The global spread of colistin or carbapenem-resistant Enterobacteriaceae (CRE) has been a pressing threat to public health. Members of Enterobacteriaceae, especially Proteus mirabilis and Escherichia coli, have been prevalent foodborne pathogens and such pathogens from fresh vegetables have triggered foodborne illness in China. However, reports about CRE, especially P. mirabilis from fresh vegetables, are still lacking. In this study, we identified five bla_NDM-positive P. mirabilis and five bla_NDM-positive generic E. coli concurrently from five fresh vegetables in two markets from China, and four of the five E. coli also carried mcr-1. The 10 isolates were characterized with methods including antimicrobial susceptibility testing, conjugation, whole-genome sequencing and phylogenetic analysis. All 10 isolates were multidrug-resistant (MDR). bla_NDM-5 in five E. coli isolates and one P. mirabilis carrying bla_NDM-5 was located on similarly transferable IncX3 plasmids, while transferably untypable plasmids were the carriers of bla_NDM-1 in four P. mirabilis isolates from different types of vegetables/markets. mcr-1 in the four bla_NDM-₅-positive E. coli was located on similarly non-conjugative IncHI2 MDR plasmids lacking transfer region. Notably, ISCR1 complex class 1 integron capable of capturing bla_NDM-1 was found on all untypable plasmids from P. mirabilis, and five copies of ISCR1 complex class 1 integron containing bla_NDM-1 even occurred in one P. mirabilis, which showed high-level carbapenem resistance. Plasmid and phylogenetic analysis revealed that the bla_NDM-positive P. mirabilis and E. coli from fresh vegetables might be derived from animals and transmitted to humans via the food chain. The concurrence of bla_NDM-positive P. mirabilis and E. coli carrying both mcr-1 and bla_NDM in different types of fresh vegetables eaten raw is alarming and threatens food safety. Sustained surveillance of these foodborne pathogens among fresh vegetables is urgent to ensure the health of food consumers. We report for the first time the concurrence of bla_NDM-positive P. mirabilis and mcr-1-bearing E. coli carrying bla_NDM from the same fresh vegetables.

Salmonella are widely distributed foodborne pathogens and are often associated with food animal products. Colistin resistance mediated by mcr-1 is an increasing threat; however, data on the characteristics of mcr-1-harboring Salmonella among retail foods are still lacking. In this study, retail meats from 24 supermarkets and eggs from nine markets in Qingdao city were investigated to determine the presence and genomic characteristics of mcr-1-harboring Salmonella. We found the retail meats and eggs were highly contaminated by Salmonella, with detection rates of 17.5% (31/177) and 12.3% (16/130), respectively. A total of 76 Salmonella isolates were obtained in this study, and 77.6% showed multidrug resistance (MDR). The MDR proportion of egg isolates (97.5%) was significantly higher than that in meat isolates (55.6%) (p < 0.05). The most prevalent Salmonella serotypes were Typhimurium (56.6%) and Enteritidis (17.1%). Of the 76 Salmonella isolates, 40 possessed mcr-1. All 40 mcr-1-positive isolates were ST34 S. Typhimurium and were from eggs of eight brands. Different mcr-1-harboring isolates existed in the same egg, and some isolates from different egg samples or brands showed clonal relationships. The mcr-1 was located on similar IncHI2/HI2A MDR non-conjugative plasmids lacking transfer region, resulting in the failure of conjugation. The phylogenetic tree using genome sequences showed that the mcr-1-positive isolates from eggs clustered together with mcr-1-positive isolates from chicken and humans in China, revealing that mcr-1-positive egg-borne Salmonella might be derived from chicken and could potentially trigger outbreaks in humans. The high occurrence of mcr-1-harboring Salmonella in fresh eggs is alarming, and there is an urgent need to monitor mcr-1-harboring Salmonella in retail meats and eggs. We report for the first time the role of retail eggs in disseminating mcr-1-positive Salmonella and the risk of transmission of these MDR pathogens from retail food to humans should be evaluated comprehensively.

The high pathogenicity rate of carbapenem-resistant Pseudomonas aeruginosa (CRPA) has resulted in substantial economic losses for humans and the breeding industry. Consequently, there is an urgent need to develop new alternatives to mitigate antibiotic use. Phage therapy has demonstrated promising results in numerous studies. In this study, lytic phages targeting CRPA were isolated from feces and river water samples in Shandong, China. A total of 94 phage strains with CRPA as hosts were obtained, exhibiting lysis rates that ranged from 29% to 76% for P. aeruginosa derived from humans and different types of animals (n = 246). We further examined five representative phages, the host bacteria of which were CRPA from clinical patients and poultry, and these phages included two myoviruses and three podoviruses. Their optimal multiplicities of infection (MOIs) ranged from 10⁻³ to 10⁻⁵, with latent periods of less than 5 to 15 min and burst durations of 140 to 175 min, resulting in burst sizes of 133 to 352 PFU/cell. All five phages exhibited the ability to survive at temperatures up to 60 °C and within pH levels of 3 to 11. Whole-genome sequencing revealed that these five phages were all double-stranded DNA phages and did not possess resistance genes or virulence factors. The two myoviruses, sharing similar sequences, were classified into the genus Pakpunavirus, with a size of 92,509 bp and 92,293 bp, 149 to 152 ORFs and 20 to 22 tRNAs. In contrast, the three similar podoviruses belong to the genus Phikmvvirus and all contained a perforin–lyase system, with a size of 43.35 kb, a GC content of 62%, 49 to 50 ORFs and 16 to 20 tRNAs. A spray disinfection experiment demonstrated that the phage cocktail exhibited a high sterilization effect after spraying and showed good efficacy against cement and metal surfaces. This study provides foundational information for further research into the elimination of CRPA in the environment.