Detection of Extended-spectrum β-lactamase-producing Escherichia coli isolates by isothermal amplification and association of their virulence genes and phylogroups with extraintestinal infection
Naeem Ullah, Thadchaporn Assawakongkarat, Yukihiro Akeda, Nuntaree Chaichanawongsaroj
Abstract
Abstract Extraintestinal pathogenic Escherichia coli (ExPEC) producing extended-spectrum β-lactamases (ESBL) cause serious human infections due to their virulence and multidrug resistance (MDR) profiles. We characterized 144 ExPEC strains (collected from a tertiary cancer institute) in terms of antimicrobial susceptibility spectrum, ESBL variants, virulence factors (VF) patterns, and Clermont’s phylogroup classification. The developed multiplex recombinase polymerase amplification and thermophilic helicase-dependent amplification (tHDA) assays for bla CTX-M , bla OXA , bla SHV , and bla TEM detection, respectively, were validated using PCR-sequencing results. All ESBL-ExPEC isolates carried bla CTX-M genes with following prevalence frequency of variants: bla CTX-M-15 (50.5%) > bla CTX-M-55 (17.9%) > bla CTX-M-27 (16.8%) > bla CTX-M-14 (14.7%). The multiplex recombinase polymerase amplification assay had 100% sensitivity, and specificity for bla CTX-M , bla OXA , bla SHV , while tHDA had 86.89% sensitivity, and 100% specificity for bla TEM . The VF genes showed the following prevalence frequency: tra T (67.4%) > omp T (52.6%) > iut A (50.5%) > fim H (47.4%) > iha (33.7%) > hly A (26.3%) > pap C (12.6%) > cva C (3.2%), in ESBL-ExPEC isolates which belonged to phylogroups A (28.4%), B2 (28.4%), and F (22.1%). The distribution of tra T, omp T, and hly A and phylogroup B2 were significantly different ( P < 0.05) between ESBL-ExPEC and non-ESBL-ExPEC isolates. Thus, these equipment-free isothermal resistance gene amplification assays contribute to effective treatment and control of virulent ExPEC, especially antimicrobial resistance strains.