Zhe Zhan

Sequencing and comparative genomics analysis of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA

Incompatibility group IncHI plasmids are important vectors of antibiotic resistance in Enterobacteriaceae. In this study, a scheme for typing IncHI into five separately clustering subgroups, including previously designated IncHI1-3 as well as IncHI4-5, was proposed based on sequenced IncHI plasmids. The complete nucleotide sequences of the IncHI2 plasmids pT5282-mphA and p112298-catA and the IncHI5 plasmid pYNKP001-dfrA from clinical Enterobacter cloacae, Citrobacter freundii and Raoultella ornithinolytica isolates, respectively, were determined and were compared with IncHI2 and IncHI5 reference plasmids. Considerable genetic conservation was observed within the backbone sequences of each of the IncHI2 and IncHI5 subgroups, but the backbone sequences of the two subgroups were dramatically different from each other. However, the conjugal transfer regions tra1 and tra2 as well as the tellurium resistance gene cluster ter were present in all five plasmids. A number of accessory regions associated with integrons, transposons and insertion sequence-based mobile elements have been inserted at various sites of the plasmid backbones, among which were several large regions harbouring genes conferring resistance to multiple classes of antibiotics. Data generated from this study provide us with a deeper understanding of the diversification of IncHI-type resistance plasmids.

Coexistence of two novel resistance plasmids, blaKPC-2-carrying p14057A and tetA(A) -carrying p14057B, in Pseudomonas aeruginosa

Lining Shia,z, Quanhui Liangb,c,z, Jiao Feng, Zhe Zhan, Yachao Zhao, Wenhui Yang, Huiying Yang, Yong Chen, Mei Huang, Yigang Tong, Xiaojun Li, Zhe Yin, Jinglin Wang, and Dongsheng Zhou Institute of Medical Laboratory Sciences, Jinling Hospital, School of Medicine, Nanjing University, Nanjing, China; State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China; Department of Clinical Laboratory, the First People’s Hospital of Foshan, Foshan, China

Comparative genomics of type 1 IncC plasmids from China.

AIM This study dealt with genomic characterization of type 1 IncC resistance plasmids, capable of spreading across taxonomic borders, from China. MATERIALS & METHODS p112298-tetA was sequenced and compared with type 1 IncC reference plasmid pR148 and two available sequenced type 1 IncC plasmids pHS36-NDM and pVAS3-1 from China. RESULTS These plasmids contained one or more exogenous resistance islands, which included the ARI-A islands, the ARI-B islands, the ISEcp1-blaCMY units and the bla KPC-2 region and were inserted at various sites in the IncC backbone and thus represented three distinct lineages. CONCLUSION Complex rearrangement and homologous recombination events have occurred during evolution of p112298-tetA, making it significantly differ modularly from the other three plasmids with respect to both plasmid backbone and exogenous resistance regions.

p1220-CTXM, a pKP048-related IncFIIK plasmid carrying bla CTX-M-14 and qnrB4

AIM This study aimed to characterize plasmid-mediated antimicrobial resistance in clinical Klebsiella pneumoniae 1220 carrying bla CTX-M-14 and qnrB4. MATERIALS & METHODS Plasmid p1220-CTXM was transformed from the 1220 isolate into Escherichia coli through conjugal transfer and then fully sequenced. Antimicrobial susceptibility was determined by VITEK. RESULTS p1220-CTXM was an IncFIIK plasmid genetically closely related to pKP048 and carried resistance markers including bla CTX-M-14, bla DHA-1, qnrB4, sul1 and qacEΔ1, all of which were harbored in a 35.7-kb multidrug-resistant region. bla CTX-M-14 was located in a truncated ISEcp1-bla CTX-M-14-orf477 transposition unit, and qnrB4 and bla DHA-1 were in a truncated qnrB4-bla DHA-1 region. CONCLUSION This study provided the insight into the co-occurrence of bla CTX-M-14 and qnrB4 and the evolution of pKP048-related IncFIIK plasmids.

Co-occurrence of three different resistance plasmids in a multi-drug resistant Cronobacter sakazakii isolate causing neonatal infections

ABSTRACT Cronobacter sakazakii 505108 was isolated from a sputum specimen of a neonate with severe pneumonia. C. sakazakii 505108 co-harbors 3 resistance plasmids of the IncHI2, IncX3, and IncFIB incomparability groups, respectively. These 3 plasmids have acquired several accessory modules, which carry an extremely large number of resistance genes, especially including those involved in resistance to carbapenems, aminoglycoside, tetracyclines, and phenicols and sulphonamide/trimethoprim. These plasmid-borne antibiotic resistance genes were associated with insertion sequences, integrons, and transposons, indicating that the assembly and mobilization of the corresponding accessory modules with complex chimera structures are facilitated by transposition and/or homologous recombination. This is the first report of fully sequence plasmids in clinical Cronobacter, which provides a deeper insight into plasmid-mediated multi-drug resistance in Cronobacter from hospital settings.

Structure genomics of two chimera plasmids p675920-1 and p675920-2 coexisting in a multi-drug resistant Klebsiella pneumoniae isolate

This study dealt with detailed genomic characterization of two multidrug resistant (MDR) plasmids p675920-1 and p675920-2 from a single clinical Klebsiella pneumoniae isolate 675920. p675920-1 was essentially a hybrid of the IncFII plasmid pHN7A8 and the IncR plasmid pKPC-LK30, and functioned as an IncFII plasmid with inactivation of the IncR replication gene. The backbone of p675920-2 was a hybrid of a novel replicon, three maintenance regions (22.0-, 2.7-, 2.6-kb in length, respectively) as found in pKPYL2, p10164-3 and pK1HV, respectively, and the entire 25.9-kb conjugal transfer region of pKPYL2. p675920-1 and p675920-2 carried a large number of resistance genes, which contributed to resistance to at least seven classes of antibiotics (β-lactams, quinolones, aminoglycosides, fosfomycins, sulphonamides, trimethoprims, and tetracyclines) and one kind of heavy mental (mercury). All of these resistance genes are associated with mobile elements such as insertion sequences, insertion sequence-based transposition units, and transposons, which constituted a total of three novel MDR regions, two in p675920-1 and another in p675920-2. Coexistence of two MDR plasmids p675920-1 and p675920-2 made K. pneumoniae 675920 tend to become extensively drug-resistant.

Plasmid and chromosomal integration of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp.

Objectives To provide detailed genetic characterization of four novel blaIMP-carrying transposons from Pseudomonas aeruginosa, Klebsiella pneumoniae and an Enterobacter sp. Methods P. aeruginosa 60512, K. pneumoniae 447, P. aeruginosa 12939 and Enterobacter sp. A1137 were subjected to genome sequencing. The complete nucleotide sequences of two plasmids (p60512-IMP from the 60512 isolate and p447-IMP from the 447 isolate) and two chromosomes (the 12939 and A1137 isolates) were determined, then a genomic comparison of p60512-IMP, p447-IMP and four novel blaIMP-carrying transposons (Tn6394, Tn6375, Tn6411 and Tn6397) with related sequences was performed. Transferability of the blaIMP gene and bacterial antimicrobial susceptibility were tested. Results Tn6394 and Tn6375 were located in p60512-IMP and p447-IMP, respectively, while Tn6411 and Tn6397 were integrated into the 12939 and A1137 chromosomes, respectively. Tn6394 was an ISPa17-based transposition unit that harboured the integron In992 (carrying blaIMP-1). In73 (carrying blaIMP-8), In73 and In992, together with the ISEcp1:IS1R-blaCTX-M-14-IS903D unit, the macAB-tolC region and the truncated aacC2-tmrB region, respectively, were integrated into the prototype transposons Tn1722, Tn1696 and Tn7, respectively, generating the Tn3-family unit transposons, Tn6375 and Tn6378, and the Tn7-family unit transposon Tn6411, respectively. Tn6397 was a large integrative and conjugative element carrying Tn6378. Conclusions Complex events of transposition and homologous recombination have occurred during the original formation and further plasmid and chromosomal integration of these four transposons, promoting accumulation and spread of antimicrobial resistance genes.

Sequencing of pT5282-CTXM, p13190-KPC and p30860-NR, and comparative genomics analysis of IncX8 plasmids

This study proposes a replicon-based scheme for typing IncX plasmids into nine separately clustering subgroups, including IncX1α, IncX1β and IncX2-8. The complete nucleotide sequences of three IncX8 plasmids, namely pT5282-CTXM and p30860-NR from Enterobacter cloacae and p13190-KPC from Klebsiella pneumoniae, were determined and were compared with two other previously sequenced IncX8 plasmids (pCAV1043-58 and pCAV1741-16). These five plasmids possessed conserved IncX8 backbones with limited genetic variation with respect to gene content and organisation, and each of them carried one or three accessory modules that harboured resistance markers and metabolic gene clusters as well as transposons, insertion sequence (IS)-based transposition units and miniature inverted repeat transposable elements (MITEs), indicating that the relatively small IncX8 backbones were able to integrate various foreign genetic contents. The resistance genes blaCTX-M-3 and blaTEM-1 (β-lactam resistance), blaKPC-2 (carbapenem resistance) and ΔblaTEM-1, and tet(A) (tetracycline resistance) and mph(E) (macrolide resistance) were found in pT5282-CTXM, p13190-KPC and pCAV1741-16, respectively, whilst p30860-NR and pCAV1043-58 carried no resistance genes. The data presented here provide an insight into the diversification and evolution history of IncX8 plasmids.

Comparative analysis of bla KPC-2 - and rmtB -carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids from Klebsiella pneumoniae CG258 strains disseminated among multiple Chinese hospitals

Background We recently reported the complete sequence of a blaKPC-2- and rmtB-carrying IncFII-family plasmid p675920-1 with the pKPC-LK30/pHN7A8 hybrid structure. Comparative genomics of additional sequenced plasmids with similar hybrid structures and their prevalence in blaKPC-carrying Klebsiella pneumoniae strains from China were investigated in this follow-up study. Methods A total of 51 blaKPC-carrying K. pneumoniae strains were isolated from 2012 to 2016 from five Chinese hospitals and genotyped by multilocus sequence typing. The blaKPC-carrying plasmids from four representative strains were sequenced and compared with p675920-1 and pCT-KPC. Plasmid transfer, carbapenemase activity determination, and bacterial antimicrobial susceptibility test were performed to characterize resistance phenotypes mediated by these plasmids. The prevalence of pCT-KPC-like plasmids in these blaKPC-carrying K. pneumoniae strains was screened by PCR. Result The six KPC-encoding plasmids p1068-KPC, p20049-KPC, p12139-KPC and p64917-KPC (sequenced in this study) and p675920-1 and pCT-KPC slightly differed from one another due to deletion and acquisition of various backbone and accessory regions. Two major accessory resistance regions, which included the blaKPC-2 region harboring blaKPC-2 (carbapenem resistance) and blaSHV-12 (β-lactam resistance), and the MDR region carrying rmtB (aminoglycoside resistance), fosA3 (fosfomycin resistance), blaTEM-1B (β-lactam resistance) and blaCTX-M-65 (β-lactam resistance), were found in each of these six plasmids and exhibited several parallel evolution routes. The pCT-KPC-like plasmids were present in all the 51 K. pneumoniae isolates, all of which belonged to CG258. Conclusion There was clonal dissemination of K. pneumoniae CG258 strains, harboring blaKPC-2- and rmtB-carrying IncFII-family pKPC-LK30/pHN7A8 hybrid plasmids, among multiple Chinese hospitals.

Dissemination of KPC-2-Encoding IncX6 Plasmids Among Multiple Enterobacteriaceae Species in a Single Chinese Hospital

Forty-five KPC-producing Enterobacteriaceae strains were isolated from multiple departments in a Chinese public hospital from 2014 to 2015. Genome sequencing of four representative strains, namely Proteus mirabilis GN2, Serratia marcescens GN26, Morganella morganii GN28, and Klebsiella aerogenes E20, indicated the presence of blaKPC-2-carrying IncX6 plasmids pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC in the four strains, respectively. These plasmids were genetically closely related to one another and to the only previously sequenced IncX6 plasmid, pKPC3_SZ. Each of the plasmids carried a single accessory module containing the blaKPC-2/3-carrying ΔTn6296 derivatives. The ΔTn6292 element from pGN26-KPC also contained qnrS, which was absent from all other plasmids. Overall, pKPC3_SZ-like blaKPC-carrying IncX6 plasmids were detected by PCR in 44.4% of the KPC-producing isolates, which included K. aerogenes, P. mirabilis, S. marcescens, M. morganii, Escherichia coli, and Klebsiella pneumoniae, and were obtained from six different departments of the hospital. Data presented herein provided insights into the genomic diversity and evolution of IncX6 plasmids, as well as the dissemination and epidemiology of blaKPC-carrying IncX6 plasmids among Enterobacteriaceae in a hospital setting.