Abstract
Bacterial strains resistant to various antibiotic drugs are frequently encountered in clinical infections, and the rapid identification of drug-resistant strains is highly essential for clinical treatment. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR (LNA-qPCR) method for the rapid detection of 13 antibiotic resistance genes and successfully used it to distinguish drug-resistant bacterial strains from positive blood culture samples. A sequence-specific primer-probe set was designed, and the specificity of the assays was assessed using 27 ATCC bacterial strains and 77 negative blood culture samples. No cross-reaction was identified among bacterial strains and in negative samples, indicating 100% specificity. The sensitivity of the assays was determined by spiking each bacterial strain into negative blood samples, and the detection limit was 1–10 colony forming units (CFU) per reaction. The LNA-qPCR assays were first applied to 72 clinical bacterial isolates for the identification of known drug resistance genes, and the results were verified by the direct sequencing of PCR products. Finally, the LNA-qPCR assays were used for the detection in 47 positive blood culture samples, 19 of which (40.4%) were positive for antibiotic resistance genes, showing 91.5% consistency with phenotypic susceptibility results. In conclusion, LNA-qPCR is a reliable method for the rapid detection of bacterial antibiotic resistance genes and can be used as a supplement to phenotypic susceptibility testing for the early detection of antimicrobial resistance to allow the selection of appropriate antimicrobial treatment and to prevent the spread of resistant isolates.
Highlights
The spread of drug-resistant bacterial strains has become a great threat to public health [1]
When the bacterial concentration was diluted to 1 colony forming units (CFU) per reaction, 8 assays showed positive signals
The rapid identification of drug resistance genes directly from positive blood cultures is critical for the selection of appropriate antibiotics for the treatment of nosocomial infections, especially sepsis
Summary
The spread of drug-resistant bacterial strains has become a great threat to public health [1]. This method is based on bacterial cultivation, which requires 1–3 days for a susceptibility report and is labor—intensive and time consuming Many molecular methods, such as multiplex PCR, real-time PCR or microarray assay, have emerged recently for the detection of antimicrobial resistance genes, including mecA [12], vanA or vanB [13,14,15], ESBLs or pAmpC [16,17,18,19,20,21,22], and carbapenemase genes such as blaKPC, blaNDM-1, blaVIM or blaIMP [23,24,25,26,27]. We developed a locked nucleic acid (LNA)-based quantitative real-time PCR assay (LNA-qPCR) [29] for the rapid detection of thirteen antibiotic resistance genes that confer drug resistance in most of the common clinical bacterial strains
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