Enrofloxacin resistance in Escherichia coli isolated from poultry: preliminary analysis of gyrA and parC sequences

Escherichia coli are usually common inhabitants of the gastrointestinal tract of birds, although avian pathogenic strains can cause several diseases. Furthermore, gut of birds can be a reservoir of zoonotic E. coli, and contamination of poultry products may occur during slaughtering and processing procedures. Enrofloxacin (ENR) is commonly used in poultry farming, although recently a prudent and rationale use of fluoroquinolone (FQ) in farm animals is recommended by the European Union. The most frequent mechanism of FQ resistance in E. coli is represented by alterations occurring in genes encoding for DNA gyrase and topoisomerase IV. The aims of this study were to evaluate the ENR resistance in E. coli isolated from poultry and to investigate mutations in the sequences of gyrA and parC genes. Swab samples collected from poultry were tested by bacteriology. Pure cultures of E. coli were used for MIC and for DNA extraction. DNA was amplified by PCR to obtain a 347 bp fragment of gyrA gene and a 964 bp fragment of parC gene. PCR products were sequenced and analysed. A total of 12 E. coli isolates resulted resistant to ENR by MIC. Further 12 ENR-susceptible E. coli were randomly selected among all susceptible isolates. Predicted amino acid sequences of gyrA and parC were aligned and analyzed blindly. Most of resistant E. coli isolates showed mutations at codons 83 and 87 of gyrA and at codon 80 of parC. In particular, 11 resistant isolates had Ser83Leu mutation, that was present also in 3 susceptible isolates. No other mutations at codons 83 and 87 of gyrA and at codon 80 of parC were found in susceptible isolates. Among resistant isolates, 10 possessed mutations at gyrA codon 87 and 11 isolates showed mutations at parC codon 80. Mutations were Asp87Asn (n=8), Asp87Gly (n=1), Asp87Tyr (n=1) in gyrA and Ser80Ile (n=9) and Ser80Arg (n=2) in parC. Evaluation of mutations at codon 80 of parC resulted the most sensitive (91.7%) and specific (100%) test for distinguishing ENR-susceptible to ENR-resistant isolates. Finally, 7 out of 12 ENR-resistant isolates showed two gyrA mutations (Ser83Leu and Asp87Asn) coupled with a substitution in parC (Ser80Ile); the same findings have been reported in E. coli isolated from poultry and resulted resistant to many FQs (ciprofloxacin, danofloxacin, difloxacin, enrofloxacin, flumequine, marbofloxacin, nalidixic acid, and sarafloxacin). These isolates showed also the highest values of MIC