Susceptibility of Neisseria meningitidis to 16 antimicrobial agents and characterization of resistance mechanisms affecting some agents

Abstract

Neisseria meningitidis represents a pathogen of great public health importance in both developed and developing countries. Resistance to some antimicrobial agents used either for therapy of invasive infections or for prophylaxis of case contacts has long been recognized, although specific guidelines for susceptibility testing have not been fully developed. We have examined the susceptibilities of a collection of 442 meningococcal clinical isolates from 15 countries to 16 antimicrobial agents. These included isolates recovered between 1917 and 2004, with representatives of all major serogroups. All isolates were tested by the Clinical and Laboratory Standards Institute (formerly NCCLS) broth microdilution method using Mueller-Hinton lysed horse blood broth, while a subset of 102 isolates was tested by agar dilution using Mueller-Hinton sheep blood agar. Most isolates provided adequate growth for MIC determinations by both broth and agar methods. Growth in broth was enhanced by CO(2) incubation and was required for two strains (1.7%). MICs of the study drugs compared favorably between the broth and agar methods (79 to 100% essential agreement), and MICs also generally agreed closely (92 to 100% essential agreement, excluding azithromycin) between broth tests incubated in the two different atmospheres. Elevated penicillin and ampicillin MICs (> or =0.12 microg/ml and > or =0.25 microg/ml, respectively) occurred in 14.3% and 8.6% of strains and were associated with polymorphisms of the penA gene encoding a modified penicillin-binding protein 2. None of the 442 isolates produced beta-lactamase. Elevated tetracycline and doxycycline (but not minocycline) MICs were associated with efflux-mediated resistance encoded by tet(B) in 13 strains. Resistance to sulfisoxazole in 21.7% of strains and to trimethoprim-sulfamethoxazole in 21.0% resulted from polymorphisms of folP encoding a modified dihydropteroate synthetase. Seven strains were resistant to rifampin due to mutations in the rpoB gene, and two strains were resistant to chloramphenicol due to production of chloramphenicol acetyltransferase mediated by catP. Two strains had reduced quinolone susceptibility due to mutations of gyrA. The determination of the susceptibilities of a large group of meningococcal strains (including strains with characterized resistance mechanisms) to 16 antimicrobial agents has served as the essential first step in defining susceptibility testing breakpoints specific for this organism.

FIG. 1.

Comparison of azithromycin MICs determined in air with MICs determined in 5% CO₂.

FIG. 2.

RFLP gel of Taq1 digest of amplified penA gene segments from 17 meningococcal isolates. Isolate numbers are indicated at the top, and the digest patterns are numbered at the bottom following the numbering scheme of Antignac et al. (1).

FIG. 3.

Frequency of penicillin (A) and ampicillin (B) MICs derived from testing 442 meningococcal clinical isolates. The numbers of strains with penA mutations divided by the number of strains examined are indicated at several MICs.

FIG. 4.

Frequency of chloramphenicol MICs derived from testing 442 meningococcal clinical isolates. Both isolates at a MIC of 16 μg/ml contained the catP gene and produced chloramphenicol acetyltransferase (34).

FIG. 5.

Frequency of tetracycline (A) and minocycline (B) MICs derived from testing 442 meningococcal clinical isolates; panel C depicts the doxycyline MICs derived from testing a subset of 125 isolates. The numbers of strains containing tet(B) divided by the number of strains examined for that gene are indicated at several MICs.

FIG. 6.

Frequency of sulfisoxazole (A) and trimethoprim-sulfamethoxazole (B) MICs derived from testing 442 meningococcal clinical isolates. The trimethoprim-sulfamethoxazole MICs are indicated only by the trimethoprim component in a fixed 1:19 ratio. The numbers of strains containing mutations of folP divided by the number of strains examined for that gene are indicated at several MICs.

FIG. 7.

Frequency of rifampin MICs derived from testing 442 meningococcal clinical isolates. All seven isolates with MICs of greater than 128 μg/ml contained rpoB gene mutations.

FIG. 8.

Frequency of ciprofloxacin (A) and nalidixic acid (B) MICs derived from testing 442 meningococcal clinical isolates. Strains with ciprofloxacin MICs of 0.06 μg/ml or higher or nalidixic acid MICs of 64 μg/ml or greater contained mutations of gyrA.