Evolution of honey resistance in experimental populations of bacteria depends on the type of honey, and has no major side effects for antibiotic susceptibility

With rising antibiotic resistance, alternative treatments for communicable diseases are increasingly relevant. One possible alternative for some types of infections is honey, used in wound care since before 2000 BCE and more recently in licensed, medical-grade products. However, it is unclear whether medical application of honey results in the evolution of bacterial honey resistance, and whether this has collateral effects on other bacterial traits such as antibiotic resistance. Here, we used single-step screening assays and serial transfer at increasing concentrations to isolate honey-resistant mutants of Escherichia coli. We only detected bacteria with consistently increased resistance to the honey they evolved in with two of the four tested honey products, and the observed increases were small (maximum two-fold increase in IC90). Genomic sequencing and experiments with single-gene knockouts showed a key mechanism by which bacteria increased their honey resistance was by mutating genes involved in detoxifying methylglyoxal, which contributes to the antibacterial activity of Leptospermum honeys. Crucially, we found no evidence that honey adaptation conferred cross-resistance or collateral sensitivity against nine antibiotics from six different classes. These results reveal constraints on bacterial adaptation to different types of honey, improving our ability to predict downstream consequences of wider honey application in medicine.

随着抗生素耐药性问题日益严峻，传染病的替代治疗方案愈发受到关注。蜂蜜作为部分感染性疾病的潜在替代疗法，早在公元前2000年就已应用于伤口护理，如今更被开发为获批上市的医用级产品。然而目前尚不清楚，蜂蜜的临床应用是否会诱导细菌产生蜂蜜耐药性的进化，以及这一过程是否会对细菌的其他性状（如抗生素耐药性）产生附带影响。本研究采用单步筛选实验法，并结合递增蜂蜜浓度下的连续传代培养策略，从大肠杆菌（Escherichia coli）中分离出耐蜂蜜突变菌株。我们仅在四种受试蜂蜜产品中的两种中，检测到对其所适应的蜂蜜耐药性持续升高的细菌，且耐药性提升幅度较小（90%抑菌浓度（IC90）最大增幅仅为2倍）。基因组测序与单基因敲除实验表明，细菌提升蜂蜜耐药性的关键机制之一，是通过突变参与降解甲基乙二醛的基因——甲基乙二醛是细子木蜂蜜（Leptospermum honeys）抗菌活性的重要贡献因子。至关重要的是，我们未发现任何证据表明，蜂蜜适应性进化会使细菌对6大类共9种抗生素产生交叉耐药性或附带敏感性。本研究结果揭示了细菌对不同类型蜂蜜产生适应性进化的限制条件，有助于我们更精准地预测蜂蜜在医学领域更广泛应用的下游效应。