Implications of differential age distribution of disease-associated meningococcal lineages for vaccine development.

New vaccines targeting meningococci expressing serogroup B polysaccharide have been developed, with some being licensed in Europe. Coverage depends on the distribution of disease-associated genotypes, which may vary by age. It is well established that a small number of hyperinvasive lineages account for most disease, and these lineages are associated with particular antigens, including vaccine candidates. A collection of 4,048 representative meningococcal disease isolates from 18 European countries, collected over a 3-year period, were characterized by multilocus sequence typing (MLST). Age data were available for 3,147 isolates. The proportions of hyperinvasive lineages, identified as particular clonal complexes (ccs) by MLST, differed among age groups. Subjects <1 year of age experienced lower risk of sequence type 11 (ST-11) cc, ST-32 cc, and ST-269 cc disease and higher risk of disease due to unassigned STs, 1- to 4-year-olds experienced lower risk of ST-11 cc and ST-32 cc disease, 5- to 14-year-olds were less likely to experience ST-11 cc and ST-269 cc disease, and ≥25-year-olds were more likely to experience disease due to less common ccs and unassigned STs. Younger and older subjects were vulnerable to a more diverse set of genotypes, indicating the more clonal nature of genotypes affecting adolescents and young adults. Knowledge of temporal and spatial diversity and the dynamics of meningococcal populations is essential for disease control by vaccines, as coverage is lineage specific. The nonrandom age distribution of hyperinvasive lineages has consequences for the design and implementation of vaccines, as different variants, or perhaps targets, may be required for different age groups.

Evolutionary changes in antimicrobial resistance of invasive Neisseria meningitidis isolates in Belgium from 2000 to 2010: increasing prevalence of penicillin nonsusceptibility.

This study was conducted to evaluate the evolution of the antimicrobial susceptibility of Neisseria meningitidis causing invasive diseases in Belgium in the period of January 2000 to December 2010. A total of 1,933 cases of N. meningitidis from invasive infections were analyzed by antimicrobial susceptibility testing at the Belgian Meningococcal Reference Centre. The majority of strains were susceptible to antibiotics that are currently used for the treatment and prophylaxis of meningococcal disease, but the prevalence of clinical isolates with reduced susceptibility to penicillin increased over the years. The phenotyping, genotyping, and determination of MICs of penicillin G were performed. The systematic shift of the curves toward higher penicillin MICs in the susceptible population indicated that this population became less sensitive to penicillin in this period. A 402-bp DNA fragment in the 3' end of penA was sequenced for the 296 nonsusceptible meningococcal strains isolated between 2000 and 2010 to examine the genetic diversity and evolution of their penA gene. In conclusion, the data obtained in our study support the statement that the position of penicillin G as a first choice in the treatment of invasive meningococcal diseases in Belgium should be reexamined. Despite an important number of isolates displaying a reduced susceptibility to penicillin, at present the expanded-spectrum cephalosporins, such as ceftriaxone, are not affected. The follow-up of the evolutionary changes in antimicrobial resistance has also proved to be essential for the recommendation of an appropriate antimicrobial treatment for invasive meningococcal diseases.

Detection of a geographical and endemic cluster of hyper-invasive meningococcal strains.

From 2006 to December 2009, 45 out of the 513 strains isolated from patients with invasive meningococcal disease in Belgium, were identified as Neisseria meningitidis serogroup B, non-serotypeable, subtype P1.14 (B:NT:P1.14). Most cases were geographically clustered in the northern part of the country. Multilocus Sequence Typing and antigen gene sequencing combined with Pulsed-Field Gel electrophoresis were used to investigate this cluster. Molecular typing showed that 39 out of these 45 N. meningitidis strains belonged to the clonal complex cc-269. The presence of the same PorA Variable Regions (VR1-VR2: 22, 14), the FetA allele (F5-1) and the highly similar Pulsed-Field Gel Electrophoresis profiles, supported genetic relatedness for 38 out of these 39 isolates. Retrospective analysis of B:NT:P1.22,14 isolates from 1999 onwards suggested that these strains belonging to the cc-269 complex, first emerged in the Belgian province of West-Flanders in 2004. This study showed that the combination of molecular tools with classical methods enabled reliable outbreak detection as well as a cluster identification.

Multicenter study for defining the breakpoint for rifampin resistance in Neisseria meningitidis by rpoB sequencing.

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 > or = 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 < or = 1 mg/liter. Rifampin-susceptible isolates could be defined as those with MICs of < or = 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.

Target gene sequencing to characterize the penicillin G susceptibility of Neisseria meningitidis.

Clinical isolates of Neisseria meningitidis with reduced susceptibility to penicillin G (intermediate isolates, Pen(I)) 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.