Distribution of Class B and Class A β-Lactamases in Clinical Strains of Pseudomonas aeruginosa : Comparison of Phenotypic Methods and High-Resolution Melting Analysis (HRMA) Assay

摘要

Background: There are various phenotypic methods for identifying class B and class A β-lactamase enzymes in Pseudomonas aeruginosa . The purpose of this study was to compare the sensitivity and specificity of different phenotypic methods with HRMA assay to detect β-lactamase-producing P. aeruginosa strains. Methods: Eighty-eight of P. aeruginosa isolates were collected from different specimens. Conventional double-disk test (DDT) and EDTA-imipenem microbiological (EIM) were performed to detect ESBL and MBL-producing strains, respectively. Meanwhile, the Modified Hodge test and Carba-NP test were performed on all carbapenem-resistant strains. HRMA method and sensitivity and specificity of primers were determined based on the melt curve temperature range. In all comparisons, PCR was considered as the gold standard. Results: Of the 402 isolates collected from different clinical specimens, 88 isolates of P. aeruginosa were identified. However, 43 strains were (48.88%) ESBL-producing, and 7 strains (7.95%) were MBL-producing. Also, using the Modified Hodge test and Carba-NP method, 11 (12.5%) and 19 (21.59%) strains were carbapenemase-producing, respectively. The results of the HRMA test revealed that genes coding for bla subSHV/sub, bla subTEM/sub, bla subKPC/sub, bla subIMP/sub, bla subVIM,/sub and bla subGES/sub were detected in 44.31%, 22.72%, 13.63%, 14.7%, 5.6%, and 2.27% of P. aeruginosa isolates. Nonetheless, for bla subKPC/sub and bla subGES/sub genes, sensitivity and specificity of the Carba-NP test were 90.47%, 94.87%, and 83.36%, 94.80%, respectively. However, sensitivity and specificity of MHT was 91.66%, 98.70%, and 77.77%, 96.42%, respectively. For bla subSHV/sub and bla subTEM/sub genes, sensitivity and specificity of DDT were 95.55%, 95.55%, and 86%, 83.50%, respectively. However, sensitivity and specificity of EMI were 77.77%, 97.59%, and 91.66%, 97.43% for bla subVIM/sub and bla subIMP/sub, respectively. Conclusion: The HRMA is a powerful, accurate, closed-tube, rapid method for detecting β-lactamase genes in P. aeruginosa . The high sensitivity and specificity of this method, along with phenotypic tests, play a useful role in increasing the predictive value of clinical reports.