Rocío Enríquez

Target Gene Sequencing To Characterize the Penicillin G Susceptibility of Neisseria meningitidis

ABSTRACT 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 PenI 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.

Multicenter Study for Defining the Breakpoint for Rifampin Resistance in Neisseria meningitidis by rpoB Sequencing

ABSTRACT 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.

Sequencing of Neisseria meningitidis penA Gene: the Key to Success in Defining Penicillin G Breakpoints

ABSTRACT Testing of susceptibility to penicillin G by E-test and sequencing of an internal fragment of the penA gene were done for 43 meningococcal strains. Those strains for which the MIC was ≥0.094 μg/ml showed mosaic alleles, so 0.094 μg/ml is suggested as the penicillin G intermediate breakpoint when E-test is used.

Sequencing of the porB gene: a step toward a true characterization of Neisseria meningitidis.

ABSTRACT Variations in class 2/3 (PorB) proteins form the basis for meningococcal serotyping. Antibodies against these proteins are bactericidal, making serotyping results useful not only for epidemiological surveillance of meningococcal disease but also for identifying potential vaccine components. A total of 20 to 60% of meningococcal B and C isolates from any given population are nontypeable (NT) using a panel of monoclonal antibodies. To analyze the mechanisms responsible for the nonserotypeability characteristic in Neisseria meningitidis, we (i) established the nucleotide sequences of porB gene in 146 meningococcal strains (95 not recognized by the serotyping panel), (ii) identified 18 new allelic variants of the porB gene, (iii) correlated allelic variants with serotypes, (iv) suggest the nontypeability characteristic in those 95 NT strains, and (v) reject the possibility of variation in the levels of PorB expression.

Interlaboratory Comparison of PCR-Based Methods for Detection of Penicillin G Susceptibility in Neisseria meningitidis

ABSTRACT We carried out a study for the nonculture detection of susceptibility of Neisseria meningitis to penicillin G in three laboratories of the European Monitoring Group on Meningococci (EMGM). Thirteen clinical samples (cerebrospinal fluids) and corresponding bacterial isolates from 13 cases of invasive meningococcal infection were distributed to the three laboratories. The MICs of penicillin G were determined for the isolates. Each laboratory used an “in-house” PCR-based method to determine alterations to the penA gene, which is associated with a reduced susceptibility to penicillin G. Nucleotide sequences from the 3′ end of the penA gene were also determined. We observed a good correlation between genotyping of penA and the phenotypic determination (MIC) of susceptibility to penicillin G. The results obtained by the three methods for penA in the samples correlated very well with those obtained in bacterial isolates and with sequence data. The kappa coefficient that was used to estimate the level of agreement between genotypic results varied between 0.65 and 1, indicating a good agreement. This suggests that genotyping can predict susceptibility of N. meningitidis to penicillin G. These data strongly suggest that genotyping of penA should be used to determine meningococcal susceptibility to penicillin G in culture-negative cases. Although the nucleotide sequence of penA may be the gold standard in genotyping of penA, the less expensive PCR-based approach reported in this study may be quicker when a large number of isolates and clinical samples need to be tested.

Target gene sequencing to define the susceptibility of Neisseria meningitidis to ciprofloxacin

ABSTRACT 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 μg/ml but not among isolates with MICs of ≤0.032 μg/ml. Experimental infection of ciprofloxacin-treated mice showed slower bacterial clearance when GyrA was altered. These data suggest a MIC of ≥0.064 μg/ml as the ciprofloxacin breakpoint for meningococci and argue for the molecular detection of ciprofloxacin resistance.

Deletion of the Correia element in the mtr gene complex of Neisseria meningitidis

The mtr gene complex in Neisseria meningitidis encodes an efflux pump that is responsible for export of antibacterial hydrophobic agents. The promoter region of the mtrCDE operon harbours an insertion sequence known as a Correia element, and a binding site for the integration host factor (IHF) is present at the centre of the Correia element. It has been suggested that the expression of the mtrCDE operon in meningococci is subject to transcriptional regulation by the IHF and post-transcriptional regulation by cleavage in the inverted repeat of the Correia element. The promoter region of the mtrCDE operon as well as the association of changes at that point with decreased susceptibility to antimicrobial drugs in 606 Neisseria meningitidis strains were analysed in this study. Two different deletions were present in the analysed region. The first one, found in seven strains, corresponded to absence of the Correia element. The second one, affecting the -10 region and first 100 bp of the mtrR gene and present in 57 isolates, was only found in ST-1624 isolates. None of the deletions were associated with decreased susceptibility to antimicrobial drugs. Although most of the meningococcal strains carry the Correia element at that position, its deletion is not an exception.

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.