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Application of pbp1A PCR in identification of penicillin-resistant Streptococcus pneumoniae
M du Plessis1, A M Smith, K P Klugman
1Pneumococcal Diseases Research Unit of MRC, SAIMR, WITS, Department of Clinical Microbiology and Infectious Diseases, South African Institute for Medical Research, Johannesburg 2000, South Africa. mignondp@hotmail.com
Abstract:
A seminested PCR assay, based on the amplification of the pneumococcal pbp1A gene, was developed for the detection of penicillin resistance in clinical isolates of Streptococcus pneumoniae. The assay was able to differentiate between intermediate (MICs = 0.25 to 0.5 microgram/ml) and higher-level (MICs = >/=1 microgram/ml) resistance. Two species-specific primers, 1A-1 and 1A-2, which amplified a 1,043-bp region of the pbp1A penicillin-binding region, were used for pneumococcal detection. Two resistance primers, 1A-R1 and 1A-R2, were designed to bind to altered areas of the pbp1A gene which, together with the downstream primer 1A-2, amplify DNA from isolates with penicillin MICs of >/=0.25 and >/=1 microgram/ml, respectively. A total of 183 clinical isolates were tested with the pbp1A assay. For 98.3% (180 of 183) of these isolates, the PCR results obtained were in agreement with the MIC data. The positive and negative predictive values of the assay were 100 and 91%, respectively, for detecting strains for which the MICs were >/=0.25 microgram/ml and were both 100% for strains for which the MICs were >/=1 microgram/ml.
Insights
A novel seminested PCR assay effectively detects penicillin resistance in Streptococcus pneumoniae by targeting the pbp1A gene. This method accurately identifies intermediate and high-level resistance in clinical isolates, aiding in antimicrobial stewardship.
Area of Science:
- Microbiology
- Molecular Biology
- Clinical Diagnostics
Background:
- Penicillin resistance in Streptococcus pneumoniae is a growing public health concern.
- Accurate and rapid detection of resistance is crucial for effective treatment and infection control.
- Existing methods for detecting penicillin resistance may require time-consuming culture-based techniques.
Purpose of the Study:
- To develop and validate a seminested PCR assay for the detection of penicillin resistance in clinical isolates of Streptococcus pneumoniae.
- To differentiate between intermediate and high-level penicillin resistance based on specific genetic markers.
- To assess the performance of the PCR assay against established antimicrobial susceptibility testing (MIC) data.
Main Methods:
- Development of a seminested PCR assay targeting the pneumococcal pbp1A gene.
- Design of species-specific primers (1A-1, 1A-2) and resistance primers (1A-R1, 1A-R2).
- Testing of 183 clinical isolates of Streptococcus pneumoniae using the developed PCR assay and determining Minimum Inhibitory Concentrations (MICs).
Main Results:
- The PCR assay demonstrated high agreement with MIC data, with 98.3% concordance for 183 clinical isolates.
- The assay achieved 100% positive and negative predictive values for detecting high-level penicillin resistance (MICs ≥1 µg/ml).
- For intermediate resistance (MICs = 0.25 to 0.5 µg/ml), positive and negative predictive values were 100% and 91%, respectively.
Conclusions:
- The developed seminested PCR assay is a reliable and accurate tool for detecting penicillin resistance in Streptococcus pneumoniae.
- The assay can effectively distinguish between different levels of penicillin resistance, aiding in clinical decision-making.
- This molecular method offers a rapid alternative to traditional susceptibility testing for identifying penicillin-resistant pneumococcal strains.