Coexistence of a novel KPC-2-encoding MDR plasmid and an NDM-1-encoding pNDM-HN380-like plasmid in a clinical isolate of Citrobacter freundii.

The objective of this study was to characterize the molecular mechanism of coproduction of KPC-2 and NDM-1 in Citrobacter freundii. C. freundii strain 112298 was isolated from a human case of septic shock in a Chinese teaching hospital. The major carbapenemase and ESBL genes were detected by PCR. The MIC values were determined by using VITEK 2 and antimicrobial susceptibility was judged by CLSI standards. The resistance plasmid was transferred into Escherichia coli by electroporation, followed by plasmid DNA isolation from the electroporant, and then fully sequenced and compared with closely related plasmids. Strain 112298 produces KPC-2 and NDM-1, encoded by the novel non-typeable plasmid p112298-KPC and an IncX3-type plasmid p112298-NDM, respectively. In p112298-KPC, a Tn1722-based blaKPC-2-carrying transposon is associated with several additional resistance modules, constituting a single MDR region. Assembly of these resistance modules is likely mediated by homologous recombination between five copies of IS26 elements at different sites within the MDR region. p112298-NDM is a very close relation of pNDM-HN380. blaNDM-1 in p112298-NDM is carried by a Tn125 variant, which differs from the prototype Tn125 as observed in pNDM-BJ01 by disruption of an upstream copy of ISAba125 by IS5 and absence of a downstream copy of ISAba125. Production of KPC-2 and NDM-1 by p112298-KPC and p112298-NDM, respectively, makes C. freundii 112298 highly resistant to carbapenems and, moreover, these two plasmids still harbour genes for resistance to cephalosporins, chloramphenicol, chromate, fosfomycin, quaternary ammonium, rifampicin and sulphonamides.

Feng J ，Qiu Y ，Yin Z ，Chen W ，Yang H ，Yang W ，Wang J ，Gao Y ，Zhou D ... -  《-》

Coexistence of NDM-1-producing Escherichia coli and Citrobacter freundii in the same patient.

Although blaNDM-1 has been widely detected in various Enterobacteriaceae clinical isolates, multiple blaNDM-1 colonisations within the same patient remain rare. The aim of the study was to describe a patient with respiratory tract colonisation with NDM-1-producing Escherichia coli and Citrobacter freundii during hospitalisation in China. Two carbapenem-resistant isolates were analysed. Antimicrobial susceptibility testing, metallo-β-lactamase production, conjugation assay, plasmid analysis and molecular typing were performed. The two clinical isolates carried the blaNDM-1 gene and showed resistance to carbapenems. S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) revealed that E. coli NB753 harboured a plasmid of ca. 80kb, and C. freundii NB865 harboured three plasmids (ca. 70, 80 and 135kb). Southern blot and Inc replicon typing further demonstrated that both isolates carried the blaNDM-1 gene on a self-transferrable IncX3 plasmid. Moreover, the two NDM-producing plasmids were conjugative and the transconjugants showed increased resistance to carbapenems. An NDM-1-encoding plasmid harboured by various clinical isolates in a single patient is worrying considering that this plasmid may be widespread in our hospital. Furthermore, the threat of carbapenemase-producing bacterial epidemics should be closely monitored. However, a limitation of this study was the extremely small sample size.

Zhu B ，Ying C ，Xu H ，Ying J ... -  《-》

Use of WGS data for investigation of a long-term NDM-1-producing Citrobacter freundii outbreak and secondary in vivo spread of blaNDM-1 to Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca.

An outbreak of NDM-1-producing Citrobacter freundii and possible secondary in vivo spread of blaNDM-1 to other Enterobacteriaceae were investigated. From October 2012 to March 2015, meropenem-resistant Enterobacteriaceae were detected in 45 samples from seven patients at Aalborg University Hospital, Aalborg, Denmark. In silico resistance genes, Inc plasmid types and STs (MLST) were obtained from WGS data from 24 meropenem-resistant isolates (13 C. freundii, 6 Klebsiella pneumoniae, 4 Escherichia coli and 1 Klebsiella oxytoca) and 1 meropenem-susceptible K. oxytoca. The sequences of the meropenem-resistant C. freundii isolates were compared by phylogenetic analyses. In vitro susceptibility to 21 antimicrobial agents was tested. Furthermore, in vitro conjugation and plasmid characterization was performed. From the seven patients, 13 highly clonal ST18 NDM-1-producing C. freundii were isolated. The ST18 NDM-1-producing C. freundii isolates were only susceptible to tetracycline, tigecycline, colistin and fosfomycin (except for the C. freundii isolates from Patient 2 and Patient 7, which were additionally resistant to tetracycline). The E. coli and K. pneumoniae from different patients belonged to different STs, indicating in vivo transfer of blaNDM-1 in the individual patients. This was further supported by in vitro conjugation and detection of a 154 kb IncA/C2 plasmid with blaNDM-1. Patient screenings failed to reveal any additional cases. None of the patients had a history of recent travel abroad and the source of the blaNDM-1 plasmid was unknown. To our knowledge, this is the first report of an NDM-1-producing C. freundii outbreak and secondary in vivo spread of an IncA/C2 plasmid with blaNDM-1 to other Enterobacteriaceae.

Hammerum AM ，Hansen F ，Nielsen HL ，Jakobsen L ，Stegger M ，Andersen PS ，Jensen P ，Nielsen TK ，Hansen LH ，Hasman H ，Fuglsang-Damgaard D ... -  《-》

Lateral dissemination and inter-patient transmission of blaKPC-3: role of a conjugative plasmid in spreading carbapenem resistance.

The objective of this study was to describe the nosocomial spread of carbapenemase-producing enterobacteria and characterize a plasmid involved in KPC dissemination. Two Klebsiella pneumoniae, one Escherichia coli and one Citrobacter freundii isolated from two patients were studied. Susceptibility profiles were obtained using Etest. Carbapenemase activity was detected using the Carba NP test. β-Lactamase gene content was screened by PCR and sequencing. K. pneumoniae isolates were genotyped by MLST and PFGE. KPC plasmid sizes were estimated by S1-DNA digestion and PFGE-Southern blot. Plasmids were sequenced using Illumina's technology and Sanger sequencing. Two patients sharing a room on a surgical unit were positive for carbapenemase-producing K. pneumoniae. One patient was also colonized with carbapenemase-producing C. freundii and E. coli. Neither patient had known risk factors for carbapenemase acquisition, although one patient had recent surgery at another Toronto hospital; the other patient's husband had surgery in New York City 3 years prior to her presentation. An extensive investigation was conducted at both hospitals, but no additional cases were identified. blaKPC-3 was detected in all clinical isolates. Variable carbapenem resistance levels were observed. Both K. pneumoniae belonged to the same clone by PFGE and MLST (ST277). pKPC-SMH (∼ 53 kb) was identified in all the clinical isolates, showing identity only with structurally similar IncN plasmids. We describe intra- and inter-patient dissemination of blaKPC. The involvement of a clone related to the successful K. pneumoniae ST258 and the blaKPC-3 gene detected in an active Tn4401 transposon carried on a conjugative broad-host-range plasmid increased the potential for this horizontal transmission.

Tijet N ，Muller MP ，Matukas LM ，Khan A ，Patel SN ，Melano RG ... -  《-》

Genetic characteristics of blaNDM-1-positive plasmid in Citrobacter freundii isolate separated from a clinical infectious patient.

Du XX ，Wang JF ，Fu Y ，Zhao F ，Chen Y ，Wang HP ，Yu YS ... -  《-》