Since the first report of the plasmid-mediated, colistin-resistant gene, mcr-1, nine mcr genes and their subvariants have been identified. The spreading scope of mcr-1~10 varies greatly, suggesting that mcr-1~10 may have different evolutionary advantages. Depending on MCR family phylogeny, mcr-6 is highly similar to mcr-1 and -2, and mcr-7~10 are highly similar to mcr-3 and -4. We compared the expression effects of MCR-1~5 on bacteria of common physiological background. The MCR-1-expressing strain showed better growth than did MCR-2~5-expressing strains in the presence of colistin. LIVE/DEAD staining analysis revealed that MCR-3~5 expression exerted more severe fitness burdens on bacteria than did MCR-1 and -2. Bacteria expressing MCRs except MCR-2 showed enhanced virulence with increased epithelial penetration ability determined by trans-well model (p < 0.05). Enhanced virulence was also observed in the Galleria mellonella model, which may have resulted from bacterial membrane damage and different levels of lipopolysaccharide (LPS) release due to MCR expression. Collectively, MCR-1-expressing strain showed the best survival advantage of MCR-1~5-expressing strains, which may partly explain the worldwide distribution of mcr-1. Our results suggested that MCR expression may cause increased bacterial virulence, which is alarming, and further attention will be needed to focus on the control of infectious diseases caused by mcr-carrying pathogens.

Carbapenem-resistant pathogens mediated by metallo-beta-lactamases (MBLs) have spread worldwide, where NDM-1 is a typical and key MBL. Here, we firstly discussed the distribution characterization of NDM-1, which produces multidrug-resistant Proteus mirabilis among broilers in China. From January to April 2019, 40 (18.1%, 40/221) bla_NDM-1-carrying P. mirabilis strains were recovered from commercial broilers in slaughterhouse B in China. All the isolates were resistant to imipenem, meropenem and other β-lactams. These isolates belong to five clusters identified via pulsed field gel electrophoresis (PFGE). Further studies on twenty representative strains revealed that seven bla_NDM-1 genes were located on plasmids with sizes of 104.5–138.9 kb. Notably, only three strains (PB72, PB96 and PB109) were successfully transferred to Escherichia coli J53, while the other four isolates were located in nontransferable plasmids. The rest were harbored in chromosomes. Ulteriorly, based on whole genome sequencing (WGS), these twenty isolates showed four typical phylogenetic clades according to single nucleotide polymorphisms (SNPs) of a core genome and presented four main genomic backbone profiles, in which type II/III strains shared a similar genetic context. All of the above is evidence of bla_NDM-1 transmission and evolution in P. mirabilis, suggesting that the prevalence may be more diverse in broiler farms. Accordingly, as intestinal and environmental symbiotic pathogens, bla_NDM-1-positive P. mirabilis will pose greater threats to the environment and public health.

Avian pathogenic Escherichia coli (APEC) poses a global threat to poultry health and public safety due to its high lethality, limited treatment options, and potential for zoonotic transmission via the food chain. However, long-term genomic surveillance remains limited, especially in countries like China where poultry farming is highly intensive. This study aimed to characterize the population structure, virulence traits, and antimicrobial resistance of 81 APEC isolates from diseased chickens collected over 16 years from Shandong and neighboring provinces in eastern China. The isolates were grouped into seven Clermont phylogroups, with A and B1 being dominant. MLST revealed 27 STs, and serotyping identified 29 O and 16 H antigens, showing high genetic diversity. The minor phylogroups (B2, C, D, E, G) encoded more virulence genes and had higher virulence-plasmid ColV carriage, with enrichment for iron-uptake, protectins, and extraintestinal toxins. In contrast, the dominant phylogroups A and B1 primarily carried adhesin and enterotoxin genes. Antimicrobial resistance was widespread: 76.5% of isolates were multidrug-resistant. The minor phylogroups exhibited higher tetracycline resistance (mediated by tet(A)), whereas the major phylogroups showed increased resistance to third- and fourth-generation cephalosporins (due to bla_CTX-M-type ESBL genes). These findings offer crucial data for APEC prevention and control, safeguarding the poultry industry and public health.

The New Delhi metallo-β-lactamase-1 (NDM-1) is a typical carbapenemase and plays a crucial role in antibiotic-resistance bacterial infection. Phylogenetic analysis, performed on known NDM-variants, classified NDM enzymes in seven clusters. Three of them include a major number of NDM-variants. In this study, we evaluated the role of the V88L substitution in NDM-24 by kinetical and structural analysis. Functional results showed that V88L did not significantly increase the resistance level in the NDM-24 transformant toward penicillins, cephalosporins, meropenem, and imipenem. Concerning ertapenem, E. coli DH5α/NDM-24 showed a MIC value 4-fold higher than that of E. coli DH5α/NDM-1. The determination of the k_cat, K_m, and k_cat/K_m values for NDM-24, compared with NDM-1 and NDM-5, demonstrated an increase of the substrate hydrolysis compared to all the β-lactams tested, except penicillins. The thermostability testing revealed that V88L generated a destabilized effect on NDM-24. The V88L substitution occurred in the β-strand and low β-sheet content in the secondary structure, as evidenced by the CD analysis data. In conclusion, the V88L substitution increases the enzyme activity and decreases the protein stability. This study characterizes the role of the V88L substitution in NDM-24 and provides insight about the NDM variants evolution.