A shared mechanism of multidrug resistance in laboratory-evolved uropathogenic <i>Escherichia coli</i>

The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms. Uropathogenic <i>Escherichia coli</i> (UPEC), the primary causative agent of urinary tract infections, is frequently associated with multidrug resistance and recurrent infections. To elucidate the mechanism of resistance of UPEC to beta-lactam antibiotics, we generated ampicillin-resistant UPEC strains through continuous exposure to low and high levels of ampicillin in the laboratory, referred to as Low Amp^R and High Amp^R, respectively. Whole-genome sequencing revealed that both Low and High Amp^R strains contained mutations in the <i>marR</i>, <i>acrR</i>, and <i>envZ</i> genes. The High Amp^R strain exhibited a single additional mutation in the <i>nlpD</i> gene. Using protein modeling and qRT-PCR analyses, we validated the contributions of each mutation in the identified genes to antibiotic resistance in the Amp^R strains, including a decrease in membrane permeability, increased expression of multidrug efflux pump, and inhibition of cell lysis. Furthermore, the Amp^R strain does not decrease the bacterial burden in the mouse bladder even after continuous antibiotic treatment <i>in vivo</i>, implicating the increasing difficulty in treating host infections caused by the Amp^R strain. Interestingly, ampicillin-induced mutations also result in multidrug resistance in UPEC, suggesting a common mechanism by which bacteria acquire cross-resistance to other classes of antibiotics.