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.

Abstract

BackgroundThere 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.MethodsEighty-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.ResultsOf 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 blaSHV, blaTEM, blaKPC, blaIMP, blaVIM, and blaGES were detected in 44.31%, 22.72%, 13.63%, 14.7%, 5.6%, and 2.27% of P. aeruginosa isolates. Nonetheless, for blaKPC and blaGES 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 blaSHV and blaTEM 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 blaVIM and blaIMP, respectively.ConclusionThe 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.