Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, PenI ) harbor alterations in the penA gene encoding the penicillin binding protein 2 (PBP2). A 402-bp DNA fragment in the 3 half of penA was sequenced from a collection of 1,670 meningococcal clinical isolates from 22 countries that spanned 60 years. Phenotyping, genotyping, and the determination of MICs of penicillin G were also performed. A total of 139 different penA alleles were detected with 38 alleles that were highly related, clustered together in maximum-likelihood analysis and corresponded to the penicillin G-susceptible isolates. The remaining 101 penA alleles were highly diverse, corresponded to different genotypes or phenotypes, and accounted for 38% of isolates, but no clonal expansion was detected. Analysis of the altered alleles that were represented by at least five isolates showed high correlation with the Pen I phenotype. The deduced amino acid sequence of the corresponding PBP2 comprised five amino acid residues that were always altered. This correlation was not complete for rare alleles, suggesting that other mechanisms may also be involved in conferring reduced susceptibility to penicillin. Evidence of mosaic structures through events of interspecies recombination was also detected in altered alleles. A new website was created based on the data from this work (http://neisseria.org/nm/typing/penA). These data argue for the use of penA sequencing to identify isolates with reduced susceptibility to penicillin G and as a tool to improve typing of meningococcal isolates, as well as to analyze DNA exchange among Neisseria species.

e Meningococcal gyrA gene sequence data, MICs, and mouse infection were used to define the ciprofloxacin breakpoint for Neisseria meningitidis. Residue T91 or D95 of GyrA was altered in all meningococcal isolates with MICs of >0.064 g/ml but not among isolates with MICs of <0.032 g/ml. Experimental infection of ciprofloxacin-treated mice showed slower bacterial clearance when GyrA was altered. These data suggest a MIC of >0.064 g/ml as the ciprofloxacin breakpoint for meningococci and argue for the molecular detection of ciprofloxacin resistance. N eisseria meningitidis is a Gram-negative encapsulated bacterium isolated only from humans, where it may provoke severe invasive infections (mainly septicemia and meningitis). Management of meningococcal disease requires prompt treatment of patients, as well as vaccination and/or chemoprophylaxis of contacts. The antibiotics currently recommended for chemoprophylaxis are rifampin, ciprofloxacin, and ceftriaxone (1). The emergence and expansion of meningococcal clones resistant to these antibiotics may jeopardize these recommendations. Ciprofloxacin resistance in meningococci was earlier linked to mutations in the quinolone resistance-determining region (QRDR) of the gyrA gene (encodes subunit A of DNA gyrase) but no mutations in the QRDR of gyrB, parC, and parE (2). Here we correlate gyrA mutations and their in vivo impact on ciprofloxacin MICs for meningococcal clinical isolates.This study examined all of the available meningococcal isolates collected from 1995 to 2011 with ciprofloxacin MICs of Ն0.064 g/ml (n ϭ 19) in four countries (France, Italy, Spain, and Sweden). Representative isolates with ciprofloxacin MICs of Յ0.032 g/ml (n ϭ 177) were also tested, as were two isolates of N. gonorrhoeae and N. cinerea with ciprofloxacin MICs of 0.250 and 0.125 g/ml, respectively. Isolate typing was performed as previously described (3), and all those data are available at the Neisseria

Identification of clinical isolates of Neisseria meningitidis that are resistant to rifampin is important to avoid prophylaxis failure in contacts of patients, but it is hindered by the absence of a breakpoint for resistance, despite many efforts toward standardization. We examined a large number (n ‫؍‬ 392) of clinical meningococcal isolates, spanning 25 years (1984 to 2009), that were collected in 11 European countries, Argentina, and the Central African Republic. The collection comprises all clinical isolates with MICs of >0.25 mg/liter (n ‫؍‬ 161) received by the national reference laboratories for meningococci in the participating countries. Representative isolates displaying rifampin MICs of <0.25 mg/liter were also examined (n ‫؍‬ 231). Typing of isolates was performed, and a 660-bp DNA fragment of the rpoB gene was sequenced. Sequences differing by at least one nucleotide were defined as unique rpoB alleles. The geometric mean of the MICs was calculated for isolates displaying the same allele. The clinical isolates displaying rifampin MICs of >1 mg/liter possessed rpoB alleles with nonsynonymous mutations at four critical amino acid residues, D542, H552, S548, and S557, that were absent in the alleles found in all isolates with MICs of <1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of <1 mg/liter. The rpoB allele sequence and isolate data have been incorporated into the PubMLST Neisseria database (http://pubmlst.org/neisseria/). The rifampin-resistant isolates belonged to diverse genetic lineages and were associated with lower levels of bacteremia and inflammatory cytokines in mice. This biological cost may explain the lack of clonal expansion of these isolates.

Single mutations in gyrA appeared to be the main mechanism involved, Thr-91-->Ile being the most frequent substitution seen. Two meningococci had four different gyrA substitutions. No mutations in the QRDRs of the parC and gyrB genes were detected, and three strains showed a His-495-->Asn substitution in the parE gene. In addition, two different alterations in the mtrR gene affecting the expression of the MtrCDE efflux system were identified which may also contribute to the reduced susceptibility to quinolones seen in three strains.

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