School of Basic Medical Sciences, Peking University, Beijing, China.
Department of Bacteriology, Capital Institute of Pediatrics, Beijing, China.
Appl Environ Microbiol. 2024 Jul 24;90(7):e0055724. doi: 10.1128/aem.00557-24. Epub 2024 Jul 2.
can enter a viable but nonculturable (VBNC) state to survive in unfavorable environments. Our research found that high-, medium-, and low-alcohol-producing strains are associated with nonalcoholic fatty liver disease. However, the presence of the three strains has not been reported in the VBNC state or during resuscitation. In this study, the effects of different strains, salt concentrations, oxygen concentrations, temperatures, and nutrients in VBNC state were evaluated. The results showed that high-alcohol-producing induced a slower VBNC state than medium-alcohol-producing , and low-alcohol-producing . A high-salt concentration and micro-oxygen environment accelerated the loss of culturability. Simultaneously, both real-time quantitative PCR and droplet digital PCR were developed to compare the quantitative comparison of three strain VBNC states by counting single-copy gene numbers. At 22°C or 37°C, the number of culturable cells decreased significantly from about 10 to 10-10 CFU/mL. In addition, imipenem, ciprofloxacin, polymyxin, and phiW14 inhibited cell resuscitation but could not kill VBNC-state cells. These results revealed that the different environments evaluated play different roles in the VBNC induction process, and new effective strategies for eliminating VBNC-state cells need to be further studied. These findings provide a better understanding of VBNC-state occurrence, maintenance, detection, and absolute quantification, as well as metabolic studies of resuscitation resistance and ethanol production.IMPORTANCEBacteria may enter VBNC state under different harsh environments. Pathogenic VBNC bacteria cells in clinical and environmental samples pose a potential threat to public health because cells cannot be found by routine culture. The alcohol-producing VBNC state was not reported, and the influencing factors were unknown. The formation and recovery of VBNC state is a complete bacterial escape process. We evaluated the influence of multiple induction conditions on the formation of VBNC state and recovery from antibiotic and bacteriophage inhibition, and established a sensitive molecular method to enumerate the VBNC cells single-copy gene. The method can improve the sensitivity of pathogen detection in clinical, food, and environmental contamination monitoring, and outbreak warning. The study of the formation and recovery of VBNC-state cells under different stress environments will also promote the microbiological research on the development, adaptation, and resuscitation in VBNC-state ecology.
可以进入存活但不可培养(VBNC)状态以在不利环境中生存。我们的研究发现,高、中、低产酒精菌株与非酒精性脂肪性肝病有关。然而,这三种菌株在 VBNC 状态或复苏过程中尚未被报道。在这项研究中,评估了不同菌株、盐浓度、氧浓度、温度和 VBNC 状态下的营养物质对产酒精菌株的影响。结果表明,高醇产菌株比中醇产菌株和低醇产菌株进入 VBNC 状态的速度较慢。高盐浓度和微氧环境加速了可培养性的丧失。同时,通过计数单拷贝基因数量,开发了实时定量 PCR 和液滴数字 PCR,以比较三种产酒精菌株 VBNC 状态的定量比较。在 22°C 或 37°C 下,可培养细胞的数量从约 10 减少到 10-10 CFU/mL,显著减少。此外,亚胺培南、环丙沙星、多粘菌素和 phiW14 抑制细胞复苏,但不能杀死 VBNC 状态的细胞。这些结果表明,评估的不同环境在 VBNC 诱导过程中发挥不同的作用,需要进一步研究消除 VBNC 状态细胞的新有效策略。这些发现为更好地了解 VBNC 状态的发生、维持、检测和绝对定量以及复苏抗性和乙醇生产的代谢研究提供了依据。
重要性
细菌可能在不同的恶劣环境下进入 VBNC 状态。临床和环境样本中致病性 VBNC 细菌细胞对公共健康构成潜在威胁,因为常规培养无法发现细胞。产酒精的 VBNC 状态尚未报道,其影响因素尚不清楚。VBNC 状态的形成和恢复是一个完整的细菌逃逸过程。我们评估了多种诱导条件对 VBNC 状态形成和从抗生素和噬菌体抑制中恢复的影响,并建立了一种敏感的分子方法来计数 VBNC 细胞的单拷贝基因。该方法可以提高临床、食品和环境污染监测中病原体检测的灵敏度,并发出爆发预警。在不同应激环境下研究 VBNC 状态细胞的形成和恢复也将促进 VBNC 状态生态学中发展、适应和复苏的微生物研究。