Target gene mutations among methicillin-resistant Staphylococcus aureus and methicillin-susceptible S. aureus with elevated MICs of enrofloxacin obtained from diseased food-producing animals or food of animal origin

Abstract

Sir, Fluoroquinolones are broad-spectrum antimicrobial agents that are effective against Gram-positive and Gram-negative bacteria. Enrofloxacin, the first fluoroquinolone approved for veterinary use, is still commonly used in veterinary practice to treat infections in food-producing, pet and companion animals, caused by a wide variety of bacterial pathogens including methicillinresistant Staphylococcus aureus (MRSA) and methicillinsusceptible S. aureus (MSSA). However, CLSI-approved clinical breakpoints for enrofloxacin applicable to Staphylococcus spp. are restricted to staphylococci from cats and dogs (susceptible, ≤0.5 mg/L; intermediate, 1–2 mg/L; resistant, ≥4 mg/L). No CLSI-approved clinical breakpoints applicable to staphylococci from pigs, cattle or poultry are currently available. DNA gyrase and DNA topoisomerase IV are the target enzymes of fluoroquinolones in S. aureus, and resistance in staphylococci is mainly due to mutations in the quinolone resistance-determining regions (QRDRs) of the genes gyrA, gyrB, grlA and grlB, which code for the corresponding subunits of these two enzymes. Another mechanism involved in fluoroquinolone resistance of S. aureus is overexpression of the norA gene, which encodes a multidrug efflux pump, by mutations in the norA promoter region. The aim of the present study was to gain insight into target gene mutations and norA regulator mutations present among MRSA and MSSA from diseased pigs, cattle and poultry, as well as food of poultry origin. For this, all MRSA and MSSA isolates that exhibited enrofloxacin MICs of ≥1 mg/L, from five different strain collections, were comparatively analysed. This cut-off was chosen based on the breakpoint for enrofloxacin-non-susceptible canine and feline Staphylococcus spp. isolates. The test population included 8/54 MRSA from diseased pigs and 9/32 MRSA from poultry meat and poultry meat products characterized in two previous studies, 2/11 MRSA from diseased cattle and 1/2 MRSA from diseased poultry collected in the GERM-Vet programme 2008–09, 1/2 MRSA and 12/24 MSSA from diseased poultry collected in the GERM-Vet programme 2006–07, and 3/27 MSSA from diseased pigs collected in the BfT-GermVet study 2006–08. In total, 36 isolates with enrofloxacin MICs of 1 to ≥32 mg/L were identified (Table 1). If not already done in previous studies, isolates were subjected to spa typing (http ://spaserver.ridom.de) as well as to two clonal complex (CC) 398-specific PCRs as described previously. Isolates that were negative in these PCRs were subjected to multilocus sequence typing (MLST; http://saureus.mlst.net). Of the 36 isolates, 24 were assigned to sequence type (ST) 398 or CC398, depending on whether MLST or the CC398-specific PCRs had been conducted. Five isolates had ST9, another five isolates had ST5 and one isolate had ST1791 (both ST5 and ST1791 belong to CC5). One avian isolate had ST2269, which is a novel ST (allelic profile 1-4-1-4-243-250-10) that has been identified for the first time during the course of this study. Nine different spa types (t002, t011, t034, t214, t899, t1197, t1419, t1430 and t2346) were detected (Table 1). The QRDRs of the gyrA, gyrB, grlA and grlB genes and the promoter region of the norA gene of all 36 S. aureus isolates were amplified by PCR using previously described primers. Mutations were identified by sequencing the PCR products, and the results are summarized in Table 1 in relation to the enrofloxacin MICs. Regardless of their origin and their methicillinresistance status, all isolates with enrofloxacin MICs of ≥4 mg/L exhibited mutations in both grlA at codon 80 (resulting in a Ser to Phe exchange) and gyrA at codon 84 (resulting in exchange of Ser to Leu or Ala) (Table 1). The Ser84Ala exchange in GyrA observed in two MRSA isolates has not been reported before in staphylococci. In contrast, isolates with enrofloxacin MICs of 1 or 2 mg/L also exhibited a mutation in grlA at codon 80 (resulting in the exchange of Ser to Phe, Tyr or Leu), but lacked the amino acid substitutions at position 84 in GyrA. Another interesting observation was the presence of the Glu422Asp exchange in GrlB in all isolates assigned to CC398/ ST398, independent of enrofloxacin MIC, the year of isolation,