Emergence of multidrug-resistant blaNDM-positive Providencia hangzhouensis from layer hens: horizontal gene transfer and virulence level

Abstract With the increasing prevalence of carbapenemase-producing bacteria, the importance of the genus Providencia has become increasingly prominent. Providencia hangzhouensis has recently been recognized as an emerging opportunistic pathogen, particularly associated with urinary tract infections, but rarely reported in poultry. We first identified and characterized a bla NDM-1 -positive P. hangzhouensis strain, designated SHZ3, isolated from laying hen feces using MacConkey agar supplemented with 2 mg/L imipenem. Whole-genome sequencing analysis was performed using Illumina and Nanopore platforms. Antimicrobial susceptibility testing was determined by broth microdilution method, virulence gene identification via the Virulence Factor database, and functional pathogenic evaluation using the Galleria mellonella infection model. The results showed that the genome size of SHZ3 strain is approximately 4.7 Mb, and bla NDM-1 is located on the plasmid pNDM-SHZ3, which possesses a unique additional composite transposon (IS 110 - sul 2 -aph (3'') -Ib-aph (6) -Id-floR -IS 6 -IS 110 ), a structure that is the first reported in P. hangzhouensis . pNDM-SHZ3 carries 17 resistance genes, including bla NDM-1 and bla OXA-10 , corresponding to multidrug-resistant phenotype, including carbapenems. Additionally, SHZ3 strain was identified as harboring 49 genes encoding proteins associated with pathogenesis and exhibited time- and dose-dependent deaths with a median lethal dose of 4.84 log 10 colony-forming units (CFU)/mL. This study is the first to report a strain of multidrug-resistant and virulent bla NDM-1 -positive P. hangzhouensis from laying hen feces. These findings emphasize the potential for zoonotic transmission and public health threat posed by P. hangzhouensis , underscoring the necessity for established surveillance mechanisms for this emerging pathogen.