Suitable disk antimicrobial susceptibility breakpoints defining Salmonella enterica serovar Typhi isolates with reduced susceptibility to fluoroquinolones

Abstract

Infections with Salmonella enterica serovar Typhi isolates that have reduced susceptibility to ofloxacin (MIC ≥ 0.25 μg/ml) or ciprofloxacin (MIC ≥ 0.125 μg/ml) have been associated with a delayed response or clinical failure following treatment with these antimicrobials. These isolates are not detected as resistant using current disk susceptibility breakpoints. We examined 816 isolates of S. Typhi from seven Asian countries. Screening for nalidixic acid resistance (MIC ≥ 16 μg/ml) identified isolates with an ofloxacin MIC of ≥0.25 μg/ml with a sensitivity of 97.3% (253/260) and specificity of 99.3% (552/556). For isolates with a ciprofloxacin MIC of ≥0.125 μg/ml, the sensitivity was 92.9% (248/267) and specificity was 98.4% (540/549). A zone of inhibition of ≤28 mm around a 5-μg ofloxacin disc detected strains with an ofloxacin MIC of ≥0.25 μg/ml with a sensitivity of 94.6% (246/260) and specificity of 94.2% (524/556). A zone of inhibition of ≤30 mm detected isolates with a ciprofloxacin MIC of ≥0.125 μg/ml with a sensitivity of 94.0% (251/267) and specificity of 94.2% (517/549). An ofloxacin MIC of ≥0.25 μg/ml and a ciprofloxacin MIC of ≥0.125 μg/ml detected 74.5% (341/460) of isolates with an identified quinolone resistance-inducing mutation and 81.5% (331/406) of the most common mutant (carrying a serine-to-phenylalanine mutation at codon 83 in the gyrA gene). Screening for nalidixic acid resistance or ciprofloxacin and ofloxacin disk inhibition zone are suitable for detecting S. Typhi isolates with reduced fluoroquinolone susceptibility.

FIG. 1.

Fluoroquinolone MIC histograms for 816 S. Typhi isolates from Asia. Histograms showing the distribution of MICs to ofloxacin (a) and ciprofloxacin (b) of 816 S. Typhi strains, isolated from patients with enteric fever. Each isolate used for analysis was isolated from an individual enteric fever patient. The MICs are plotted on the x axis, and the numbers of isolates corresponding with particular MICs are plotted on the y axis. The white proportion of the columns indicates the nalidixic acid-susceptible isolates (n = 563). The black proportion of the columns indicates the nalidixic acid-resistant isolates (n = 253). Both histograms show a bimodal distribution, which is partly differentiated by nalidixic acid resistance.

FIG. 2.

Scatter plots relating ofloxacin and ciprofloxacin MICs to nalidixic acid MIC for 816 Asian S. Typhi isolates. Scatter plots comprised of MIC data from 816 S. Typhi isolates from Nepal (n = 104), India (n = 25), Indonesia (n = 27), Bangladesh (n = 40), Pakistan (n = 53), China (n = 51), and Vietnam (n = 516). Plots show the relationship between the MIC to nalidixic acid (y axis) and the MIC to ofloxacin (a) and ciprofloxacin (b) (x axis). The vertical and horizontal shading in each scatter plot indicates the current CLSI recommendations for breakpoints between susceptibility (white), intermediate (light gray, nalidixic acid; light red, ofloxacin and ciprofloxacin), and resistance (dark gray, nalidixic acid; dark red, ofloxacin and ciprofloxacin) (nalidixic acid MIC, ≤8 μg/ml and ≥32 μg/ml; ofloxacin MIC, ≤2 μg/ml and ≥8 μg/ml; and ciprofloxacin MIC, ≤1 μg/ml and ≥4 μg/ml). The red broken line corresponds to the proposed MIC breakpoint identifying strains with reduced susceptibility to fluoroquinolones (ofloxacin MIC of ≥0.25 μg/ml and ciprofloxacin MIC of ≥0.125 μg/ml).

FIG. 3.

Scatter plots relating ofloxacin and ciprofloxacin MIC to inhibition zone diameter for 816 Asian S. Typhi isolates. Scatter plots for 816 S. Typhi isolates comparing the inhibition zone diameters using a 5-μg ciprofloxacin disc (a) and a 5-μg ofloxacin disc (b) (x axis) and the corresponding MIC of ciprofloxacin (a) and ofloxacin (b) (y axis). The numbers in brackets relate to the 253 nalidixic acid-resistant isolates. The vertical red shading in each scatter plot is the current CLSI disc zone breakpoint for resistance (ofloxacin inhibition zone diameter, ≤16 mm; ciprofloxacin inhibition zone diameter, ≤21 mm). The horizontal red shading distinguishes strains with an MIC of ≥2 μg/ml for ofloxacin or an MIC of ≥1 μg/ml for ciprofloxacin. The gray shading is the proposed breakpoint for S. Typhi isolates with reduced susceptibility (ofloxacin MIC, ≥0.25 μg/ml; ciprofloxacin MIC, ≥0.125 μg/ml). The red broken line corresponds with the proposed breakpoints for strains with reduced susceptibility (ofloxacin inhibition zone diameter, ≤28 mm; ciprofloxacin inhibition zone diameter, ≤30 mm).

FIG. 4.

The relationship of gyrA and parC mutations and the MICs to ofloxacin and ciprofloxacin in the S. Typhi strain isolated in Asia. Box plots (boxes relate to the 25th and 75th percentiles) relating mutations in the gyrA and the parC genes (the S80I mutation is in the parC gene, and the remainder are in the gyrA gene) to the MICs of ofloxacin (a) and ciprofloxacin (b) in 475 Asian clinical isolates of S. Typhi. The MICs to ofloxacin and ciprofloxacin are plotted on the y axis. The MICs to ofloxacin ranged from 0.016 to 12 μg/ml, and those to ciprofloxacin from 0.008 to 6 μg/ml. Median values for each mutant group are indentified by a solid line in the box. Bars demonstrate the 95% confidence interval for the groups with sufficient numbers; dots correspond to outliers. The x axis is subdivided into the eight different groups of S. Typhi strains identified and assayed, characterized as follows: no mutations in gyrA or parC (n = 15), D87A (n = 2), S83Y (n = 46), S83F (n = 406), D87G (n = 2), S83F/D87N (n = 1), S83F/D87G (n = 1), and S83F/D87G/S80I (n = 2). The upper broken lines indicate the current CLSI breakpoint recommendations for ofloxacin and ciprofloxacin. The lower broken lines correspond with the proposed breakpoints for strains with reduced susceptibility to ofloxacin and ciprofloxacin. Statistical significance was calculated between the nonmutant group and the single mutant group and between the single mutant group and the double/triple mutant group using the Student's t test.