Detection of decreased fluoroquinolone susceptibility in Salmonellas and validation of nalidixic acid screening test

Abstract

We evaluated 1,010 Salmonella isolates classified as fluoroquinolone susceptible according to the National Committee for Clinical Laboratory Standards guidelines for susceptibility to nalidixic acid and three fluoroquinolones. These isolates were divided into two distinct subpopulations, with the great majority (n = 960) being fully ciprofloxacin susceptible and a minority (n = 50) exhibiting reduced ciprofloxacin susceptibility (MICs ranging between 0.125 and 0.5 microg/ml). The less ciprofloxacin-susceptible isolates were uniformly resistant to nalidixic acid, while only 12 (1.3%) of the fully susceptible isolates were nalidixic acid resistant. A similar association was observed between resistance to nalidixic acid and decreased susceptibility to ofloxacin or norfloxacin. A mutation of the gyrA gene could be demonstrated in all isolates for which the ciprofloxacin MICs were >/= 0.125 microg/ml and in 94% of the nalidixic acid-resistant isolates but in none of the nalidixic acid-susceptible isolates analyzed. Identification of nalidixic acid resistance by the disk diffusion method provided a sensitivity of 100% and a specificity of 87.3% as tools to screen for isolates for which the MICs of ciprofloxacin were >/= 0.125 microg/ml. We regard it as important that microbiology laboratories endeavor to recognize these less susceptible Salmonella strains, in order to reveal their clinical importance and to survey their epidemic spread.

FIG. 1

Ciprofloxacin (CIP) MIC histogram for 1,010 epidemiologically unrelated Salmonella isolates collected in Finland from 1995 to 1998. The MICs of ciprofloxacin are plotted on the x axis, and the number of isolates are plotted on the y axis. White columns indicate nalidixic acid-susceptible (n = 948) isolates, and black columns indicate nalidixic acid-resistant (n = 62) isolates.

FIG. 2

Scattergrams for 1,010 Salmonella isolates correlating the MICs of nalidixic acid to those of ciprofloxacin (a), ofloxacin (b), and norfloxacin (c). The vertical dashed lines of panels a to c indicate the NCCLS breakpoint recommendations for susceptibility and resistance, respectively, to ciprofloxacin (MIC, ≤1 and ≥4 μg/ml), ofloxacin (MIC, ≤2 and ≥8 μg/ml), and norfloxacin (MIC, ≤4 and ≥16 μg/ml). The horizontal solid lines indicate the respective NCCLS breakpoint recommendations for nalidixic acid (MIC, ≤16 and ≥32 μg/ml). The numbers within the graph indicate the numbers of Salmonella isolates. CIP, ciprofloxacin; OFL, ofloxacin; NOR, norfloxacin; NAL, nalidixic acid.

FIG. 3

Scattergram plotting the MICs of ciprofloxacin (x axis) and the inhibition zone diameters around a 5-μg ciprofloxacin disk (y axis) for 62 nalidixic acid-resistant and 67 nalidixic acid-susceptible Salmonella isolates. Nalidixic acid-resistant isolates (MIC, ≥32 μg/ml) are indicated with an asterisk, and the resistant isolates exhibiting a mutation in the gyrA gene are indicated with a double asterisk. The dashed lines are discussed in the text.

FIG. 4

Scattergram plotting the MICs of ciprofloxacin (x axis) and the inhibition zone diameters around a 30-μg nalidixic acid disk (y axis) for 62 nalidixic acid-resistant and 67 nalidixic acid-susceptible Salmonella isolates. The horizontal dashed lines indicate the NCCLS recommendations for susceptibility (inhibition zone diameter, ≥19 mm) and resistance (inhibition zone diameter, ≤13 mm) to nalidixic acid. Isolates designated as resistant to nalidixic acid based on MIC (≥32 μg/ml) determinations are indicated with an asterisk, and the resistant isolates exhibiting a mutation in the gyrA gene are indicated with a double asterisk.