- Email: [email protected]
Whole genome sequence of an MCR-1-carrying, extended-spectrum β-lactamase (ESBL)-producing Escherichia coli ST746 isolate recovered from a community-acquired urinary tract infection
Journal of Global Antimicrobial Resistance 13 (2018) 171–173
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Genome Note
Whole genome sequence of an MCR-1-carrying, extended-spectrum b-lactamase (ESBL)-producing Escherichia coli ST746 isolate recovered from a community-acquired urinary tract infection Lingjiao Wua,* , Jing Chenb , Li Wangb , Zhe Wub a Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China b The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 December 2017 Received in revised form 22 February 2018 Accepted 27 March 2018 Available online 4 April 2018
Objectives: Colistin is regarded as one of the last-resort antimicrobials for severe infections. Isolates carrying the plasmid-borne mobile colistin resistance gene mcr-1 were rarely reported in communityacquired infections. Here we report the draft genome sequence of an MCR-1-carrying, extendedspectrum b-lactamase (ESBL)-producing Escherichia coli isolate from community-acquired urinary tract infection. Methods: Antimicrobial susceptibility testing (AST) was performed by the broth microdilution method. Transferability of the mcr-bearing plasmid was determined by filter mating using E. coli EC600 as recipient strain. Multilocus sequence typing (MLST) was undertaken using the E. coli MLST database. The draft genome sequence of isolate LX13 was obtained using an Illumina HiSeq X-Ten platform. The genome was assembled using SOAPdenovo. Acquired antimicrobial resistance genes were identified using ResFinder 2.1. Results: AST showed that LX13 was resistant to ampicillin, amoxicillin/clavulanic acid, piperacillin/ tazobactam, cefazolin, cefepime and polymyxins. MLST showed that isolate LX13 belongs to ST746. The MCR-1-producing plasmid was conjugative and conferred increased resistance to colistin the transconjugant. The draft genome of E. coli LX13 was 4 914 035 bp in size. In silico analysis revealed the presence of eight putative acquired resistance genes, including blaCTX-M-14, blaTEM-1B, aadA5, mcr-1, dfrA17, sul2, tet34 and tetA. plasmidSPAdes revealed that the mcr-1 gene was harboured by a plasmid of replicon type IncI2. Conclusions: This study highlights the potential risk of spread of MCR-1-carrying, ESBL-producing E. coli in the community. The genome sequence of E. coli LX13 will facilitate the understanding of colistin resistance mechanisms and genomic features of clinically isolated colistin-resistant E. coli. © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Escherichia coli mcr-1 blaCTX-M-14 ST746 Urinary tract infection
The progressive global increase of extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae is worrisome, and adding to the concern is the recent discovery of the plasmidmediated mobile colistin resistance gene mcr-1 [1–3]. MCR-1 confers resistance to polymyxins, a group of polypeptide antibiotics that are currently considered the ‘last-resort’ therapeutics against lethal challenges by multidrug-resistant (MDR) Gramnegative pathogens [3]. The wide spread of mcr-1 and the finding that this resistance marker is often associated with MDR
* Corresponding author. E-mail address: [email protected] (L. Wu).
Enterobacteriaceae, e.g. ESBL- or carbapenemase-producers, is of great concern [4]. Since its discovery, mcr genes have been identified almost worldwide, mostly in animal and food samples and to a lesser extent in human clinical samples [2]. However, MCR-1-producing isolates have rarely been reported in community-acquired infections [2]. In this study, whole-genome sequencing (WGS) and microbiological analysis were performed to elucidate the antimicrobial resistance mechanisms of a MCR-1-carrying, ESBL-producing E. coli strain recovered from a communityacquired urinary tract infection (UTI). Escherichia coli isolate LX13 was recovered from a urine sample of a 72-year-old female admitted to the Department of Infectious Diseases of the First Affiliated Hospital of Zhejiang University
https://doi.org/10.1016/j.jgar.2018.03.014 2213-7165/© 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
172
L. Wu et al. / Journal of Global Antimicrobial Resistance 13 (2018) 171–173
Table 1 Acquired antimicrobial resistance genes of Escherichia coli LX13. Antibiotic class
Resistance gene
HSP length/query
Contig
Position in contig
% identity
Accession no.
b-Lactams
blaCTX-M-14 blaTEM-1B mcr-1 dfrA17 aadA5 sul2 tet34 tetA
876/876 861/861 1626/1626 474/474 789/789 816/816 356/465 1200/1200
98 56 49 94 94 76 19 89
819–1695 286–1146 18 126–19 751 1152–1625 233–1021 3901–4716 53 125–53 480 1544–2743
100 100 100 100 100 100 75 100
AF252622 JF910132 KP347127 FJ460238 AF137361 GQ421466 AB061440 AJ517790
Colistin Trimethoprim Aminoglycosides Sulphonamides Tetracycline
(Hangzhou, China) with community-acquired UTI in August 2016. The urine sample was cultivated aerobically on a Columbia blood agar plate for bacterial isolation. Escherichia coli isolate LX13 was subjected to antimicrobial susceptibility testing by the microdilution broth method for the following antimicrobial agents: ampicillin; amoxicillin/clavulanic acid (AMC); piperacillin/tazobactam (TZP); cefazolin; cefepime; aztreonam; ciprofloxacin; levofloxacin; ertapenem; imipenem; meropenem; tigecycline; colistin; and polymyxin B (PMB). The results were interrupted according to Clinical and Laboratory Standards Institute (CLSI) guidelines (https://clsi.org/), except for tigecycline, colistin and PMB, which were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Multilocus sequence typing (MLST) was performed according to protocols described in the E. coli MLST database (http://mlst.ucc.ie/mlst/dbs/Ecoli). LX13 was further characterised by conjugation experiments to assess its ability to transfer colistin resistance. The conjugation experiment was carried out by the filter mating method using E. coli EC600 as the recipient strain [5]. The resulting transconjugants were selected on brain–heart infusion agar plates supplemented with colistin (2 mg/L). The presence of mcr-1 was further confirmed by PCR and sequencing. The transconjugants was further confirmed by pulsed-field gel electrophoresis (PFGE). Genomic DNA was extracted from a pure culture using an E.Z.N. A.1 Bacterial DNA Kit (Omega Bio-Tek Inc., Norcross, GA) according to the manufacturer’s instructions. Bacterial identification of the isolate was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonik GmbH, Bremen, Germany) and 16S rRNA gene sequencing. WGS was performed on an Illumina HiSeq X-Ten platform (Illumina Inc., San Diego, CA). All reads with adaptor contamination and with unknown nucleotides comprising >5% were removed. De novo assembly was performed using SOAPdenovo v.2.01 (http://soap.genomics.org.cn/soapdenovo.html). Open reading frames (ORFs) were identified and classified using the Rapid Annotation using Subsystem Technology (RAST) server (http://rast. nmpdr.org/). Plasmid classification was performed using PlasmidFinder 1.3 (https://cge.cbs.dtu.dk/services/PlasmidFinder/). Acquired resistance genes were identified by ResFinder 2.1 (https://cge.cbs.dtu.dk/services/ResFinder/). All of the putative antibiotic resistance genes were further verified through a BLAST search against the Comprehensive Antibiotic Resistance Database (https://card.mcmaster.ca/). Antimicrobial susceptibility testing showed that LX13 was susceptible to aztreonam, ciprofloxacin, levofloxacin, carbapenems and tigecycline but was resistant to the other tested antibiotics, including ampicillin, AMC, TZP, cefazolin, cefepime, colistin and PMB. The conjugation experiment demonstrated that the mcr-1 gene was located on a conjugative plasmid (Supplementary Table S1). MLST analysis showed that isolate LX13 belongs to ST746.
The draft genome of E. coli LX13 was assembled into 149 contigs comprising a size of 4 914 035 bp, and the overall GC content was 50.36%. The assembled genome covered an average depth of 120fold and contained 149 contigs, of which the N50 contig length was 91120 bp. The genome consists of one linear chromosome with 8 rRNA operons, 76 tRNA genes, 9 ncRNAs, 4850 protein-coding genes (CDSs) among the 5130 genes, and 3 CRISPR (clustered regularly interspaced short palindromic repeats) arrays. The antibiotic resistance genes were screened on a genomewide scale in order to further explore the genetic basis of multidrug resistance in this strain (Table 1). In silico bioinformatics analysis showed that isolate LX13 contained multiple genes conferring resistance to b-lactams (blaCTX-M-14 and blaTEM-1B), colistin (mcr-1), trimethoprim (dfrA17), aminoglycosides (aadA5), sulfonamides (sul2) and tetracycline (tet34 and tetA), which is consistent with the phenotypic results (Supplementary Table S1). An IncI1 and an IncI2 plasmid were identified using plasmidSPAdes assembler with high coverage. In addition, a 28-kb mcr-1-carrying contig was identified in the genome. Querying this contig against the nr/nt database revealed 99% homology with the mcr-1-positive IncI2 plasmid p5CRE51-MCR-1 (GenBank accession no. CP021176) from a clinical isolate and the IncI2 plasmid pHNGDF93 (GenBank MF978388) from an animal isolate. Taken together, here we report the first draft genome sequence of a clinical colistin-resistant E. coli LX13 isolate from a community-acquired UTI. This study highlights the potential risk of spread of MCR-1-carrying E. coli in the community. The genome sequence of E. coli LX13 will facilitate the understanding of colistin resistance mechanisms and genomic features of clinically isolated colistin-resistant, ESBL-producing E. coli. Further studies involving more MCR-1-producing isolates from healthy people and community-acquired infections are urgently needed to investigate the dissemination dynamics of mcr genes in the community. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. PJIY00000000. The version described in this paper is version PJIY01000000. Funding This work was financially supported by a grant from the Zhejiang Provincial Medical & Hygienic Science and Technology Project of China [No. 2012RCB019]. Competing interests None declared. Ethical approval Not required.
L. Wu et al. / Journal of Global Antimicrobial Resistance 13 (2018) 171–173
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jgar.2018.03.014. References [1] Woerther P.L., Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of extended-spectrum b-lactamases in the community: toward the globalization of CTX-M. Clin Microbiol Rev 2013;26:744–58.
173
[2] Prim N, Turbau M, Rivera A, Rodriguez-Navarro J, Coll P, Mirelis B. Prevalence of colistin resistance in clinical isolates of Enterobacteriaceae: a four-year crosssectional study. J Infect 2017;75:493–8. [3] Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmidmediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016;16:161–8. [4] Zheng B, Dong H, Xu H, Lv J, Zhang J, Jiang X, et al. Coexistence of MCR-1 and NDM-1 in clinical Escherichia coli isolates. Clin Infect Dis 2016;63:1393–5. [5] Zheng B, Lv T, Xu H, Yu X, Chen Y, Li J, et al. Discovery and characterisation of an Escherichia coli ST206 strain producing NDM-5 and MCR-1 from a patient with acute diarrhoea in China. Int J Antimicrob Agents 2018;51:273–5.
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Genome Note
Whole genome sequence of an MCR-1-carrying, extended-spectrum b-lactamase (ESBL)-producing Escherichia coli ST746 isolate recovered from a community-acquired urinary tract infection Lingjiao Wua,* , Jing Chenb , Li Wangb , Zhe Wub a Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China b The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310003, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 26 December 2017 Received in revised form 22 February 2018 Accepted 27 March 2018 Available online 4 April 2018
Objectives: Colistin is regarded as one of the last-resort antimicrobials for severe infections. Isolates carrying the plasmid-borne mobile colistin resistance gene mcr-1 were rarely reported in communityacquired infections. Here we report the draft genome sequence of an MCR-1-carrying, extendedspectrum b-lactamase (ESBL)-producing Escherichia coli isolate from community-acquired urinary tract infection. Methods: Antimicrobial susceptibility testing (AST) was performed by the broth microdilution method. Transferability of the mcr-bearing plasmid was determined by filter mating using E. coli EC600 as recipient strain. Multilocus sequence typing (MLST) was undertaken using the E. coli MLST database. The draft genome sequence of isolate LX13 was obtained using an Illumina HiSeq X-Ten platform. The genome was assembled using SOAPdenovo. Acquired antimicrobial resistance genes were identified using ResFinder 2.1. Results: AST showed that LX13 was resistant to ampicillin, amoxicillin/clavulanic acid, piperacillin/ tazobactam, cefazolin, cefepime and polymyxins. MLST showed that isolate LX13 belongs to ST746. The MCR-1-producing plasmid was conjugative and conferred increased resistance to colistin the transconjugant. The draft genome of E. coli LX13 was 4 914 035 bp in size. In silico analysis revealed the presence of eight putative acquired resistance genes, including blaCTX-M-14, blaTEM-1B, aadA5, mcr-1, dfrA17, sul2, tet34 and tetA. plasmidSPAdes revealed that the mcr-1 gene was harboured by a plasmid of replicon type IncI2. Conclusions: This study highlights the potential risk of spread of MCR-1-carrying, ESBL-producing E. coli in the community. The genome sequence of E. coli LX13 will facilitate the understanding of colistin resistance mechanisms and genomic features of clinically isolated colistin-resistant E. coli. © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Escherichia coli mcr-1 blaCTX-M-14 ST746 Urinary tract infection
The progressive global increase of extended-spectrum b-lactamase (ESBL)-producing Enterobacteriaceae is worrisome, and adding to the concern is the recent discovery of the plasmidmediated mobile colistin resistance gene mcr-1 [1–3]. MCR-1 confers resistance to polymyxins, a group of polypeptide antibiotics that are currently considered the ‘last-resort’ therapeutics against lethal challenges by multidrug-resistant (MDR) Gramnegative pathogens [3]. The wide spread of mcr-1 and the finding that this resistance marker is often associated with MDR
* Corresponding author. E-mail address: [email protected] (L. Wu).
Enterobacteriaceae, e.g. ESBL- or carbapenemase-producers, is of great concern [4]. Since its discovery, mcr genes have been identified almost worldwide, mostly in animal and food samples and to a lesser extent in human clinical samples [2]. However, MCR-1-producing isolates have rarely been reported in community-acquired infections [2]. In this study, whole-genome sequencing (WGS) and microbiological analysis were performed to elucidate the antimicrobial resistance mechanisms of a MCR-1-carrying, ESBL-producing E. coli strain recovered from a communityacquired urinary tract infection (UTI). Escherichia coli isolate LX13 was recovered from a urine sample of a 72-year-old female admitted to the Department of Infectious Diseases of the First Affiliated Hospital of Zhejiang University
https://doi.org/10.1016/j.jgar.2018.03.014 2213-7165/© 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
172
L. Wu et al. / Journal of Global Antimicrobial Resistance 13 (2018) 171–173
Table 1 Acquired antimicrobial resistance genes of Escherichia coli LX13. Antibiotic class
Resistance gene
HSP length/query
Contig
Position in contig
% identity
Accession no.
b-Lactams
blaCTX-M-14 blaTEM-1B mcr-1 dfrA17 aadA5 sul2 tet34 tetA
876/876 861/861 1626/1626 474/474 789/789 816/816 356/465 1200/1200
98 56 49 94 94 76 19 89
819–1695 286–1146 18 126–19 751 1152–1625 233–1021 3901–4716 53 125–53 480 1544–2743
100 100 100 100 100 100 75 100
AF252622 JF910132 KP347127 FJ460238 AF137361 GQ421466 AB061440 AJ517790
Colistin Trimethoprim Aminoglycosides Sulphonamides Tetracycline
(Hangzhou, China) with community-acquired UTI in August 2016. The urine sample was cultivated aerobically on a Columbia blood agar plate for bacterial isolation. Escherichia coli isolate LX13 was subjected to antimicrobial susceptibility testing by the microdilution broth method for the following antimicrobial agents: ampicillin; amoxicillin/clavulanic acid (AMC); piperacillin/tazobactam (TZP); cefazolin; cefepime; aztreonam; ciprofloxacin; levofloxacin; ertapenem; imipenem; meropenem; tigecycline; colistin; and polymyxin B (PMB). The results were interrupted according to Clinical and Laboratory Standards Institute (CLSI) guidelines (https://clsi.org/), except for tigecycline, colistin and PMB, which were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Multilocus sequence typing (MLST) was performed according to protocols described in the E. coli MLST database (http://mlst.ucc.ie/mlst/dbs/Ecoli). LX13 was further characterised by conjugation experiments to assess its ability to transfer colistin resistance. The conjugation experiment was carried out by the filter mating method using E. coli EC600 as the recipient strain [5]. The resulting transconjugants were selected on brain–heart infusion agar plates supplemented with colistin (2 mg/L). The presence of mcr-1 was further confirmed by PCR and sequencing. The transconjugants was further confirmed by pulsed-field gel electrophoresis (PFGE). Genomic DNA was extracted from a pure culture using an E.Z.N. A.1 Bacterial DNA Kit (Omega Bio-Tek Inc., Norcross, GA) according to the manufacturer’s instructions. Bacterial identification of the isolate was determined by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS) (Bruker Daltonik GmbH, Bremen, Germany) and 16S rRNA gene sequencing. WGS was performed on an Illumina HiSeq X-Ten platform (Illumina Inc., San Diego, CA). All reads with adaptor contamination and with unknown nucleotides comprising >5% were removed. De novo assembly was performed using SOAPdenovo v.2.01 (http://soap.genomics.org.cn/soapdenovo.html). Open reading frames (ORFs) were identified and classified using the Rapid Annotation using Subsystem Technology (RAST) server (http://rast. nmpdr.org/). Plasmid classification was performed using PlasmidFinder 1.3 (https://cge.cbs.dtu.dk/services/PlasmidFinder/). Acquired resistance genes were identified by ResFinder 2.1 (https://cge.cbs.dtu.dk/services/ResFinder/). All of the putative antibiotic resistance genes were further verified through a BLAST search against the Comprehensive Antibiotic Resistance Database (https://card.mcmaster.ca/). Antimicrobial susceptibility testing showed that LX13 was susceptible to aztreonam, ciprofloxacin, levofloxacin, carbapenems and tigecycline but was resistant to the other tested antibiotics, including ampicillin, AMC, TZP, cefazolin, cefepime, colistin and PMB. The conjugation experiment demonstrated that the mcr-1 gene was located on a conjugative plasmid (Supplementary Table S1). MLST analysis showed that isolate LX13 belongs to ST746.
The draft genome of E. coli LX13 was assembled into 149 contigs comprising a size of 4 914 035 bp, and the overall GC content was 50.36%. The assembled genome covered an average depth of 120fold and contained 149 contigs, of which the N50 contig length was 91120 bp. The genome consists of one linear chromosome with 8 rRNA operons, 76 tRNA genes, 9 ncRNAs, 4850 protein-coding genes (CDSs) among the 5130 genes, and 3 CRISPR (clustered regularly interspaced short palindromic repeats) arrays. The antibiotic resistance genes were screened on a genomewide scale in order to further explore the genetic basis of multidrug resistance in this strain (Table 1). In silico bioinformatics analysis showed that isolate LX13 contained multiple genes conferring resistance to b-lactams (blaCTX-M-14 and blaTEM-1B), colistin (mcr-1), trimethoprim (dfrA17), aminoglycosides (aadA5), sulfonamides (sul2) and tetracycline (tet34 and tetA), which is consistent with the phenotypic results (Supplementary Table S1). An IncI1 and an IncI2 plasmid were identified using plasmidSPAdes assembler with high coverage. In addition, a 28-kb mcr-1-carrying contig was identified in the genome. Querying this contig against the nr/nt database revealed 99% homology with the mcr-1-positive IncI2 plasmid p5CRE51-MCR-1 (GenBank accession no. CP021176) from a clinical isolate and the IncI2 plasmid pHNGDF93 (GenBank MF978388) from an animal isolate. Taken together, here we report the first draft genome sequence of a clinical colistin-resistant E. coli LX13 isolate from a community-acquired UTI. This study highlights the potential risk of spread of MCR-1-carrying E. coli in the community. The genome sequence of E. coli LX13 will facilitate the understanding of colistin resistance mechanisms and genomic features of clinically isolated colistin-resistant, ESBL-producing E. coli. Further studies involving more MCR-1-producing isolates from healthy people and community-acquired infections are urgently needed to investigate the dissemination dynamics of mcr genes in the community. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. PJIY00000000. The version described in this paper is version PJIY01000000. Funding This work was financially supported by a grant from the Zhejiang Provincial Medical & Hygienic Science and Technology Project of China [No. 2012RCB019]. Competing interests None declared. Ethical approval Not required.
L. Wu et al. / Journal of Global Antimicrobial Resistance 13 (2018) 171–173
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jgar.2018.03.014. References [1] Woerther P.L., Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of extended-spectrum b-lactamases in the community: toward the globalization of CTX-M. Clin Microbiol Rev 2013;26:744–58.
173
[2] Prim N, Turbau M, Rivera A, Rodriguez-Navarro J, Coll P, Mirelis B. Prevalence of colistin resistance in clinical isolates of Enterobacteriaceae: a four-year crosssectional study. J Infect 2017;75:493–8. [3] Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R, Spencer J, et al. Emergence of plasmidmediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016;16:161–8. [4] Zheng B, Dong H, Xu H, Lv J, Zhang J, Jiang X, et al. Coexistence of MCR-1 and NDM-1 in clinical Escherichia coli isolates. Clin Infect Dis 2016;63:1393–5. [5] Zheng B, Lv T, Xu H, Yu X, Chen Y, Li J, et al. Discovery and characterisation of an Escherichia coli ST206 strain producing NDM-5 and MCR-1 from a patient with acute diarrhoea in China. Int J Antimicrob Agents 2018;51:273–5.