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

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_SHV, bla_TEM, bla_KPC, bla_IMP, bla_VIM, and bla_GES were detected in 44.31%, 22.72%, 13.63%, 14.7%, 5.6%, and 2.27% of P. aeruginosa isolates. Nonetheless, for bla_KPC and bla_GES 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_SHV and bla_TEM 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_VIM and bla_IMP, 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

Acknowledgments

The authors of this article are grateful to Hamadan University of Medical Sciences for their financial support in conducting the research.

Abbreviations

PPV, positive predictive values; TEM, temoneira; VEB, Verona integron-encoded metallo-β-lactamase; OXA, oxacillin hydrolyzing capabilities; MBL, metallo-β-lactamase; bla_GES, Guiana extended-spectrum; bla_IMP, imipenem; bla_KPC, Klebsiella pneumoniae carbapenemase; PER, Pseudomonas extended resistant; ESBL, extended-spectrum beta-lactamase; PSE, Pseudomonas-specific enzymes; HRMA, high-Resolution Melt.

Data Sharing Statement

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Ethics Approval and Consent to Participate

This study was approved by the Ethics Committee of Hamadan University of Medical Sciences (CodeNo: IR.UMSHA.REC.1398.573).

Consent for Publication

All the authors agree to publish the manuscript in Infection and Drug Resistance journal.

Author Contributions

HT and SD performed microbiological and molecular tests and wrote the manuscript. MYA and FK play a role in Project Administration. MA supervised all of the stages of designing the study, conducting the research, and writing the manuscript. All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that they have no competing interests.