The emergence of quinolone-resistant isolates of salmonellae has been related to the use of antibiotics in veterinary medicine, which causes an important impact on the selection of resistance (2, 3, 8). It has also been demonstrated that veterinary clones possess the same mechanism of resistance as human clinical isolates (10). Quinolone resistance in salmonellae is mainly associated with mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes (6, 10).
However, little is known about what the incidence of resistance in wild animals is and which of the mechanisms of resistance these strains have, although it is thought that wild animals are also integrated in the same epidemiologic cycle (1). The aim of this study was to analyze the prevalence of quinolone-resistant isolates of salmonellae from wild birds and determine the mechanisms involved.
A total of 45 Salmonella enterica subsp. enterica isolates were obtained from stool samples from wild birds just arrived to GREFA Wild Life Hospital. Seven strains (15.5%) were selected on the basis of resistance to nalidixic acid. The MICs of nalidixic acid (Sigma Aldrich, Madrid, Spain), ciprofloxacin, and enrofloxacin (Bayer, Leverkusen, Germany) were measured by a twofold agar dilution method and interpreted according to the recommendations of the National Committee for Clinical Laboratory Standards guidelines (4). To identify gyrA and parC mutations in resistant isolates, PCR and direct DNA sequencing were performed as follows. A 312-bp fragment of the QRDR of the gyrA gene was amplified from a genomic DNA template by using specific primers gyr A-1 (5′-GGTACACCGTGCCGTACTTT-3′) and gyr A-2 (5′-TCCACGAAATCCACCGTC-3′) corresponding to positions 17 to 137 and 311 to 328, respectively. These salmonella-specific primers were constructed on the basis of Salmonella enterica serotype Typhimurium gyrA gene sequence data (GenBank accession number X78977). The primers for the amplification of the QRDR of the parC gene have been described elsewhere (9). The mutations in the gyrA gene leading to the amino acid substitutions and MICs are shown in Table 1.
TABLE 1.
Susceptibility to quinolone and GyrA substitutions of S. enterica subsp. enterica isolates from wild birds
| Isolate | Serovar | Origin | MIC (μg/ml)a
|
Position | Mutation (amino acid) | ||
|---|---|---|---|---|---|---|---|
| NAL | CIP | ENR | |||||
| S2 | Havana | Black vulture | 256 | 0.25 | 1 | 83 | TCC (Ser)\TAC (Tyr) |
| S9 | Adelaide | Sparrow hawk | 256 | 0.25 | 1 | 83 | TCC (Ser)\TAC (Tyr) |
| S14 | Adelaide | Imperial eagle | 256 | 0.25 | 1 | 83 | TCC (Ser)\TAC (Tyr) |
| S20 | Adelaide | Little owl | 256 | 0.25 | 1 | 83 | TCC (Ser)\TAC (Tyr) |
| S10 | Brandenburg | Kestrel | 512 | 0.25 | 1 | 83 | TCC (Ser)\TAC (Tyr) |
| S57 | Hadar | Kestrel | 512 | 0.25 | 1 | 87 | GAC (Asp)\CAC (Asn) |
| S60 | Hadar | Jay | 512 | 0.25 | 1 | 87 | GAC (Asp)\CAC (Asn) |
NAL, nalidixic acid; CIP, ciprofloxacin; ENR, enrofloxacin.
Strains S2, S9, S10, S14, and S20 carried a Cys-284→Ala transversion, resulting in a Ser-83→Tyr substitution in the GyrA subunit. This mutation has been described in Salmonella serotype Typhimurium strains resistant to nalidixic acid and reduced the susceptibility to fluoroquinolones isolated from food-producing animals (10). Strains S57 and S60 carried a Gly-259→Cys transition, resulting in an Asp-87→Asn substitution in the GyrA subunit. This mutation has also been described in Salmonella isolates of human and animal origin (2, 5).
No mutations in the QRDR of parC were detected. These results may confirm that parC mutations are not necessary to obtain a high level of resistance to nalidixic acid (7).
The mechanisms of resistance described here are the same as the ones described for humans and domestic animals. Therefore, it is possible that wild animals be included in the epidemiologic cycle of the spread of resistance and the dissemination of resistant bacteria among animals and the environment. Further studies are necessary to obtain more information about what is the real role of wild animals in these facts.
Nucleotide sequence accession numbers.
The GenBank accession numbers for the partial sequences of the quinolone-resistant strains are AF447053, AF447054, AF447055, AF447056, AF447057, AF447058, and AF447059.
Acknowledgments
This work has been supported by grant 13/99 of the Universidad San Pablo CEU.
We are grateful to GREFA Wild Life Hospital for supplying the samples, A. Echeita and M. A. Usera for the serotyping of the isolates, and Linda Hamalainen for linguistic assistance.
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