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Interlaboratory comparison of PCR-based methods for detection of penicillin G susceptibility in Neisseria
Muhamed-Kheir Taha1, Maria Leticia Zarantonelli, Arianna Neri
1Neisseria Unit and French National Reference Center for Meningococci, Institut Pasteur, 28 Rue du Dr Roux, Paris, France. mktaha@pasteur.fr
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.
Insights
Genotyping the penA gene accurately predicts Neisseria meningitidis susceptibility to penicillin G, even in culture-negative samples. This PCR-based method offers a rapid, reliable alternative for antimicrobial susceptibility testing.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- Neisseria meningitidis is a significant cause of bacterial meningitis.
- Accurate antimicrobial susceptibility testing is crucial for effective treatment.
- Penicillin G remains a key antibiotic, but resistance is a concern.
Purpose of the Study:
- To evaluate a nonculture method for detecting penicillin G susceptibility in Neisseria meningitidis.
- To assess the correlation between penA gene genotyping and phenotypic susceptibility testing.
- To determine the utility of PCR-based penA genotyping for culture-negative samples.
Main Methods:
- Multi-laboratory study involving 13 clinical cerebrospinal fluid samples and isolates.
- Determination of penicillin G Minimum Inhibitory Concentrations (MICs) for isolates.
- In-house PCR-based genotyping of the penA gene and nucleotide sequencing.
Main Results:
- Good correlation observed between penA genotyping and phenotypic penicillin G susceptibility (MICs).
- PCR results for penA showed high agreement (kappa coefficient 0.65-1) with isolate data and sequence analysis.
- Genotyping successfully predicted antimicrobial susceptibility in both isolates and clinical samples.
Conclusions:
- PenA gene genotyping is a reliable method for predicting Neisseria meningitidis susceptibility to penicillin G.
- PCR-based genotyping offers a rapid and cost-effective approach for large-scale testing.
- Genotyping is particularly valuable for determining susceptibility in culture-negative cases, guiding antimicrobial therapy.