The First Egyptian Report Showing the Co-Existence of bla _NDM-25, bla _OXA-23, bla _OXA-181, and bla _GES-1 Among Carbapenem-Resistant K. pneumoniae Clinical Isolates Genotyped by BOX-PCR

Abstract

Background and Objective

The emergence of carbapenem-resistant K. pneumoniae (CRKP) continues to escalate and is alarming because of the emergence of pan drug-resistant strains. The objective of this study was to investigate the existence of 12 carbapenemase genes among CRKP clinical isolates.

Methods

Ninety-six Klebsiella spp. clinical isolates were collected. The isolates were identified phenotypically and genotypically. These isolates were screened for susceptibility to 24 different antibiotics. The modified Hodge test (MHT) and the Carba Nordmann/Poirel (NP) test were used to phenotypically screen carbapenem-resistant strains for carbapenemase production. Phenotypic characterization of carbapenemases was performed using the combined disk synergy test (CDST). Additionally, the presence of 12 carbapenemase genes in CRKP isolates was investigated. The DNA sequence of bla _NDM and bla _GES genes was determined. The BOX-PCR technique was used to determine the clonal relationship between CRKP isolates.

Results

All carbapenem-resistant isolates were related to K. pneumoniae. Susceptibility testing showed that 19.79% (19/96) of the collected isolates were carbapenem-resistant. Of the CRKP isolates, 68.42% (13/19) tested positive for the MHT and Carba NP test. CDST showed that 42.11% (8/19), 63.16% (12/19), 47.37% (9/19), and 73.68% (14/19) of the CRKP isolates tested positive for the inhibitory effect of clavulanic acid, sulbactam, phenylboronic acid, and tazobactam, respectively, while 84.21% (16/19) and 68.42% (13/16) tested positive for the inhibitory effect of EDTA and mercaptopropionic acid, respectively. It was found that 10.53% (2/19) of the isolates tested positive for the inhibitory effect of sodium chloride. Molecular investigation of carbapenemases showed that 26.32% (5/19), 73.68% (14/19), 21.05% (4/19), 10.53% (2/19), and 5.26% (1/19) of the isolates tested positive for bla _NDM, bla _OXA-48, bla _OXA-181, bla _OXA-51, and bla _OXA-23, respectively. None of the isolates tested positive for bla _OXA-40 and bla _OXA-58. Two allelic variants of bla _NDM ( bla _NDM-1 and bla _NDM-25) were detected. BOX-PCR revealed high clonal relatedness between CRKP isolates.

Conclusion

MHT was more sensitive than Carba NP test for evaluating carbapenemase production and class D carbapenemase genes were the most prevalent of the 12 carbapenemase genes that were evaluated.