Combined real-time PCR and pyrosequencing strategy for objective, sensitive, specific, and high-throughput identification of reduced susceptibility to penicillins in Neisseria meningitidis

Abstract

A segment of penA in Neisseria meningitidis strains (n = 127), including two nucleotide sites closely associated to reduced susceptibility to penicillins, was amplified and pyrosequenced. All results were in concordance with Sanger sequencing, and a high correlation between alterations in the two Pen(i)-specific sites and reduced susceptibility to penicillins was identified.

FIG. 1.

Multiple-sequence alignment of the 17 different partial penA sequences, which corresponds to nucleotides 1460 to 1600 of MC58 penA (17), that were identified among the N. meningitidis isolates included for development of the pyrosequencing strategy (n = 79). The positions of the used PCR primers (penA-F, 5′-GTGCGGTAGATGGTTTCGA-3′; penA-R, 5′-TTACCGCCACAATCACACG-3′) and pyrosequencing primers (penA-s1-3, 5′-GTAGCGATGTGTTTGT-3′; penA-s1-4, GTGGCAACGTGTTTGT) are shown. Accordingly, due to genetic heterogeneity different sequencing primers were used for penA wild-type alleles and penA mosaic alleles. The two boxes indicate the two Penⁱ-specific sites that are altered only in penA mosaic alleles and, accordingly, N. meningitidis isolates with reduced susceptibility to penicillins. WT, wild-type penA sequence; M, penA mosaic allele; B, primer biotinylated at the 5′ end.

FIG. 2.

MICs of penicillin G, ampicillin, penicillin V, and cefuroxime using Etest (20, 21) and presence of altered Penⁱ-specific sites in N. meningitidis isolates (n = 127). Three N. meningitidis serogroup Y isolates did not grow on Mueller-Hinton agar supplemented with 5% sheep blood, and, accordingly, these were analyzed on medium B (Swedish Reference Group for Antibiotics; http://www.srga.org; accessed 12 November 2007). Gray bars indicate wild-type penA genes, and black bars indicate the presence of altered Penⁱ-specific sites. The broken lines in panels a and b indicate the breakpoints suggested for isolates with reduced susceptibility (15, 18).