Supplemental Material for Phillips et al., 2026

The rapid emergence of antimicrobial resistance in bacterial pathogens threatens the efficacy of nearly all available antibiotics. One evolution-informed strategy to limit the emergence of resistance is the exploitation of collateral sensitivity, whereby resistance to one compound results in increased sensitivity to another. Here, we investigate the collateral sensitivity relationship between the cephalosporin antibiotic ceftazidime and the natural product borrelidin A in Escherichia coli. Previously, we evolved replicate populations of E. coli under four drug regimens: ceftazidime alone, ceftazidime with norfloxacin, and ceftazidime with either 32 or 128µM borrelidin A, and found that borrelidin A prevented the evolution of clinical ceftazidime resistance. Here, we isolated 36 evolved strains, nine per treatment, measuring their resistances and susceptibilities to additional β-lactam and quinolone antibiotics, their relative fitness, and performed whole-genome sequencing. Co-dosing with 128µM borrelidin A significantly reduced the evolution of ceftazidime resistance, while preserving fitness in the absence of drug. Overall co-dosed strains had reduced resistance and cross-resistance to all tested antibiotics. Whole-genome sequencing revealed that co-dosing suppressed the accumulation of mutations in known resistance-associated genes; however, in some cases, selected for a possible novel borrelidin A resistance mutation. Our results show that ceftazidime-borrelidin A co-dosing limits both resistance and cross-resistance through selection against costly resistance mutations. While borrelidin A itself is cytotoxic, our findings highlight the promise of targeting bacteria-specific vulnerabilities to curb the emergence of multidrug resistance. These findings contribute to the growing body of evidence supporting collateral sensitivity-informed approaches as practical strategies to mitigate antimicrobial resistance in bacterial populations.细菌病原体的抗菌素耐药性（antimicrobial resistance）快速兴起，几乎威胁到所有现有抗生素的临床疗效。限制耐药性产生的一种基于进化原理的策略是利用附带敏感性（collateral sensitivity）——即对一种化合物产生耐药性后，会对另一种化合物的敏感性升高。本研究以大肠杆菌（Escherichia coli）为模型，探究头孢菌素类（cephalosporin）抗生素头孢他啶（ceftazidime）与天然产物波雷菌素A（borrelidin A）之间的附带敏感性关联。此前我们通过四种给药方案对大肠杆菌进行传代进化：单独使用头孢他啶、头孢他啶联合诺氟沙星（norfloxacin），以及头孢他啶分别联合32或128微摩尔（µM）的波雷菌素A，并发现波雷菌素A可阻止临床相关头孢他啶耐药性的进化。本研究共分离得到36株进化菌株，每个给药组各9株。我们测定了这些菌株对其他β-内酰胺类（β-lactam）与喹诺酮类（quinolone）抗生素的耐药性与敏感性，检测了它们的相对适合度（relative fitness），并完成了全基因组测序（whole-genome sequencing）。联合使用128µM波雷菌素A可显著降低头孢他啶耐药性的进化速率，且在无药物环境下保留菌株的适合度。整体而言，联合给药组菌株对所有受试抗生素的耐药性与交叉耐药性（cross-resistance）均有所降低。全基因组测序结果显示，联合给药抑制了已知耐药相关基因的突变积累；但在部分菌株中，却筛选出了可能的新型波雷菌素A耐药突变。本研究结果表明，头孢他啶与波雷菌素A联合给药可通过筛选代价高昂的耐药突变，同时限制耐药性与交叉耐药性的产生。尽管波雷菌素A本身具有细胞毒性（cytotoxic），但本研究结果凸显了靶向细菌特异性脆弱位点以遏制细菌群体多重耐药性（multidrug resistance）兴起的潜力。本研究结果进一步丰富了相关证据，支持基于附带敏感性的策略可作为缓解细菌抗菌素耐药性的实用方案。