Prediction of Decreased Susceptibility to Penicillin of Neisseria meningitidis Strains by Real-Time PCR

Abstract

Sequence analysis of the penA gene, encoding penicillin-binding protein 2 (PBP2), in 30 penicillin-intermediate (PenI) Neisseria meningitidis strains showed altered gene sequences due to the translocation of exogenous DNA blocks derived from commensal neisseriae, which are known to have PBP2 proteins with decreased affinity for the antibiotic. In order to obtain a rapid and reproducible method for predicting the PenI phenotype, a real-time PCR assay was set up with primers and probes designed on the basis of the penA gene. The A3G mutation at codon 566, in the transpeptidase domain of the penA gene (which is present in the whole sample of 30 PenI strains and in all the 41 sequences of PenI meningococci isolated worldwide and has been deposited in the sequence databank), was chosen as a marker of penA translocations. Two hybridization probes were designed to distinguish the wild-type penA gene in penicillin-susceptible (PenS) meningococci from the mutated penA gene at codon 566 in PenI strains. Thermal analysis of probe hybridization revealed a melting temperature difference of at least 6°C between PenI and PenS strains. This real-time PCR protocol characterizes the penicillin phenotype of N. meningitidis in a few hours without DNA sequencing and is useful for rapid screening of the penicillin-intermediate genotype among meningococcal isolates.

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

“…Laboratory 2 (L2) used real-time PCR and oligonucleotide thermal analysis. Primers, fluorescent resonance energy transfer probes, and PCR parameters were as previously described (22), and primers and probes are listed in Table 1. The real-time PCR mixture contained either 1 l of purified chromosomal DNA (100 ng) extracted from the cultured isolates (22) or 10 l of boiled CSF added directly to the mixture with no additional purification step.…”

“…The first method amplifies the 3Ј part of the penA gene and then uses restriction fragment length polymorphism analysis to reveal alterations to penA and thus predict decreased susceptibility to penicillin G (2). The second method uses a real-time PCR assay to detect one of the alterations to the penA gene, which has been chosen as a marker of penA modifications: a different melting temperature between Pen I and Pen S isolates (22). The third method uses a differential PCR, in which oligonucleotides were designed to obtain a positive PCR only when penA is not altered, thus indicating an isolate susceptible to penicillin G. These methods were applied to meningococcal isolates for the rapid screening of the penicillin-intermediate genotype (2,22,24).…”

“…The second method uses a real-time PCR assay to detect one of the alterations to the penA gene, which has been chosen as a marker of penA modifications: a different melting temperature between Pen I and Pen S isolates (22). The third method uses a differential PCR, in which oligonucleotides were designed to obtain a positive PCR only when penA is not altered, thus indicating an isolate susceptible to penicillin G. These methods were applied to meningococcal isolates for the rapid screening of the penicillin-intermediate genotype (2,22,24). Moreover, these methods can be directly applied to clinical samples, such as cerebrospinal fluid and blood, and allow a rapid and easily interpretable detection of Pen I isolates without requiring cul-turing.…”

Use of Applied Biosystems 7900HT Sequence Detection System and Taqman Assay for Detection of Quinolone-Resistant Neisseria gonorrhoeae

“…With widespread use of nucleic acid amplification tests (NAATs), antimicrobial resistance detection will require molecular methods, as has been described for other organisms (1,7,14,(16)(17)(18)25). QRNG detection is a good model for this approach since the resistance mechanisms are based on stepwise accumulation of point mutations correlating with increased MICs (20,22,23).…”

A Novel Real-Time Duplex PCR Assay for Detecting penA and ponA Genotypes in Neisseria gonorrhoeae: Comparison with Phenotypes Determined by the E-Test

“…Recent advances in PCR have made it possible to use clinical samples to characterize both the pathogenic agent and its susceptibility to antimicrobial drugs (7)(8)(9)(10). A recently validated real-time PCR assay (10 ) has been used for rapid detection of a ponA variation associated with a decreased susceptibility to penicillin.…”