Department of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.
Balgrist University Hospital, University of Zurich, Zurich, Switzerland.
Clin Microbiol Infect. 2022 Jul;28(7):1022.e1-1022.e7. doi: 10.1016/j.cmi.2022.01.021. Epub 2022 Feb 3.
Difficult-to-treat infections caused by antibiotic-susceptible strains have been linked to the occurrence of persisters, a subpopulation of dormant bacteria that tolerate antibiotic exposure despite lacking genetic resistance. These persisters can be identified phenotypically by plating on nutrient agar because of their altered growth dynamics, resulting in colony-size heterogeneity. The occurrence of within-patient bacterial phenotypic heterogeneity in various infections and clinical determinants of persister formation remains unknown.
We plated bacteria derived from 132 patient samples of difficult-to-treat infections directly on nutrient-rich agar and monitored colony growth by time-lapse imaging. We retained 36 Staphylococcus aureus monocultures for further analysis. We investigated clinical factors associated with increased colony growth-delay with regression analyses. We corroborated the clinical findings using in vitro grown static biofilms exposed to distinct antibiotics.
The extent of phenotypic heterogeneity of patient-derived S. aureus varied substantially between patients (from no delay to a maximum of 57.6 hours). Increased heterogeneity coincided with increased median colony growth-delay. Multivariable regression showed that rifampicin treatment was significantly associated with increased median growth-delay (13.3 hours; 95% CI 7.13-19.6 hours; p < 0.001). S. aureus grown in biofilms and exposed to high concentrations of rifampicin or a combination of rifampicin with clindamycin or levofloxacin exhibited prolonged growth-delay (p < 0.05 for 11 of 12 comparisons), correlating with a strain-dependent increase in antibiotic tolerance.
Colony-size heterogeneity upon direct sampling of difficult-to-treat S. aureus infections was frequently observed. Hence, future studies are needed to assess the potential benefit of phenotypic heterogeneity quantification for staphylococcal infection prognosis and treatment guidelines.
抗生素敏感株引起的难治性感染与休眠菌（一种耐受抗生素暴露但缺乏遗传抗性的亚群）的产生有关。这些休眠菌可以通过在营养琼脂平板上进行表型鉴定，因为它们的生长动力学发生了改变，导致菌落大小异质性。在各种感染中，患者内细菌表型异质性的发生和休眠菌形成的临床决定因素仍不清楚。
我们直接将来自 132 名难治性感染患者样本的细菌接种在营养丰富的琼脂平板上，并通过延时成像监测菌落生长。我们保留了 36 株金黄色葡萄球菌单培养物进行进一步分析。我们通过回归分析研究了与菌落生长延迟增加相关的临床因素。我们使用暴露于不同抗生素的体外静态生物膜进一步验证了临床发现。
患者来源的金黄色葡萄球菌的表型异质性程度在患者之间差异很大（从无延迟到最大 57.6 小时）。异质性增加与菌落生长延迟增加一致。多变量回归显示，利福平治疗与中位生长延迟显著相关（13.3 小时；95%CI7.13-19.6 小时；p<0.001）。在生物膜中生长并暴露于高浓度利福平或利福平与克林霉素或左氧氟沙星联合的金黄色葡萄球菌表现出延长的生长延迟（12 次比较中的 11 次比较，p<0.05），这与抗生素耐药性的菌株依赖性增加相关。
我们经常观察到直接采样治疗困难的金黄色葡萄球菌感染时的菌落大小异质性。因此，需要进一步研究来评估表型异质性量化对葡萄球菌感染预后和治疗指南的潜在益处。
