Biofilms are surface-associated communities of bacteria that grow in a self-produced matrix of polysaccharides, proteins, and extracellular DNA (eDNA). Sub-minimal inhibitory concentrations (sub-MIC) of antibiotics induce biofilm formation, potentially as a defensive response to antibiotic stress. However, the mechanisms behind sub-MIC antibiotic-induced biofilm formation are unclear. We show that treatment of Pseudomonas aeruginosa with multiple classes of sub-MIC antibiotics with distinct targets induces biofilm formation. Further, addition of exogenous eDNA or cell lysate failed to increase biofilm formation to the same extent as antibiotics, suggesting that the release of cellular contents by antibiotic-driven bacteriolysis is insufficient. Using a genetic screen for stimulation-deficient mutants, we identified the outer membrane porin OprF and the ECF sigma factor SigX as important. Similarly, loss of OmpA – the Escherichia coli OprF homolog – prevented sub-MIC antibiotic stimulation of E. coli biofilms. Our screen also identified the periplasmic disulfide bond-forming enzyme DsbA and a predicted cyclic-di-GMP phosphodiesterase encoded by PA2200 as essential for biofilm stimulation. The phosphodiesterase activity of PA2200 is likely controlled by a disulfide bond in its regulatory domain, and folding of OprF is influenced by disulfide bond formation, connecting the mutant phenotypes. Addition of reducing agent dithiothreitol prevented sub-MIC antibiotic biofilm stimulation. Finally, activation of a c-di-GMP-responsive promoter follows treatment with sub-MIC antibiotics in the wild-type but not an oprF mutant. Together, these results show that antibiotic-induced biofilm formation is likely driven by a signaling pathway that translates changes in periplasmic redox state into elevated biofilm formation through increases in c-di-GMP.

生物膜是与表面相关的细菌群落，在自产的多糖、蛋白质和细胞外 DNA (eDNA) 基质中生长。抗生素的次最小抑菌浓度 (sub-MIC) 会诱导生物膜形成，可能是对抗生素应激的防御反应。然而，亚 MIC 抗生素诱导生物膜形成的机制尚不清楚。我们发现，用多种具有不同靶标的亚 MIC 抗生素治疗铜绿假单胞菌可诱导生物膜形成。此外，添加外源 eDNA 或细胞裂解液未能将生物膜形成增加到与抗生素相同的程度，这表明抗生素驱动的溶菌作用释放的细胞内容物不足。通过对刺激缺陷突变体进行遗传筛选，我们发现外膜孔蛋白 OprF 和 ECF 西格玛因子 SigX 很重要。同样，OmpA（大肠杆菌OprF 同源物）的缺失可以防止大肠杆菌生物膜的亚 MIC 抗生素刺激。我们的筛选还确定了周质二硫键形成酶 DsbA 和 PA2200 编码的预测的环二 GMP 磷酸二酯酶对于生物膜刺激至关重要。 PA2200 的磷酸二酯酶活性可能受其调节域中的二硫键控制，OprF 的折叠受二硫键形成的影响，连接突变表型。添加还原剂二硫苏糖醇可防止亚 MIC 抗生素生物膜刺激。最后，在野生型中用亚 MIC 抗生素处理后，c-di-GMP 响应启动子被激活，但oprF突变体则不然。总之，这些结果表明，抗生素诱导的生物膜形成可能是由信号通路驱动的，该信号通路通过 c-di-GMP 的增加将周质氧化还原状态的变化转化为生物膜形成的增加。