Molecular Characterization Of Plasmid-encoded Extended Spectrum Beta-lactamases CTX-M-55and Carbapencmases NDM-1and IMP-1in Pathogenic Bacteria

Objectives: The wide use of β-lactam antibiotics has resulted in theemergence of resistant strains of Gram-negative pathogenic bacteria, especiallyincluding Enterobacteriaceae and non-fermenters. Horizontal gene transfer ofmobile genetic elements, such as conjugative plasmids, transposons and integrons,provides a significant contribution to bacterial genome innovation in terms ofdrug resistance. The purpose of this study was to characterize plasmid-encodingextended-spectrum beta-lactamase CTX-M-55in Shigella sonnei#1081,carbapenemase NDM-1in Enterobacter aerogenes3-SP and carbapenemaseIMP-1in Achromobacter xylosoxidans22732, which were multidrug resistanceisolates from Chinese hospitals.Methods: Species identification was carried out by VITEK2, BrukerMALDI Biotyper and16S ribosomal RNA gene sequencing. The presence ofESBLs and carbapenemases was detected by the double-disk synergy test and amodified Carba NP test, respectively. All the known carbapenemase and ESBLgenes were screened by PCR. Conjugal transfer experiments were carried out toanswer whether the drug resistance genes were harbored on conjugative plasmids.The minimum inhibitory concentration (MIC) values were determined for thewild-type and transconjugant strains to judge antimicrobial susceptibility. Thecomplete nucleotide sequences of drug resistance plasmids were determined andfurther compared with those of the genetically closest plasmids deposited inGenBank database. The promoters of drug resistance genes were defined on thebasis of transcription start sites as determined by primer extension assay.Results: Strains#1081,3-SP and A22732were identified as S. sonneiharboring blaCTX-M-55, E. aerogenes harboring blaNDM-1and A. xylosoxidansharboring blaIMP-1.All of the above β-lactamase genes were located on conjugativeplasmids. blaCTX-M-55was the only known antibiotic resistance gene and located in a transposition unit of ISEcp1-blaCTX-M-55-orf477on plasmid p1081-CTXM.Plasmid pHN1122-1, harbored in a faecal Escherichia strain recovered from a dogin China, was a close variant of p1081-CTXM indicating potential transfer ofCTX-M-55-harboring plasmids between nonpathogenic E. coli and pathogenic S.sonnei. blaNDM-1was located in a transposition unit with two insert sequences ofISAba125in plasmid p3SP-NDM. Plasmid p3SP-NDM was almost identical topNDM-BJ01from a Acinetobacter lwoffii causing urinary tract infection inBeijing. A. xylosoxidans A22732had a conjugative plasmid pA22732-IMP, whichcontained a novel class I integron of intI1-aacA7-blaIMP-1-tniA. PlasmidpA22732-IMP and pB8(from sewage treatment plant, Russia) had evolved toacquire class I integrons through site-specific homologous recombination,compared to pAKD31(from agricultural soils, Norway) and plasmids pDB1(isolated from agricultural soils, Republic of Korea). The MIC analysesdemonstrated that NDM-1had higher carbapenemase activity than IMP-1and thatCTX-M-55had the narrowest β-lactam hydrolyses activity compared to the abovetwo.Conclusions: This study provided the evidence of increasing β-lactamhydrolyses activity of CTX-M-55, IMP-1and NDM-1. Data presented here notonly demonstrated that mobile genetic elements contributed to horizontal transferof β-lactamase genes, but revealed that β-lactamase genes could be transferredbetween bacteria of different origins.