Survival characteristics of vancomycin-resistant Enterococcus isolated from sludge compost under heat and drying stress: Implications for pathogen inactivation during composting

The limitation of traditional culturing methods often obscure signals from certain bacterial subpopulations, hindering accurate identification of microbial heterogeneity. Understanding the heterogeneity of these different subpopulations is crucial for optimizing microbial control strategies in various applications. Sludge composting, a primary method for resource recovery from sludge, plays a vital role in reducing contaminants such as pathogens, with enterococci regarded as a superior indicator organism for pathogen inactivation efficacy. Our prior research revealed that heat and moisture content during composting primarily impact the culturable and non-culturable Enterococcus subpopulations, respectively. This study is the first to systematically investigate the survival mechanisms of a vancomycin-resistant Enterococcus (VRE) strain, isolated from sludge compost, under simulated heat and drying stress to evaluate the response of distinct subpopulations. Using dual staining with propidium iodide (PI) and carboxyfluorescein diacetate (CFDA), coupled with flow cytometry, distinct VRE subpopulations were effectively distinguished. The results revealed that among the three stressed subpopulations - the injured, dormant, and metabolically active but non-culturable (ABNC) subpopulation - the dormant subpopulation exhibited superior resistance to adverse conditions. Heat stress effectively reduced culturable Enterococcus, while decreasing moisture content to below 11% notably reduced the active cells. Prolonged low-moisture periods further decreased the proportion of dormant subpopulations. These findings highlight the potential for enhancing pathogen inactivation by optimizing composting parameters. Extending the thermophilic phase can effectively reduce culturable subpopulations, whereas maintaining low moisture levels during the cooling phase can facilitate dormant subpopulation inactivation. This study provides valuable insights for improving sludge composting practices and advancing public health safety.