; these differences were minimal or not evident among the nalidixic acid-susceptible strains, which suggested overexpression of an efflux pump inhibited by Phe-Arg-β-naphthylamide (likely to be AcrAB) as a mechanism of resistance. When the QRDR of the gyrA gene of each strain was amplified and sequenced, the nalidixic acid-resistant strains presented a mutation in codon Ser-83 of the gyrA gene (TCC →TTC), resulting in an amino acid change of Ser to Phe. All strains were susceptible to the remaining antimicrobial agents tested.
The NALr isolates presented identical ERIC-PCR profiles, which differed from those of the NALs isolates. The technique showed that whereas the NALr isolates belonged to the same clone, they did not have an epidemiological relationship with the NALs isolates. In addition, there were some differences between the two NALs isolates. Nalidixic acid resistance and decreased susceptibility to ciprofloxacin among Salmonella isolates is an increasing concern in several countries, even in Argentina, where 4% of human isolates are resistant to nalidixic acid8-10. The emergence of multiple resistance mechanisms in Salmonella enterica requires constant surveillance among the frequent and unusual serotypes.
Acknowledgments
This work was supported in part by grants from Fundación Alberto J. Roemmers and SGCYT (U.N.N.E.). We are grateful to the Biology Faculty of Universidad de Barcelona for amplifying and sequencing the genes, and to Ana María Pato for providing the Salmonella isolates.
