The <i>cnf1</i> gene is associated with an expanding <i>Escherichia coli</i> ST131 <i>H</i>30Rx/C2 subclade and confers a competitive advantage for gut colonization.

Epidemiological projections point to acquisition of ever-expanding multidrug resistance (MDR) by <i>Escherichia coli</i>, a commensal of the digestive tract and a source of urinary tract pathogens. Bioinformatics analyses of a large collection of <i>E. coli</i> genomes from EnteroBase, enriched in clinical isolates of worldwide origins, suggest the Cytotoxic Necrotizing Factor 1 (CNF1)-toxin encoding gene, <i>cnf1</i>, is preferentially distributed in four common sequence types (ST) encompassing the pandemic <i>E. coli</i> MDR lineage ST131. This lineage is responsible for a majority of extraintestinal infections that escape first-line antibiotic treatment, with known enhanced capacities to colonize the gastrointestinal tract. Statistical projections based on this dataset point to a global expansion of <i>cnf1</i>-positive multidrug-resistant ST131 strains from subclade <i>H</i>30Rx/C2, accounting for a rising prevalence of <i>cnf1</i>-positive strains in ST131. Despite the absence of phylogeographical signals, <i>cnf1</i>-positive isolates segregated into clusters in the ST131-<i>H</i>30Rx/C2 phylogeny, sharing a similar profile of virulence factors and the same <i>cnf1</i> allele. The suggested dominant expansion of <i>cnf1</i>-positive strains in ST131-<i>H</i>30Rx/C2 led us to uncover the competitive advantage conferred by <i>cnf1</i> for gut colonization to the clinical strain EC131GY ST131-<i>H</i>30Rx/C2 versus <i>cnf1</i>-deleted isogenic strain. Complementation experiments showed that colon tissue invasion was compromised in the absence of deamidase activity on Rho GTPases by CNF1. Hence, gut colonization factor function of <i>cnf1</i> was confirmed for another clinical strain ST131-<i>H</i>30Rx/C2. In addition, functional analysis of the <i>cnf1</i>-positive clinical strain EC131GY ST131-<i>H</i>30Rx/C2 and a <i>cnf1</i>-deleted isogenic strain showed no detectable impact of the CNF1 gene on bacterial fitness and inflammation during the acute phase of bladder monoinfection. Together these data argue for an absence of role of CNF1 in virulence during UTI, while enhancing gut colonization capacities of ST131-<i>H</i>30Rx/C2 and suggested expansion of <i>cnf1</i>-positive MDR isolates in subclade ST131-<i>H</i>30Rx/C2.