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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

Authors Dehbashi S, Tahmasebi H, Alikhani MY, Keramat F, Arabestani MR

Received 30 March 2020

Accepted for publication 18 June 2020

Published 30 June 2020 Volume 2020:13 Pages 2037—2052


Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 3

Editor who approved publication: Professor Suresh Antony

Sanaz Dehbashi,1 Hamed Tahmasebi,2 Mohammad Yousef Alikhani,1 Fariba Keramat,3 Mohammad Reza Arabestani1,4

1Microbiology Department, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran; 2Microbiology Department, Faculty of Medicine, Zahedan University of Medical Sciences, Zahedan, Iran; 3Brucellosis Research Center, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran; 4Nutrition Health Research Center, Hamadan University of Medical Sciences, Hamadan, Iran

Correspondence: Mohammad Reza Arabestani
Department of Microbiology, Faculty of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran
Tel +989 188662009

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 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.
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.

Keywords: Pseudomonas aeruginosa, high-resolution melting curve analysis, HRMA, β-lactamases, drug resistance

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