Detection of Extended-spectrum β-lactamase-producing Escherichia coli isolates by isothermal amplification and association of their virulence genes and phylogroups with extraintestinal infection

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: traT (67.4%) > ompT (52.6%) > iutA (50.5%) > fimH (47.4%) > iha (33.7%) > hlyA (26.3%) > papC (12.6%) > cvaC (3.2%), in ESBL-ExPEC isolates which belonged to phylogroups A (28.4%), B2 (28.4%), and F (22.1%). The distribution of traT, ompT, and hlyA 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.

Figure 1

Optimization of the RPA assay. Agarose gel electrophoresis shows the bla_CTX-M, bla_OXA, and bla_SHV amplicons (A) when 0.2 µM CTX-M, 0.1 µM OXA, 0.1 µM SHV and 0.2 µM CTX-M, 0.05 µM OXA and 0.05 µM SHV were used; (B) at 37–41 °C; (C) at 10–30 min incubation; and (D) at different DNA template concentrations (1.5–25 ng) [M, 100 bp Marker; C+, positive DNA control; C−, no template control].

Figure 2

Optimization of the tHDA assay. Agarose gel electrophoresis showed the bla_TEM amplicons (A) at 59–67 °C; (B) at 15–90 min; (C) at different DNA template concentrations (0.05–100 ng); and (D) at different primers concentrations (0.025–0.075 µM) [M, 100 bp Marker; C+, positive DNA control; C−, no template control].

Figure 3

(A–C) LODs for bla_CTX-M, bla_OXA, and bla_SHV detection by RPA assay; (D) LODs for bla_TEM detection by tHDA; (E) Specificity of the RPA assay for bla_CTX-M, bla_OXA, and bla_SHV genes; and (F) Specificity of the tHDA assay for bla_TEM gene [M, 100 bp Marker; C+, positive DNA control; C−, no template control].