Modulation of Membrane Influx and Efflux in Escherichia coli Sequence Type 131 Has an Impact on Bacterial Motility, Biofilm Formation, and Virulence in a Caenorhabditis elegans Model

Abstract

Energy-dependent efflux overexpression and altered outer membrane permeability (influx) can promote multidrug resistance (MDR). The present study clarifies the regulatory pathways that control membrane permeability in the pandemic clone Escherichia coli sequence type 131 (ST131) and evaluates the impact of efflux and influx modulations on biofilm formation, motility, and virulence in the Caenorhabditis elegans model. Mutants of two uropathogenic E. coli (UPEC) strains, MECB5 (ST131; H30-Rx) and CFT073 (ST73), as well as a fecal strain, S250 (ST131; H22), were in vitro selected using continuous subculture in subinhibitory concentrations of ertapenem (ETP), chloramphenicol (CMP), and cefoxitin (FOX). Mutations in genes known to control permeability were shown for the two UPEC strains: MECB5-FOX (deletion of 127 bp in marR; deletion of 1 bp and insertion of an IS1 element in acrR) and CFT073-CMP (a 1-bp deletion causing a premature stop in marR). We also demonstrated that efflux phenotypes in the mutants selected with CMP and FOX were related to the AcrAB-TolC pump, but also to other efflux systems. Alteration of membrane permeability, caused by underexpression of the two major porins, OmpF and OmpC, was shown in MECB5-ETP and mutants selected with FOX. Lastly, our findings suggest that efflux pump-overproducing isolates (CMP mutants) pose a serious threat in terms of virulence (significant reduction in worm median survival) and host colonization. Lack of porins (ETP and FOX mutants) led to a high level of antibiotic resistance in an H30-Rx subclone. Nevertheless, this adaptation created a physiological disadvantage (decreased motility and ability to form biofilm) associated with a low potential for virulence. Uropathogenic Escherichia coli (UPEC) is the leading causative agent of urinary tract infection (UTI) in both communities and hospitals worldwide (1). The therapeutic management of UTI is particularly problematic because of the increasingly widespread resistance to all classes of antibiotics, including in communityacquired infections (2). With the dissemination of the novel CTX-M family, E. coli has become the species most frequently associated with the production of extended-spectrum ␤-lactamase (ESBL) (3). In 2008, a globally disseminated and multidrugresistant (MDR) clone of E. coli O25b:H4-ST131 was identified in clinical samples from three continents (4). Since this first description, sequence type 131 (ST131) was shown to be a successful clone, causing the majority of MDR E. coli infections while exhibiting multiple virulence factors (5, 6). Moreover, it was described as one of the most prevalent clones among phylogenetic group B2 in the digestive carriage of healthy subjects in France (7). Within the ST131 group, various subclonal lineages have been identified. Indeed, a dominant subclone named H30-Rx has extensively expanded. This subgroup harbors allele 30 of fimH, the type 1 fimbrial adhesin gene, and is associated with both bla CTX-M-15 carriage and fluoroquinolone (FQ) resistance by specific topoi...

The ST131 Escherichia coli H22 subclone from human intestinal microbiota: Comparison of genomic and phenotypic traits with those of the globally successful H30 subclone

“…Comparison of the genomes of H 22 strain S250, H 30-Rx strain JJ1886, and H 41 strain SE15 showed high similarity in terms of the number and types of VF genes from the VFDB. This may explain the similar level of virulence that we found previously between H 22 strain S250 and an H 30-Rx strain in the C. elegans model [19, 42]. Nevertheless, strains S250 and JJ1886 displayed two different virotypes: D for strain S250 and C for strain JJ1886.…”

“…Biofilm formation was significantly delayed in the H 30-R strains. This is the first study to compare the formation of biofilms by H 22 and non-ESBL-producing H 30-R strains [4, 42, 44–46]. Further studies are required to verify the involvement of the fimB gene in the two different biofilm phenotypes.…”

Success of Escherichia coli O25b:H4 ST131 clade C associated with a decrease in virulence

“…As previously described, our clade B strains were more frequently early biofilm producers than clade C strains (23, 24, 42). Knocking out the fimB gene in clade B strains resulted in loss of both early biofilm production capacity and early type 1 fimbriae expression.…”

Biofilm formation by ESBL-producing strains of Escherichia coli and Klebsiella pneumoniae

“…In another study, the prevalence of biofilm production was significantly higher among ST131 than non-ST131 isolates regardless ESBL-production and antibiotic resistance (Kudinha et al, 2013). More refined analysis from Pantel et al has demonstrated largely diverse biofilm activity in E. coli ST131 clones (Pantel et al, 2016), as substantiated by others (Olesen et al, 2014;Shin and Ko., 2015). In this study, we also observed a strong relationship between ST131 strains and two virulence factors, fyuA and papGII, both of which, when absent, are significantly associated with biofilm production.…”