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.多重耐药细菌的出现对人类健康构成重大威胁，亟需全面阐明其潜在耐药机制。尿路致病性大肠埃希菌（Uropathogenic Escherichia coli, UPEC）作为尿路感染的主要致病菌，常与多重耐药及复发感染密切相关。为阐明UPEC对β-内酰胺类抗生素（beta-lactam antibiotics）的耐药机制，本研究通过在实验室中持续暴露于低、高浓度氨苄青霉素，构建了氨苄青霉素耐药UPEC菌株，分别命名为低浓度氨苄青霉素耐药株（Low Amp^R）与高浓度氨苄青霉素耐药株（High Amp^R）。全基因组测序结果显示，Low Amp^R与High Amp^R菌株均携带marR、acrR及envZ基因的突变；其中High Amp^R菌株额外存在nlpD基因的单点突变。本研究通过蛋白质建模与实时荧光定量PCR（qRT-PCR）分析，验证了上述各基因突变对氨苄青霉素耐药株耐药性的贡献，包括膜通透性降低、多药外排泵表达上调以及细胞裂解受抑制。进一步的体内（in vivo）实验表明，即使经过持续抗生素治疗，氨苄青霉素耐药株仍不会降低小鼠膀胱内的细菌载量，这意味着该耐药株引发的宿主感染治疗难度进一步提升。值得注意的是，氨苄青霉素诱导产生的突变同样可赋予UPEC多重耐药表型，提示细菌存在获得其他类别抗生素交叉耐药性的通用机制。