Detection of Escherichia coli and Associated β-Lactamases Genes from Diabetic Foot Ulcers by Multiplex PCR and Molecular Modeling and Docking of SHV-1, TEM-1, and OXA-1 β-Lactamases with Clindamycin and Piperacillin-Tazobactam

Abstract

Diabetic foot ulcer (DFU) is a common and devastating complication in diabetes. Antimicrobial resistance mediated by extended-spectrum β-lactamases (ESBLs) production by bacteria is considered to be a major threat for foot amputation. The present study deals with the detection of Escherichia coli and the prevalence of bla TEM, bla SHV and bla OXA genes directly from biopsy and swab of foot ulcers of diabetic patients. In total, 116 DFU patients were screened, of which 42 suffering with severe DFUs were selected for this study. Altogether 16 E. coli strains were successfully isolated from biopsy and/or swab samples of 15 (35.71%) patients. ESBL production was noted in 12 (75%) strains. Amplification of β-lactamase genes by multiplex PCR showed the presence of bla CTX-M like genes in 10 strains, bla TEM and bla OXA in 9 strains each, and bla SHV in 8 of the total 16 strains of E. coli. Out of the ten antibiotics tested, E. coli strains were found to be resistant to ampicillin (75%), cefoxitin (56.25%), cefazolin (50%), meropenem (37.5%), cefoperazone (25%), cefepime (31.25%), ceftazidime (56.25%), and cefotaxime (68.75%) but all showed sensitivity (100%) to clindamycin and piperacillin-tazobactam. 3D models of the most prevalent variants of β-lactamases namely TEM-1, SHV-1, OXA-1, and ESBL namely CTX-M-15 were predicted and docking was performed with clindamycin and piperacillin-tazobactam to reveal the molecular basis of drug sensitivity. Docking showed the best docking score with significant interactions, forming hydrogen bond, Van der Waals and polar level interaction with active site residues. Findings of the present study may provide useful insights for the development of new antibiotic drugs and may also prevent ESBLs-mediated resistance problem in DFU. The novel multiplex PCR assay designed in this study may be routinely used in clinical diagnostics of E. coli and associated bla TEM, bla SHV, and bla OXA like genes.

Isolation and Genetic Analysis of Multidrug Resistant Bacteria from Diabetic Foot Ulcers

“…Resistance to β-lactam antibiotics in Gram-negative bacteria is primarily mediated by β-lactamases, which hydrolyze the β-lactam ring and thus inactivate the antibiotic (Livermore and Woodford, 2006 ). Different β-lactamases have been described, but bla TEM , bla SHV , bla OXA , and bla CTX−M like genes are the most predominant in Gram-negative bacteria (Livermore and Woodford, 2006 ; Ahmed et al, 2007 ; Shahi et al, 2013 ). Our findings are in agreement with above reports as β-lactamase genes conferring resistance to β-lactam antibiotics were noted in 35 out of 38 MDR isolates.…”

A study on the relation of the severity of diabetic foot ulcers with the type of bacterial flora isolated from the wounds

“…Detection of ESBL producing strains is of vital importance as they are responsible for the spread of resistance among different bacteria. 20,21 A combination of factors such as co-selection due to MDR phenotypes, virulence factors, mobile genetic elements, clonal spread of virulent strains and the acquisition of diverse ESBL-bearing plasmids may facilitate the spread of ESBL strains. ESBL producing bacteria are emerging as a worldwide clinical threat.…”

Molecular screening of bovine raw milk for the presence of Shiga toxin-producing Escherichia coli (STEC) on dairy farms

“…The development of PCR-based methods for the detection of pathogens or virulence factors (Shahi et al, 2013;Branquinho & Ferreira, 2012;Gandra et al, 2011) can be used for the initial screening of the presence of microorganisms in samples of milk and dairy products. Furthermore, the present study, through direct isolation of bacterial DNA in milk samples, aimed to develop the multiplex PCR to assess the prevalence of E. coli and STEC in samples of fresh milk.…”