In Vivo Transfer and Microevolution of Avian Native IncA/C ₂ bla _NDM-1 -Carrying Plasmid pRH-1238 during a Broiler Chicken Infection Study

Abstract

The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) in wildlife and livestock animals pose an important safety concern for public health. With our in vivo broiler chicken infection study, we investigated the transfer and experimental microevolution of the blaNDM-1-carrying IncA/C2 plasmid (pRH-1238) introduced by avian native Salmonella enterica subsp. enterica serovar Corvallis without inducing antibiotic selection pressure. We evaluated the dependency of the time point of inoculation on donor (S. Corvallis [12-SA01738]) and plasmid-free Salmonella recipient [d-tartrate-fermenting (d-Ta+) S. Paratyphi B (13-SA01617), referred to here as S. Paratyphi B (d-Ta+)] excretion by quantifying their excretion dynamics. Using plasmid profiling by S1 nuclease-restricted pulsed-field gel electrophoresis, we gained insight into the variability of the native plasmid content among S. Corvallis reisolates as well as plasmid acquisition in S. Paratyphi B (d-Ta+) and the enterobacterial gut microflora. Whole-genome sequencing enabled us to gain an in-depth insight into the microevolution of plasmid pRH-1238 in S. Corvallis and enterobacterial recipient isolates. Our study revealed that the fecal excretion of avian native carbapenemase-producing S. Corvallis is significantly higher than that of S. Paratyphi (d-Ta+) and is not hampered by S. Paratyphi (d-Ta+). Acquisition of pRH-1238 in other Enterobacteriaceae and several events of plasmid pRH-1238 transfer to different Escherichia coli sequence types and Klebsiella pneumoniae demonstrated an interspecies broad host range. Regardless of the microevolutionary structural deletions in pRH-1238, the single carbapenem resistance marker blaNDM-1 was maintained on pRH-1238 throughout the trial. Furthermore, we showed the importance of the gut E. coli population as a vector of pRH-1238. In a potential scenario of the introduction of NDM-1-producing S. Corvallis into a broiler flock, the pRH-1238 plasmid could persist and spread to a broad host range even in the absence of antibiotic pressure.

Complete Genome Sequence of an Avian Native NDM-1-Producing Salmonella enterica subsp. enterica Serovar Corvallis Strain

“…The pSE12-01738-2 plasmid is a derivative of the bla NDM-1 -carrying IncA/C 2 multiresistance plasmid pRH-1238 (GenBank accession number KR091911), previously described by Villa et al (7) but lacking a genetic element including genes for chromate and macrolide resistance as well as a class I integron carrying dfrA7-aadA5-sul1 resistance gene cassettes. This, along with previously observed structural deletions in pRH-1238 after an in vivo passage, indicates certain evolutionary plasticity, still enabling maintenance of the bla NDM-1 gene (8). …”

“…In recent in vivo infection studies, we demonstrated the persistence of this strain in broiler chickens and dissemination of its IncA/C 2 bla NDM-1 -carrying plasmid to different Enterobacteriaceae , both without antibiotic pressure (8). Such observations are noteworthy due to a possible scenario of bla NDM-1 introduction into commercial broiler production and downstream in the production chain, posing a subsequent risk for human exposure.…”

Occurrence of NDM-1-producing Morganella morganii and Proteus mirabilis in a single patient in Portugal: probable in vivo transfer by conjugation

“…plasmid within different enterobacterial species was recently reported in infected chickens, but also in hospitalized patients, with a single IncC plasmid (published as IncA/C 2 ) being identified in Citrobacter freundii, E. coli, K. pneumoniae and Klebsiella oxytoca. 16,17 NDM-1-producing P. mirabilis have been previously reported in different parts of the world, but usually the bla NDM-1 gene was identified within an SGI-type genomic island, hence at a chromosomal location. 18 Here, we showed it may also be found as plasmid located, on a broad host range IncC plasmid co-encoding b-lactamases CMY and TEM, in addition to the 16S rRNA methylase ArmA, as previously found.…”

Characterization of mcr-5 -Harboring Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Animal and Food Origin in Germany

“…Selected mcr-5carrying strains were used as a donor for filter mating conjugation studies using sodium azide-resistant E. coli K-12 J53 recipient cells. Filter mating conjugation was performed as previously described (33). The reaction mixtures were plated on transconjugant selective plates containing 1 mg/liter colistin sulfate and 100 mg/liter sodium azide (Sigma-Aldrich, Darmstadt, Germany) and incubated for approximately 42 h at 37°C.…”