Emergence of a ST2570 Klebsiella pneumoniae isolate carrying mcr-1 and blaCTX-M-14 recovered from a bloodstream infection in China
Authors: Z. Ruan, Q. Sun, H. Jia, C. Huang, W. Zhou, X. Xie, J. Zhang
Journal: Clinical Microbiology and Infection (2019)

The worldwide emergence of the plasmid-borne colistin resistance mediated by mcr-1 gene not only extended our knowledge on colistin resistance, but also poses a serious threat to clinical and public health [1Schwarz S. Johnson A.P. Transferable resistance to colistin: a new but old threat.J Antimicrob Chemother. 2016; 71: 2066-2070Crossref PubMed Scopus (237) Google Scholar, 2Nordmann P. Poirel L. Plasmid-mediated colistin resistance: an additional antibiotic resistance menace.Clin Microbiol Infect. 2016; 22: 398-400Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar]. Since its first discovery, mcr-1-carrying Enterobacteriaceae from human, animal, food, and environmental origins have been widely identified, but few mcr-1-positive clinical strains of Klebsiella pneumoniae have been reported so far, especially when associated with community-acquired infections [3Liu Y.Y. Wang Y. Walsh T.R. Yi L.X. Zhang R. Spencer J. et al.Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect Dis. 2016; 16: 161-168Abstract Full Text Full Text PDF PubMed Scopus (3284) Google Scholar, 4Wang Y. Tian G.B. Zhang R. Shen Y. Tyrrell J.M. Huang X. et al.Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study.Lancet Infect Dis. 2017; 17: 390-399Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar]. Here, we report the emergence of a colistin-resistant K. pneumoniae isolate, which belonged to a rare sporadic clone, co-carrying mcr-1 and blaCTX-M-14 genes simultaneously recovered from a community-acquired bloodstream infection in China. Whole-genome sequencing and microbiological analysis were performed to elucidate its antimicrobial resistance mechanisms. K. pneumoniae isolate 121 was cultured from the blood sample of a male patient with symptoms indicative of abdominal pain, nausea, vomiting, and fever. The patient had no recent travel history, but lived in the countryside, close to livestock. Initial species identification was performed using Vitek 2 (bioMérieux, Marcy-l’Étoile, France) and MALDI-TOF (Bruker, Billerica, MA, USA). K. pneumoniae 121 was subjected to antimicrobial susceptibility testing by the microdilution broth method for the following antimicrobial agents: amikacin, aztreonam, cefepime, cefotaxime, cefoxitin, ceftazidime, ciprofloxacin, colistin, ertapenem, fosfomycin, gentamicin, imipenem, levofloxacin, meropenem, tetracycline, and tigecycline. The results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines, except for tigecycline and colistin, which were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. To determine whether the mcr-1 is located on the chromosome or plasmid, Southern blot analysis was performed, i.e. genomic DNA was digested with S1 nuclease, separated by pulsed-field gel electrophoresis (PFGE), and subjected to Southern blot using a probe for mcr-1 as previously described [[3]Liu Y.Y. Wang Y. Walsh T.R. Yi L.X. Zhang R. Spencer J. et al.Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect Dis. 2016; 16: 161-168Abstract Full Text Full Text PDF PubMed Scopus (3284) Google Scholar]. The transferability of the mcr-1-carrying plasmid was assessed by conjugation assay, with Escherichia coli J53 as the recipient strain. The presence of mcr-1 in the transconjugant was confirmed by PCR and Sanger sequencing. Whole-genome sequencing (WGS) of K. pneumoniae 121 was performed using both the HiSeq X10 (Illumina, San Diego, CA, USA), with the 150-bp paired-end protocol, and the PacBio RS II (Pacific Biosciences, Menlo Park, CA, USA) platforms. Hybrid assembly of both short Illumina reads and long PacBio reads was constructed using Unicycler v. 0.3.0 with the Pilon (v1.22) option on for the modification of the assembled reads [[5]Wick R.R. Judd L.M. Gorrie C.L. Holt K.E. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads.PLoS Comput Biol. 2017; 13e1005595Crossref PubMed Scopus (2793) Google Scholar]. Antibiotic resistance genes and plasmid associations were queried using the ResFinder and PlasmidFinder databases at the Center for Genomic Epidemiology (http://www.genomicepidemiology.org/) [6Zankari E. Hasman H. Cosentino S. Vestergaard M. Rasmussen S. Lund O. et al.Identification of acquired antimicrobial resistance genes.J Antimicrob Chemother. 2012; 67: 2640-2644Crossref PubMed Scopus (3144) Google Scholar, 7Carattoli A. Zankari E. Garcia-Fernandez A. Voldby Larsen M. Lund O. Villa L. et al.In silico detection and typing of plasmids using PlasmidFinder and plasmid multilocus sequence typing.Antimicrob Agents Chemother. 2014; 58: 3895-3903Crossref PubMed Scopus (2160) Google Scholar]. In silico multilocus sequence typing (MLST) analysis and bacterial source tracking for implementing both cgMLST and single nucleotide polymorphisms (SNP) strategy were performed by our recently updated BacWGSTdb server [[8]Ruan Z. Feng Y. BacWGSTdb, a database for genotyping and source tracking bacterial pathogens.Nucleic Acids Res. 2016; 44: D682-D687Crossref PubMed Scopus (130) Google Scholar]. Antimicrobial susceptibility testing showed that K. pneumoniae 121 was resistant to colistin, cefotaxime, gentamicin, and fosfomycin, but it remained susceptible to several other antimicrobial agents, including carbapenems (Table 1). In particular, MIC of colistin for the isolate was 8 mg/L for the broth microdilution method. It contained multiple genes conferring resistance to β-lactams (blaCTX-M-14 and blaSHV-11), colistin (mcr-1), aminoglycosides (aac(3)-Iva, aadA1, aadA2, aph(3′)-Ia, aph(4)-Ia), sulphonamides (sul2, sul3), phenicol (cmlA1, floR), fosfomycin (fosA3), macrolide (mph(A)), and quinolone (oqxA, oqxB), which is consistent with the phenotypic data. To determine the possible chromosomal colistin resistance determinants, sequences of pmrAB, phoPQ and its negative regulator mgrB were extracted from the WGS data and evaluated [[9]Cannatelli A. D’Andrea M.M. Giani T. Di Pilato V. Arena F. Ambretti S. et al.In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator.Antimicrob Agents Chemother. 2013; 57: 5521-5526Crossref PubMed Scopus (258) Google Scholar]. No mutations or insertion of IS elements that were predicted to alter the function of gene products were detected.Table 1Minimum inhibitory concentration (MIC) for parental, recipient, and transconjugant strainsStrainsMIC (mg/L)AMKATMFEPCTXFOXCAZCIPCSTETPFOFGENIPMLVXMEMTETTGCKlebsiella pneumoniae 12112416420.062580.03256160.250.030.062520.5Escherichia coli J53 + pKP121-1-mcr1248420.0380.01525680.250.030.062520.25E. coli J5310.06250.06250.12540.250.030.1250.01520.250.1250.030.062520.25Antimicrobial susceptibilities, reported as MIC (mg/L), were interpreted as resistant (bold) or susceptible (plain text) in accordance with established breakpoints. AMK, amikacin; ATM, aztreonam; FEP, cefepime; CTX, cefotaxime; FOX, cefoxitin; CAZ, ceftazidime; CIP, ciprofloxacin; CST, colistin; ETP, ertapenem; FOF, fosfomycin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; MEM, meropenem; TET, tetracycline; TGC tigecycline. Open table in a new tab Antimicrobial susceptibilities, reported as MIC (mg/L), were interpreted as resistant (bold) or susceptible (plain text) in accordance with established breakpoints. AMK, amikacin; ATM, aztreonam; FEP, cefepime; CTX, cefotaxime; FOX, cefoxitin; CAZ, ceftazidime; CIP, ciprofloxacin; CST, colistin; ETP, ertapenem; FOF, fosfomycin; GEN, gentamicin; IPM, imipenem; LVX, levofloxacin; MEM, meropenem; TET, tetracycline; TGC tigecycline. Whole-genome sequencing showed that the K. pneumoniae 121 belonged to sequence type (ST) 2570 and also confirmed the presence of the mcr-1 gene located on a novel 239,020-bp plasmid pKP121-1-mcr of incompatibility group IncHI2. The plasmid backbone of pKP121-1-mcr is almost identical to that of pHNSHP45-2, a mcr-1-carrying plasmid in E. coli recovered from an intensive pig farm in Shanghai (90% coverage and 99% nucleotide identity), but the variable region was distinct [3Liu Y.Y. Wang Y. Walsh T.R. Yi L.X. Zhang R. Spencer J. et al.Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study.Lancet Infect Dis. 2016; 16: 161-168Abstract Full Text Full Text PDF PubMed Scopus (3284) Google Scholar, 10Zhi C. Lv L. Yu L.F. Doi Y. Liu J.H. Dissemination of the mcr-1 colistin resistance gene.Lancet Infect Dis. 2016; 16: 292-293Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar]. The shared sequence contains mcr-1 in association with ISApl1, but in pKP121-1-mcr it is in a different location and orientation. pKP121-1-mcr also carried 12 additional antibiotic resistance genes, including the extended spectrum beta-lactamase (ESBL) gene blaCTX-M-14, and all the resistance genes except mcr-1 were embedded in a variable region (Fig. 1). mcr-1 has previously been reported to be carried on IncP plasmids and IncX4 plasmids in K. pneumoniae [11Tian G.B. Doi Y. Shen J. Walsh T.R. Wang Y. Zhang R. et al.MCR-1-producing Klebsiella pneumoniae outbreak in China.Lancet Infect Dis. 2017; 17: 577Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 12Zhao F. Feng Y. Lu X. McNally A. Zong Z. IncP plasmid carrying colistin resistance gene mcr-1 in Klebsiella pneumoniae from hospital sewage.Antimicrob Agents Chemother. 2017; 61 (e02229-16)Crossref Scopus (57) Google Scholar]. Our study highlight that dissemination of mcr-1 among K. pneumoniae is occurring via different types of plasmids. A conjugation assay showed that the colistin resistance determinant mcr-1 in K. pneumoniae 121 can be transferred into the recipient E. coli J53 (Fig. 2). In addition to colistin, the transconjugant also showed elevated minimum inhibitory concentrations to aztreonam, cefepime, cefotaxime, ceftazidime, fosfomycin, and gentamicin (Table 1). These data confirmed the presence of the colistin resistance determinant mcr-1 located on a conjugative plasmid in K. pneumoniae 121.Fig. 2S1-PFGE and Southern blot analysis of Klebsiella pneumoniae 121 and its transconjugant. Lane M, marker (Salmonella serotype Braender up strain H9812); lane 1, K. pneumoniae 121; lane 2, transconjugant Escherichia coli J53 + pKP121-1-mcr; lane 3, recipient E. coli J53.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The origin of the K. pneumoniae 121 remains unknown. We analysed the phylogenetic relationship between K. pneumoniae 121 and a total of 6273 K. pneumoniae strains currently deposited in the NCBI GenBank database by two bacterial source tracking strategies: SNP and cgMLST. Both suggested that K. pneumoniae 121 belonged to a rare sporadic clone and the closest relatives differed by over 5000 SNPs and 1700 loci, which were associated with both clinical and non-clinical settings (food animals). One possible explanation is that the patient acquired this strain from the livestock nearby, but this hypothesis needs further verification to obtain the direct evidence. In summary, we report the first identification of a clinical colistin-resistant K. pneumoniae isolate belonging to a rare sporadic clone recovered from the community-acquired bloodstream infection in China. Our study highlights the potential transmission opportunity of MCR-1 and ESBL-producing K. pneumoniae in the community. Further studies involving more MCR-1-producing isolates from healthy people and community-acquired infections are warranted to identify reservoirs and monitor the transmission dynamics of mcr genes in the community. The nucleotide sequence of the chromosome and plasmids of K. pneumoniae isolate 121 have been deposited in GenBank under accession numbers CP031849-CP031853. None to declare. This work was supported by the National Natural Science Foundation of China (81871696). The authors would like to thank Dr Ying Fu for her excellent technical assistance in the bacterial conjugation assay.