Identification of DNA Gyrase A Mutations in Ciprofloxacin-Resistant Isolates ofSalmonella typhimuriumfrom Men and Cattle in Germany

Abstract

Six multiply resistant isolates of Salmonella typhimurium var. copenhagen with high-level resistance to fluoroquinolones (e.g., MIC of ciprofloxacin: 32 micrograms/ml) were isolated from human patients (n = 3) and from cattle (n = 3). The isolates were examined by complementation tests using a set of broad-host-range plasmids, which carry either the gyrA+ or the gyrB+ genes or a combination of both from Escherichia coli K-12. The results indicated a combination of gyrA and gyrB mutations in all isolates. Subsequent direct sequencing of PCR-generated internal DNA fragments of gyrA revealed an identical double mutation in all six isolates (Ser-83-->Ala and Asp-87-->Asn). In addition, the results of phenotypic (i.e., phagetype, biotype, serotype) and genotypic characterization [i.e., ribotyping and polymerase chain reaction fingerprinting (PCR-fingerprinting)] were identical for all six isolates and were distinguishable from a quinolone-susceptible strain of the same serovar and an unrelated isolate of S. typhimurium. These data indicate the clonal identity of the fluoroquinolone-resistant strains of S. typhimurium isolated from men and cattle in Germany.

Mechanisms of quinolone resistance in Salmonella

“…This concerned a particular clone named Copenhagen variant encountered in Germany during the early 1990s. The double mutation was identical in several isolates (Ser-83 to Ala and Asp-87 to Asn) thereby suggesting the clonality of the strains [25]. These isolates additionally had an altered gyrB gene coding for the B subunit of gyrase [23].…”

“…High-level fluoroquinolone resistance in clinical isolates of Salmonella has only been reported for the Copenhagen variant clone of S. enterica serovar Typhimurium which appeared during the early 1990s in Germany [23,25] and probably also in Belgium [27]. As described above only mutations in the gyrA and gyrB target genes have been investigated [23,25].…”

“…To date, only one example has been recorded with the emergence and probable clonal diffusion of the S. enterica Typhimurium variant Copenhagen in limited areas of Europe. These strains, which are highly resistant to fluoroquinolones, were mainly isolated between 1991 and 1995 from animals and humans [23,25,27]. Since then, at least in France, only certain strains with a decreased susceptibility to fluoroquinolones have been isolated [17].…”

Emergence of Salmonella epidemics: The problems related to Salmonella enterica serotyp Enteritidis and multiple antibiotic resistance in other major serotypes

“…Amino acid changes at Ser-83 (to Phe, Tyr, or Ala) or at Asp-87 (to Gly, Asn, or Tyr) are the most frequently observed in nalidixic acid (Nal)-resistant strains [30]. Double mutations at both residues 83 and 87 have been identified in clinical isolates of an S. Typhimurium DT204 clone showing high-level resistance to FQ [67]. These strains were mainly isolated between 1991 and 1995 from animals and humans in limited areas in Europe and are highly resistant to ciprofloxacin (Cip) (MIC of 32 µg/mL).…”

Epidemiology of resistance to quinolones in Salmonella

“…As early as 1994, Lontie et al [17] suggested that the occurrence of highly fluoroquinolone-resistant Salmonella Typhimurium in three patients who had not received fluoroquinolones might be explained by its transmission from the animal reservoir. Using a number of phenotypic and genotypic characterisation criteria, Heisig et al [12] failed to differentiate between isolates with high-level resistance to fluoroquinolones from cattle and humans. More recently, Mølbak et al [20] demonstrated that quinolone-resistant Salmonella Typhimurium DT104 responsible for a foodborne outbreak resulting in two fatal cases could be traced back to two pig farms.…”