Characterization of blaNDM-positive Enterobacteriaceae reveals the clonal dissemination of Enterobacter hormaechei coharboring blaNDM and tet(X4) along the pork production chain

The convergence of blaNDM and tet(X4) in a single strain has been sporadically detected in diverse pathogens from different sources, causing widespread concerns. However, the genomic characterization of blaNDM-positive or blaNDM- and tet(X4)-coharboring Enterobacteriaceae in the pork production chain, as well as how they emerged, remains mostly unclear. In this study, forty blaNDM-positive Enterobacteriaceae, including eleven tet(X4)- and blaNDM-coharboring isolates, were isolated from a pig slaughterhouse and a retail pork market in Yangzhou, China. This is the first report presenting the coexistence of blaNDM and tet(X4) in Enterobacter hormaechei, and it was demonstrated that they could spread clonally from the slaughterhouse to the retail market. Furthermore, we also discovered that the slaughterhouse served as a possible reservoir for blaNDM-positive ST48 E. coli strains. The blaNDM-bearing IncX3 plasmid was found in twenty blaNDM-positive strains as well as all blaNDM-5- and tet(X4)-coharboring strains, and most of them (30/31) were conjugative. In Klebsiella aerogenes NTT31XS, blaNDM was found on a nonconjugative ~17 kb IncX3 plasmid. When compared to the typical IncX3 plasmid, it deleted large-scale regions, which might imply a low fitness cost dissemination strategy for the IncX3 plasmid. Despite being from different species, tet(X4)-bearing plasmids carried by all blaNDM-5- and tet(X4)-coharboring strains were nonconjugative and had identical architectures. Phylogenetic analysis suggested that the formation of K. aerogenes NTT31XS may have been mediated by translocation of tet(X4)-positive circular intermediates and recombination events of regions in typical blaNDM-bearing IncX3 plasmids. Overall, this study expounded the prevalence and transmission characteristics of blaNDM-positive Enterobacteriaceae strains in the two links of pork production chain before they were exposed to humans, and expanded our knowledge of the molecular features and evolutionary history of tet(X4)- and blaNDM-5-coharboring strains. Strict control measures should be implemented to prevent the spread of such pathogens along food production chains.