A vancomycin-resistant Enterococcus faecium isolate from a Danish healthy volunteer, detected 7 years after the ban of avoparcin, is possibly related to pig isolates.

ParC proteins. The results show that the use of 20 mg/L of Phe-Arg-β-naphthylamide decreased the MIC of nalidixic acid (from two to five serial twofold dilution steps) in all isolates tested, even in those with the nalidixic acid-susceptible phenotype. This high reduction in the MIC values was observed independent of the number of amino acid changes in GyrA and ParC. This effect was especially noticeable among isolates carrying one single GyrA substitution, in which the MIC 50 of nalidixic acid decreased from resistant to susceptible levels (from 128 to 8 mg/L). This result indicates that one amino acid change in GyrA by itself (without an additional mechanism of resistance) is unable to confer a resistant phenotype to nalidixic acid in E. coli. On the other hand, almost no decreases in the MIC 50 were detected in the fluoroquinolones tested. Nevertheless, Phe-Arg-β-naphthylamide affected the MIC of ciprofloxacin in 10 of the 60 E. coli isolates whose MIC values were lowered by at least two serial dilution steps. Eight of these 10 isolates also decreased the MIC of norfloxacin (in both cases including mutant Nor5). Interestingly, fluoroquinolone MIC values for these 10 isolates were higher than the MIC 50 that corresponds with the number of substitutions present in the targets. The addition of the inhibitor decreased these high MIC values to expected MIC 50 values (for example, the MIC of ciprofloxacin was decreased from 1 to 0.25 mg/L in one isolate with only a mutation in GyrA). The presence of 20 mg/L of Phe-Arg-β-naphthylamide also affected the MIC of chloramphenicol in 51 isolates whose MICs decreased by two or three serial dilution steps. Finally, no effect was observed when the MIC of tetracycline was analysed. The effect of different concentrations of Phe-Arg-β-naphthylamide (40, 80 or 160 mg/L) on nalidixic acid, ciprofloxacin and norfloxacin MICs was also studied in 25 of the E. coli isolates. The influence of the Phe-Arg-β-naphthylamide was homogeneous in the range 20–160 mg/L of the inhibitor in all the isolates tested. These results might be explained by the activity of the inhibitor against two or more efflux systems. The effect on the MIC of nalidixic acid in all the strains (both susceptible and resistant) suggests the presence of a constitutive efflux pump able to discharge the anti-bacterial agent and confer a basal level of resistance. Moreover, chloramphenicol might be another substrate of this hypothetical efflux pump. The Phe-Arg-β-naphthylamide only …