The risk of viable but non-culturable (VBNC) enterococci and antibiotic resistance transmission during simulated municipal sludge composting.

Sludge composting is a sludge resource utilization method that can reduce pollutants, such as pathogens. Enterococci are regarded as more reliable and conservative indicators of pathogen inactivation than fecal coliforms, which are typically used as indicators of fecal pollution. Non-spore pathogenic bacteria may enter a viable but non-culturable (VBNC) state during composting, leading to residual risk. The VBNC status of bacteria is related to their survival during composting. However, the survival mechanisms of enterococci during sludge composting remain unclear. Therefore, this study aimed to investigate the VBNC state of enterococci in different phases of simulated sludge composting and the fate of antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) during the composting process. This study is expected to provide a basis for subsequent exploration of possible methods to completely inactivate enterococci and reduce ARGs during sludge composting. Culturable enterococci were reduced in the thermophilic phase of sludge composting, but the proportion of VBNC subpopulation increased. It was reported for the first time that most VBNC enterococci were killed by extending the cooling phase of sludge compost, and by prolonging the cooling phase the types of ARG were reduced. However, there was a certain quantity (approximately 104/g dry weight) of culturable and VBNC enterococci in the compost products. In addition, MGEs and ARGs exist in both bacteria and compost products, leading to the risk of spreading antibiotic-resistant bacteria and antibiotic resistance when sludge compost products are used.

Neglected Drivers of Antibiotic Resistance: Survival of Extended-Spectrum ß-Lactamase-Producing Pathogenic Escherichia coli from Livestock Waste through Dormancy and Release of Transformable Extracellular Antibiotic Resistance Genes under Heat Treatment.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae has caused a global pandemic with high prevalence in livestock and poultry, which could disseminate into the environment and humans. To curb this risk, heat-based harmless treatment of livestock waste was carried out. However, some risks of the bacterial persistence have not been thoroughly assessed. This study demonstrated that antibiotic-resistant bacteria (ARB) could survive at 55 °C through dormancy, and simultaneously transformable extracellular antibiotic resistance genes (eARGs) would be released. The ESBL-producing pathogenic Escherichia coli CM1 from chicken manure could enter a dormant state at 55 °C and reactivate at 37 °C. Dormant CM1 had stronger ß-lactam resistance, which was associated with high expression of ß-lactamase genes and low expression of outer membrane porin genes. Resuscitated CM1 maintained its virulence expression and multidrug resistance and even had stronger cephalosporin resistance, which might be due to the ultra-low expression of the porin genes. Besides, heat at 55 °C promoted the release of eARGs, some of which possessed a certain nuclease stability and heat persistence, and even maintained their transformability to an Acinetobacter baylyi strain. Therefore, dormant multidrug-resistant pathogens from livestock waste will still pose a direct health risk to humans, while the resuscitation of dormant ARB and the transformation of released eARGs will jointly promote the proliferation of ARGs and the spread of antibiotic resistance.

Multidrug-resistant enteroaggregative Escherichia coli (EAEC) enters dormant state during heat treatment: A potential hazard in municipal sludge.

Reuse of sewage sludge is a general trend and land application is an essential way to reuse sludge. The outbreak of coronavirus disease has raised concerns about human pathogens and their serious threat to public health. The risk of pathogenic bacterial contamination from land application of municipal sludge has not been well assessed. The purpose of this study was to investigate the presence of pathogenic bacteria in municipal sewage sludge and to examine the survival potential of certain multidrug-resistant enteroaggregative Escherichia coli (EAEC) strain isolated from sewage sludge during heat treatment. The sewage sludge produced in the two wastewater treatment plants contained pathogenic bacteria such as pathogenic E. coli, Shigella flexneri, and Citrobacter freundii. The environmental strain of EAEC isolated from the sludge was resistant to eight types of antibiotics. It could also enter the dormant state after 4.5 h of treatment at 55 °C and regrow at 37 °C, while maintaining its antibiotic resistance. Our results indicate that the dormancy of EAEC might be why it is heat-resistant and could not be killed completely during the sludge heat treatment process. Owing to the regrowth of the dormant pathogenic bacteria, it is risky to apply the sludge to land even if the sludge is heat-treated, and there is also a risk of spreading antibiotic resistance.