The physiological and ecological properties of bacterial persisters discovered from municipal sewage sludge and the potential risk

Bacterial persisters are a special microbial population and are considered to be the bacterial reservoir of antibiotic-resistant bacteria. They can survive antibiotic treatment even in high concentrations of antibiotics and revive in the appropriate conditions. However, the characteristics of bacterial persisters in the municipal sewage sludge and their potential environmental risks have not yet been paid much attention to. In this study, bacterial persisters were discovered from the sludge of wastewater treatment plants in four different regions (Jilin, Lhasa, Shenzhen, and Yili), and the metagenomic analysis confirmed that bacterial persisters were ubiquitous in all four municipal sewage sludge and positively related to the protobacterium populations. At the taxonomic genus level, a total of 57 genera of bacterial persisters were shared by the four sewage sludge, and the genera with abundance exceeding 2% were Acinetobacter, Lysinibacillus, Aeromonas, Brevundimonas, Pseudomonas, and Alcaligenes, among which Acinetobacter accounted for 57.24%. Genus Lysinibacillus and Aeromonas were significant in Jilin and Lhasa, respectively. The persistence mechanism of bacterial persisters derived from sludge was also clarified, among which, Aeromonas, Brevundimonas, and Alcaligenes rely on the hipBA toxin-antitoxin system, while Acinetobacter enters the persistence state mainly through the stringent response system based on (p)ppGpp. Moreover, it was found that a typical bacterial persister originated from Acinetobacter, named T9-9, could tolerate a variety of antibiotics, such as 1000 μg/mL of kanamycin, 160 μg/mL of tetracycline, and 30 μg/mL of ciprofloxacin. Even if the ultraviolet intensity was 6-36 times the usual dosage of ultraviolet disinfection in wastewater treatment plants, it could not completely kill T9-9, but the killing efficiency by chlorine disinfection technology could reach 100%. This study pointed out an environmental risk of bacterial persisters that existed in sewage sludge that had been neglected and strongly recommended to improve the disinfection process in the wastewater treatment plant.