- Email: [email protected]
First genome sequence of a blaKPC-2-carrying Citrobacter koseri isolate collected from a patient with diarrhoea
Journal of Global Antimicrobial Resistance 15 (2018) 166–168
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Genome Note
First genome sequence of a blaKPC-2-carrying Citrobacter koseri isolate collected from a patient with diarrhoea Xinjun Hua,b , Hao Xub , Yibing Shanga , Lihua Guob , Li Songa , Huping Zhanga , Feng Yuea , Hongwei Jianga,* , Beiwen Zhengb,* a Department of Infectious Diseases, The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology, Luoyang, China b 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, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 May 2018 Received in revised form 7 August 2018 Accepted 26 September 2018 Available online 3 October 2018
Objectives: The blaKPC gene is rarely reported in Citrobacter koseri. Here we report the first draft genome sequence of a blaKPC-2-carrying C. koseri isolate from a patient with diarrhoea. Methods: Transferability of the blaKPC-2-bearing plasmid was determined by the filter mating method. The whole genome sequence of C. koseri L168 was determined using an Illumina HiSeq platform. The genome was de novo assembled using Velvet 1.2.10. Acquired antimicrobial resistance genes and plasmid replicons were identified using ResFinder 2.1 and PlasmidFinder 1.3, respectively. Results: Antimicrobial susceptibility testing (AST) showed that C. koseri L168 was resistant to multiple antibiotics but was susceptible to ciprofloxacin, gentamicin, tobramycin, amikacin, tigecycline and colistin. A KPC-2-harbouring plasmid was conjugative and the transconjugants conferred increased resistance to carbapenems confirmed by conjugation experiments and AST. In silico analysis revealed the presence of the β-lactam resistance genes blaKPC-2 and blaMAL-1. Additionally, plasmids of incompatibility groups IncFII and IncX4 were identified in the genome by PlasmidFinder. BLAST analysis revealed that blaKPC-2 was located on a Tn3 transposon element in C. koseri L168 with the conserved linear structure ISKpn27–blaKPC-2–DISKpn6–korC–klcA. Conclusions: To our knowledge, this is only the second report of C. koseri producing KPC-2, and we report the first draft genome sequence of a blaKPC-2-carrying C. koseri isolate from a patient with diarrhoea in China. This work may facilitate our understanding of the pathogenesis, multidrug resistance mechanisms and genomic features of this species. Further monitoring of bacteria carrying carbapenemase genes in patients’ gut microbiota is warranted. © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Citrobacter koseri blaKPC-2 blaMAL-1 Resistome Whole-genome sequencing
The global increase in Enterobacteriaceae producing Klebsiella pneumoniae carbapenemase (KPC)-type carbapenemases is a major clinical challenge and a public-health concern. KPC enzymes confer resistance to carbapenems, which are members of the βlactam class of antibiotics, that are currently considered the ‘last resort’ therapeutics against infections caused by Gram-negative pathogens with multidrug resistance [1]. More importantly, KPC enzymes are typically transposon-encoded determinants and
* Corresponding authors. E-mail addresses: [email protected] (H. Jiang), [email protected] (B. Zheng).
therefore have the ability to move between plasmids and across bacterial species. Citrobacter koseri is a facultative, anaerobic, Gram-negative, non-spore-forming bacterium. It is usually found in the environment and in the digestive tract of humans and animals [1]. Occasionally, it acts as an opportunistic pathogen in a variety of human infections, mainly in children [2]. Although previous studies have documented that some C. koseri isolates harbour extended-spectrum β-lactamase (ESBL) genes, detection of C. koseri isolates producing KPC enzymes remains rare [2]. To elucidate the mechanisms of antimicrobial resistance of the blaKPC-2-carrying C. koseri isolate, here we report the first genome sequence of a blaKPC-2-carrying C. koseri isolate recovered from gut colonisation.
https://doi.org/10.1016/j.jgar.2018.09.016 2213-7165/© 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
X. Hu et al. / Journal of Global Antimicrobial Resistance 15 (2018) 166–168
Citrobacter koseri isolate L168 was isolated in 2016 from a stool sample collected from a female patient with diarrhoea in Hangzhou, China. Minimum inhibitory concentrations (MICs) for the isolate were determined by the broth microdilution method. Conjugation experiments were performed by filter mating with Escherichia coli EC600 as the recipient strain [3]. The resulting transconjugant strains were selected on brain–heart infusion agar plates containing 2 mg/L imipenem and 150 mg/L sodium azide. Presence of the blaKPC-2 gene in the transconjugants was subsequently analysed by PCR and sequencing. Genomic DNA was extracted from 2 mL of pure culture cells using a Genomic DNA Isolation Kit (QIAGEN, Hilden, Germany) and was sequenced using an Illumina HiSeq platform (Illumina Inc., San Diego, CA). De novo genomic assembly was conducted using Velvet 1.2.10. (https://www.ebi.ac.uk/zerbino/velvet/) with the default settings. Short contigs less than 300 nucleotides in length were discarded. Genome annotation was done using the Rapid Annotation using Subsystem Technology (RAST) server (http://rast.nmpdr.org/), and specific genes were manually curated. Plasmid classification and acquired antimicrobial resistance genes were identified using PlasmidFinder 1.3 (https://cge.cbs.dtu.dk/services/PlasmidFinder/)
167
and ResFinder 2.1 (https://cge.cbs.dtu.dk/services/ResFinder/), respectively, available from the Center for Genomic Epidemiology. The Comprehensive Antibiotic Resistance Database (CARD) (https:// card.mcmaster.ca/) was used to verify all putative antimicrobial resistance genes through a BLAST search. Identification of the putative KPC-carrying plasmid was done using the plasmidSPAdes assembly tool (https://omictools.com/plasmidspades-tool). Antimicrobial susceptibility testing showed that C. koseri L168 was resistant to imipenem, meropenem, piperacillin/tazobactam, cefotaxime, ceftazidime, cefpirome and aztreonam but remained susceptible to ciprofloxacin, gentamicin, tobramycin, amikacin, tigecycline and colistin (see Supplementary Table S1 in the online version, at DOI:10.1016/j.jgar.2018.09.016). Conjugation experiments were performed and the KPC-producing plasmid was confirmed as conjugative. The transconjugants isolated were shown to confer increased resistance to carbapenems (see Supplementary Table S1 in the online version, at DOI:10.1016/j. jgar.2018.09.016). PCR and sequencing confirmed the presence of the blaKPC-2 gene in the transconjugants. Whole-genome sequencing analysis revealed the presence of 4636 genes comprising 4535 coding sequences, 88 tRNAs and 12
Fig. 1. (a). Circular representation of isolate Citrobacter koseri L168. Circles from inside to outside represent: (G + C) skew (purple); gc bias (orange); rRNA (green); tRNA (blue); CDS from negative strand (yellow); CDS from positive strand (red); and contigs (black), respectively. (b). Schematic illustration showing the genetic environment of the blaKPC2 gene in C. koseri L168. CDS, coding sequence.
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X. Hu et al. / Journal of Global Antimicrobial Resistance 15 (2018) 166–168
rRNA. The G + C content of isolate C. koseri L168 was 53.8%. The draft genome of C. koseri L168 was assembled into 52 contigs with a mean contig length (N50) of 4 655 100 bp and a total size of 4 845 100 bp (Fig. 1A). Resistome analysis revealed that C. koseri L168 contained the βlactam resistance genes blaKPC-2 and blaMAL-1 (see Supplementary Table S2 in the online version, at DOI:10.1016/j.jgar.2018.09.016). Plasmids of incompatibility (Inc) groups IncFII and IncX4 were identified in the genome by PlasmidFinder. In addition, a 5075 bplength contig harbouring the blaKPC-2 gene was identified by plasmidSPAdes. BLAST analysis of the blaKPC-2 gene sequence showed 100% sequence identity and 100% query coverage with a 146.8-kb plasmid pKPC2_020079 (CP029381) isolated from a clinical blaKPC2-carrying K. pneumoniae strain in China. In silico analysis revealed that the blaKPC-2 gene was located on a Tn3 transposon element in C. koseri L168 with the linear structure ISKpn27–blaKPC-2–DISKpn6– korC–klcA (Fig. 1B). In China, the first case of clinical infection caused by a KPCproducing isolate was identified in 2007 in our hospital [1]. Since then, a high prevalence of blaKPC-2-carrying K. pneumoniae isolates in nosocomial infections was observed [4]. However, the occurrence of blaKPC-2-carrying C. koseri had not been reported in China. To our knowledge, this is only the second report of C. koseri producing KPC-2 [2]. Here we report the first draft genome sequence of a clinical carbapenem-resistant C. koseri isolate. The conserved structure of the blaKPC-2 gene found on a Tn3 transposon element (ISKpn27–blaKPC-2–DISKpn6–korC–klcA) is identical to the blaKPC-2 gene structures previously identified in Citrobacter freundii [5] and Klebsiella michiganensis [6]. These findings suggest that a common KPC-harbouring plasmid was transferred among different host species in our hospital. The fact that C. koseri L168 was recovered from a faecal sample suggests the potential in vivo transfer of blaKPC-2 to C. koseri. This work underscores the further spread and increasing clinical importance of KPC-producing micro-organisms in China. The genome sequence of C. koseri L168 will facilitate our understanding of the pathogenesis, multidrug resistance mechanisms and genomic features of clinically isolated blaKPC-2-carrying
C. koseri isolates. Further monitoring of bacteria carrying carbapenemase genes from patients’ gut microbiota is warranted. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. QHHL00000000. The version described in this paper is version QHHL01000000. Funding This work was supported by the National Natural Science Foundation of China [nos. 81600512 and 81741098], the National Key Research and Development Program of China [no. 2016YFD0501105] and the Zhejiang Provincial Key Research and Development Program [no. 2015C03032]. Competing interests None declared. Ethical approval Not required. References [1] Wei Z.Q., Du XX, Yu YS, Shen P, Chen YG, Li LJ. Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China. Antimicrob Agents Chemother 2007;51:763–5. [2] Mavroidi A, Neonakis I, Liakopoulos A, Papaioannou A, Ntala M, Tryposkiadis F, et al. Detection of Citrobacter koseri carrying β-lactamase KPC-2 in a hospitalised patient, Greece, July 2011. Euro Surveill 2011;16: pii: 19990. [3] Xu H, Wang X, Yu X, Zhang J, Guo L, Huang C, et al. First detection and genomics analysis of KPC-2-producing Citrobacter isolates from river sediments. Environ Pollut 2018;235:931–7. [4] Zhang R, Liu L, Zhou H, Chan EW, Li J, Fang Y, et al. Nationwide surveillance of clinical carbapenem-resistant Enterobacteriaceae (CRE) strains in China. EBioMedicine 2017;19:98–106. [5] Zheng B, Huang C, Xu H, Yu X, Zhang J, Wang X, et al. Complete nucleotide sequences of two KPC-2-encoding plasmids from the same Citrobacter freundii isolate. J Antimicrob Chemother 2018;73:531–3. [6] Zheng B, Xu H, Yu X, Lv T, Jiang X, Cheng H, et al. Identification and genomic characterization of a KPC-2-, NDM-1- and NDM-5-producing Klebsiella michiganensis isolate. J Antimicrob Chemother 2018;73:536–8.
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Genome Note
First genome sequence of a blaKPC-2-carrying Citrobacter koseri isolate collected from a patient with diarrhoea Xinjun Hua,b , Hao Xub , Yibing Shanga , Lihua Guob , Li Songa , Huping Zhanga , Feng Yuea , Hongwei Jianga,* , Beiwen Zhengb,* a Department of Infectious Diseases, The First Affiliated Hospital, and College of Clinical Medicine of Henan University of Science and Technology, Luoyang, China b 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, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 31 May 2018 Received in revised form 7 August 2018 Accepted 26 September 2018 Available online 3 October 2018
Objectives: The blaKPC gene is rarely reported in Citrobacter koseri. Here we report the first draft genome sequence of a blaKPC-2-carrying C. koseri isolate from a patient with diarrhoea. Methods: Transferability of the blaKPC-2-bearing plasmid was determined by the filter mating method. The whole genome sequence of C. koseri L168 was determined using an Illumina HiSeq platform. The genome was de novo assembled using Velvet 1.2.10. Acquired antimicrobial resistance genes and plasmid replicons were identified using ResFinder 2.1 and PlasmidFinder 1.3, respectively. Results: Antimicrobial susceptibility testing (AST) showed that C. koseri L168 was resistant to multiple antibiotics but was susceptible to ciprofloxacin, gentamicin, tobramycin, amikacin, tigecycline and colistin. A KPC-2-harbouring plasmid was conjugative and the transconjugants conferred increased resistance to carbapenems confirmed by conjugation experiments and AST. In silico analysis revealed the presence of the β-lactam resistance genes blaKPC-2 and blaMAL-1. Additionally, plasmids of incompatibility groups IncFII and IncX4 were identified in the genome by PlasmidFinder. BLAST analysis revealed that blaKPC-2 was located on a Tn3 transposon element in C. koseri L168 with the conserved linear structure ISKpn27–blaKPC-2–DISKpn6–korC–klcA. Conclusions: To our knowledge, this is only the second report of C. koseri producing KPC-2, and we report the first draft genome sequence of a blaKPC-2-carrying C. koseri isolate from a patient with diarrhoea in China. This work may facilitate our understanding of the pathogenesis, multidrug resistance mechanisms and genomic features of this species. Further monitoring of bacteria carrying carbapenemase genes in patients’ gut microbiota is warranted. © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Citrobacter koseri blaKPC-2 blaMAL-1 Resistome Whole-genome sequencing
The global increase in Enterobacteriaceae producing Klebsiella pneumoniae carbapenemase (KPC)-type carbapenemases is a major clinical challenge and a public-health concern. KPC enzymes confer resistance to carbapenems, which are members of the βlactam class of antibiotics, that are currently considered the ‘last resort’ therapeutics against infections caused by Gram-negative pathogens with multidrug resistance [1]. More importantly, KPC enzymes are typically transposon-encoded determinants and
* Corresponding authors. E-mail addresses: [email protected] (H. Jiang), [email protected] (B. Zheng).
therefore have the ability to move between plasmids and across bacterial species. Citrobacter koseri is a facultative, anaerobic, Gram-negative, non-spore-forming bacterium. It is usually found in the environment and in the digestive tract of humans and animals [1]. Occasionally, it acts as an opportunistic pathogen in a variety of human infections, mainly in children [2]. Although previous studies have documented that some C. koseri isolates harbour extended-spectrum β-lactamase (ESBL) genes, detection of C. koseri isolates producing KPC enzymes remains rare [2]. To elucidate the mechanisms of antimicrobial resistance of the blaKPC-2-carrying C. koseri isolate, here we report the first genome sequence of a blaKPC-2-carrying C. koseri isolate recovered from gut colonisation.
https://doi.org/10.1016/j.jgar.2018.09.016 2213-7165/© 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
X. Hu et al. / Journal of Global Antimicrobial Resistance 15 (2018) 166–168
Citrobacter koseri isolate L168 was isolated in 2016 from a stool sample collected from a female patient with diarrhoea in Hangzhou, China. Minimum inhibitory concentrations (MICs) for the isolate were determined by the broth microdilution method. Conjugation experiments were performed by filter mating with Escherichia coli EC600 as the recipient strain [3]. The resulting transconjugant strains were selected on brain–heart infusion agar plates containing 2 mg/L imipenem and 150 mg/L sodium azide. Presence of the blaKPC-2 gene in the transconjugants was subsequently analysed by PCR and sequencing. Genomic DNA was extracted from 2 mL of pure culture cells using a Genomic DNA Isolation Kit (QIAGEN, Hilden, Germany) and was sequenced using an Illumina HiSeq platform (Illumina Inc., San Diego, CA). De novo genomic assembly was conducted using Velvet 1.2.10. (https://www.ebi.ac.uk/zerbino/velvet/) with the default settings. Short contigs less than 300 nucleotides in length were discarded. Genome annotation was done using the Rapid Annotation using Subsystem Technology (RAST) server (http://rast.nmpdr.org/), and specific genes were manually curated. Plasmid classification and acquired antimicrobial resistance genes were identified using PlasmidFinder 1.3 (https://cge.cbs.dtu.dk/services/PlasmidFinder/)
167
and ResFinder 2.1 (https://cge.cbs.dtu.dk/services/ResFinder/), respectively, available from the Center for Genomic Epidemiology. The Comprehensive Antibiotic Resistance Database (CARD) (https:// card.mcmaster.ca/) was used to verify all putative antimicrobial resistance genes through a BLAST search. Identification of the putative KPC-carrying plasmid was done using the plasmidSPAdes assembly tool (https://omictools.com/plasmidspades-tool). Antimicrobial susceptibility testing showed that C. koseri L168 was resistant to imipenem, meropenem, piperacillin/tazobactam, cefotaxime, ceftazidime, cefpirome and aztreonam but remained susceptible to ciprofloxacin, gentamicin, tobramycin, amikacin, tigecycline and colistin (see Supplementary Table S1 in the online version, at DOI:10.1016/j.jgar.2018.09.016). Conjugation experiments were performed and the KPC-producing plasmid was confirmed as conjugative. The transconjugants isolated were shown to confer increased resistance to carbapenems (see Supplementary Table S1 in the online version, at DOI:10.1016/j. jgar.2018.09.016). PCR and sequencing confirmed the presence of the blaKPC-2 gene in the transconjugants. Whole-genome sequencing analysis revealed the presence of 4636 genes comprising 4535 coding sequences, 88 tRNAs and 12
Fig. 1. (a). Circular representation of isolate Citrobacter koseri L168. Circles from inside to outside represent: (G + C) skew (purple); gc bias (orange); rRNA (green); tRNA (blue); CDS from negative strand (yellow); CDS from positive strand (red); and contigs (black), respectively. (b). Schematic illustration showing the genetic environment of the blaKPC2 gene in C. koseri L168. CDS, coding sequence.
168
X. Hu et al. / Journal of Global Antimicrobial Resistance 15 (2018) 166–168
rRNA. The G + C content of isolate C. koseri L168 was 53.8%. The draft genome of C. koseri L168 was assembled into 52 contigs with a mean contig length (N50) of 4 655 100 bp and a total size of 4 845 100 bp (Fig. 1A). Resistome analysis revealed that C. koseri L168 contained the βlactam resistance genes blaKPC-2 and blaMAL-1 (see Supplementary Table S2 in the online version, at DOI:10.1016/j.jgar.2018.09.016). Plasmids of incompatibility (Inc) groups IncFII and IncX4 were identified in the genome by PlasmidFinder. In addition, a 5075 bplength contig harbouring the blaKPC-2 gene was identified by plasmidSPAdes. BLAST analysis of the blaKPC-2 gene sequence showed 100% sequence identity and 100% query coverage with a 146.8-kb plasmid pKPC2_020079 (CP029381) isolated from a clinical blaKPC2-carrying K. pneumoniae strain in China. In silico analysis revealed that the blaKPC-2 gene was located on a Tn3 transposon element in C. koseri L168 with the linear structure ISKpn27–blaKPC-2–DISKpn6– korC–klcA (Fig. 1B). In China, the first case of clinical infection caused by a KPCproducing isolate was identified in 2007 in our hospital [1]. Since then, a high prevalence of blaKPC-2-carrying K. pneumoniae isolates in nosocomial infections was observed [4]. However, the occurrence of blaKPC-2-carrying C. koseri had not been reported in China. To our knowledge, this is only the second report of C. koseri producing KPC-2 [2]. Here we report the first draft genome sequence of a clinical carbapenem-resistant C. koseri isolate. The conserved structure of the blaKPC-2 gene found on a Tn3 transposon element (ISKpn27–blaKPC-2–DISKpn6–korC–klcA) is identical to the blaKPC-2 gene structures previously identified in Citrobacter freundii [5] and Klebsiella michiganensis [6]. These findings suggest that a common KPC-harbouring plasmid was transferred among different host species in our hospital. The fact that C. koseri L168 was recovered from a faecal sample suggests the potential in vivo transfer of blaKPC-2 to C. koseri. This work underscores the further spread and increasing clinical importance of KPC-producing micro-organisms in China. The genome sequence of C. koseri L168 will facilitate our understanding of the pathogenesis, multidrug resistance mechanisms and genomic features of clinically isolated blaKPC-2-carrying
C. koseri isolates. Further monitoring of bacteria carrying carbapenemase genes from patients’ gut microbiota is warranted. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. QHHL00000000. The version described in this paper is version QHHL01000000. Funding This work was supported by the National Natural Science Foundation of China [nos. 81600512 and 81741098], the National Key Research and Development Program of China [no. 2016YFD0501105] and the Zhejiang Provincial Key Research and Development Program [no. 2015C03032]. Competing interests None declared. Ethical approval Not required. References [1] Wei Z.Q., Du XX, Yu YS, Shen P, Chen YG, Li LJ. Plasmid-mediated KPC-2 in a Klebsiella pneumoniae isolate from China. Antimicrob Agents Chemother 2007;51:763–5. [2] Mavroidi A, Neonakis I, Liakopoulos A, Papaioannou A, Ntala M, Tryposkiadis F, et al. Detection of Citrobacter koseri carrying β-lactamase KPC-2 in a hospitalised patient, Greece, July 2011. Euro Surveill 2011;16: pii: 19990. [3] Xu H, Wang X, Yu X, Zhang J, Guo L, Huang C, et al. First detection and genomics analysis of KPC-2-producing Citrobacter isolates from river sediments. Environ Pollut 2018;235:931–7. [4] Zhang R, Liu L, Zhou H, Chan EW, Li J, Fang Y, et al. Nationwide surveillance of clinical carbapenem-resistant Enterobacteriaceae (CRE) strains in China. EBioMedicine 2017;19:98–106. [5] Zheng B, Huang C, Xu H, Yu X, Zhang J, Wang X, et al. Complete nucleotide sequences of two KPC-2-encoding plasmids from the same Citrobacter freundii isolate. J Antimicrob Chemother 2018;73:531–3. [6] Zheng B, Xu H, Yu X, Lv T, Jiang X, Cheng H, et al. Identification and genomic characterization of a KPC-2-, NDM-1- and NDM-5-producing Klebsiella michiganensis isolate. J Antimicrob Chemother 2018;73:536–8.