Nalidixic Acid Disk for Laboratory Detection of Ciprofloxacin Resistance in Neisseria meningitidis

Abstract

Recently the CLSI recommended a disk diffusion method and breakpoints for meningococci which include breakpoints derived for nalidixic acid which serve as surrogate markers for gyrase A mutations associated with diminished fluoroquinolone susceptibility. This study presents the application of this methodology to a panel of 57 meningococcal strains isolated in Spain that include all levels of susceptibility to ciprofloxacin. In conclusion, the most useful method to predict isolates with gyrA mutations that decrease the activity of fluoroquinolones is the use of 30-μg nalidixic acid disks.

Although the emergence of meningococcal strains showing decreased susceptibility to ciprofloxacin seems still to be a rare event, reports of this type of isolate are becoming more frequent (1, 2, 6) and widespread. Continuous surveillance is necessary to monitor the emergence and spread of ciprofloxacin-resistant strains.

Disk diffusion testing is one of the preferred methods for defining susceptibility levels because it is fast and needs few resources. After some controversial studies, the CLSI-recommended method (3), only recently and with strict rules to obtain reproducible results, now includes interpretive criteria for disk diffusion testing of the drugs most widely used (with the exception of penicillin and ampicillin) for therapy and prophylaxis of meningococcal disease.

The CLSI has recommended the disk diffusion method and breakpoints for meningococci after an interesting study (5) in which they suggested that disk diffusion breakpoints derived for nalidixic acid could serve as surrogate markers for gyrase A mutations associated with diminished fluoroquinolone susceptibility. Nevertheless, the study included only two well-characterized strains showing resistance to ciprofloxacin and six additional isolates with some level of decreased susceptibility that was apparently determined during the study (5). Moreover, there were several gaps in the MICs of ciprofloxacin: strains showing MICs of ≤0.0015, 0.03, or 0.12 mg/liter were not included in the study.

Now, we present data from 57 meningococcal strains isolated in Spain from 2000 to 2007. MICs for ciprofloxacin ranged from ≤0.0007 to 0.5 mg/liter, including some representative of all dilutions of the antibiotic (Fig. 1). The MIC was determined using the previously described agar dilution method (4), and the disk diffusion methodology was applied following the suggestions of Jorgensen et al. (5). All strains defined as intermediate or fully resistant to ciprofloxacin showed gyrA mutations associated with reduced susceptibility to the drug (4, 7).

FIG. 1.

(A) Combined ciprofloxacin MICs and 5-μg ciprofloxacin disk zone diameters for the 57 strains tested. (B) Combined ciprofloxacin MICs and 30-μg nalidixic acid disk zone diameters for the 57 strains tested. Boldface indicates discrepancies resulting from application of the CLSI-recommended breakpoints.

The use of 5-μg ciprofloxacin disks has been suggested, with resistant strains being defined as those with a single zone diameter less than 32 mm (3, 5). In our study, using ciprofloxacin disks, all susceptible strains showed single zone diameters ranging from 34 to 47 mm. Only one strain with a ciprofloxacin MIC of 0.06 mg/liter (defined as intermediate) was included, showing a single zone diameter of 35 mm, and finally, those isolates defined as resistant (MIC, ≥0.12 mg/liter) presented single zone diameters ranging from 29 to 35 mm (Fig. 1A). In our opinion the use of 5-μg ciprofloxacin disks is not a reliable method for assessing the susceptibility of Neisseria meningitidis to this antibiotic.

Most of the strains susceptible to ciprofloxacin (MICs, ≤0.03 mg/liter), using 30-μg nalidixic acid disks, showed single zone diameters ranging from 28 to 40 mm (Fig. 1B). However, three strains, with a MIC of 0.03 mg/liter, displayed zone diameters of 15, 16, and 25 mm. According to the CLSI recommendations for nalidixic acid disks, the three strains might be considered resistant but the MIC defined them as susceptible. In fact, the gyrA sequence in these three strains did not show differences from those of the wild-type susceptible strains. For all meningococci with MICs higher than 0.03 mg/liter, all of which showed gyrA mutations associated with diminished susceptibility to ciprofloxacin, no single zone was observed at all. From this observation we suggest a very simple algorithm, defining meningococcal quinolone-resistant strains associated with gyrA mutations as those with single zone diameters of <15 mm with 30-μg nalidixic acid disks. Those strains showing single zone diameters ranging from 15 to 25 mm should be checked carefully for ciprofloxacin susceptibility.

The MIC of nalidixic acid was also checked. All the isolates with a ciprofloxacin MIC of <0.03 mg/liter showed MICs for nalidixic acid ranging from 0.25 to 1 mg/liter. Four strains with ciprofloxacin MICs of 0.03 mg/liter were included in the study: two of them presented nalidixic acid MICs of 1 and 2 mg/liter, but the other two showed a nalidixic acid MIC of 16 mg/liter. All the isolates with MICs of >0.06 mg/liter presented nalidixic acid MICs of >256 mg/liter, with the sole exception of an isolate with a MIC of 16 mg/liter. In general terms, the MIC for nalidixic acid might predict the level of susceptibility to ciprofloxacin. However, the three exceptions found in this study (two with MICs of 0.03 mg/liter and one with a MIC of 0.25 mg/liter) could be an obstacle to following this suggestion.

In conclusion, the most useful method to predict isolates with gyrA mutations that decrease the activity of fluoroquinolones is the use of 30-μg nalidixic acid disks. In light of these results, further studies are necessary for checking if the current breakpoint recommendations need to be revised.