- Email: [email protected]
Draft genome sequence of carbapenem-resistant Shewanella algae strain AC isolated from small abalone (Haliotis diversicolor)
Journal of Global Antimicrobial Resistance 14 (2018) 65–67
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Genome Note
Draft genome sequence of carbapenem-resistant Shewanella algae strain AC isolated from small abalone (Haliotis diversicolor) Yao-Ting Huanga , Jan-Fang Chengb , Shi-Yu Chena , Yu-Kai Honga , Zong-Yen Wub,c , Po-Yu Liud,e,f,* a
Department of Computer Science and Information Engineering, National Chung Cheng University, Chia-Yi, Taiwan Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan d Department of Nursing, Shu-Zen Junior College of Medicine and Management, Kaohsiung City, Taiwan e Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, Taiwan f Division of Infectious Diseases, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 March 2018 Received in revised form 17 May 2018 Accepted 11 June 2018 Available online 19 June 2018
Objectives: Shewanella algae is an environmental marine bacterium and an emerging opportunistic human pathogen. Moreover, there are increasing reports of strains showing multidrug resistance, particularly carbapenem-resistant isolates. Although S. algae has been found in bivalve shellfish aquaculture, there is very little genome-wide data on resistance determinants in S. algae from shellfish. The aim of this study was to determine the whole genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone in Taiwan. Methods: Bacterial genomic DNA was sequenced using an Illumina MiSeq platform with 250-bp pairedend reads. De novo genome assembly was performed using Velvet v.1.2.07. The whole genome was annotated and several candidate genes for antimicrobial resistance were identified. Results: The genome size was calculated at 4 751156 bp, with a mean G + C content of 53.09%. A total of 4164 protein-coding sequences, 7 rRNAs, 85 tRNAs and 5 non-coding RNAs were identified. The genome contains genes associated with resistance to β-lactams, trimethoprim, tetracycline, colistin and quinolones. Multiple efflux pump genes were also detected. Conclusion: Small abalone is a potential source of foodborne drug-resistant S. algae. The genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone will provide valuable information for further study of the dissemination of resistance genes at the human–animal interface. © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Shewanella algae Whole-genome sequencing Carbapenem resistance
Shewanella algae is a non-fermentative Gram-negative bacterium that is widely distributed in nature, in particular in the marine environment [1]. The organism is regarded as an emerging opportunistic human pathogen that is associated with rare but life-threatening cases of bacteraemia, soft tissue infection, bone and joint infection, lower respiratory tract infection and gastroenteritis [2]. Shewanella algae infection is associated with exposure to contaminated water [2]. Although this bacterial species is
* Corresponding author. Present address: Division of Infectious Diseases, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan. E-mail address: [email protected] (P.-Y. Liu).
generally susceptible to broad-spectrum cephalosporins, carbapenems and quinolones, there are reports of carbapenem-resistant S. algae in clinical isolates [1]. Antibiotic-resistant Shewanella have been found in bivalve shellfish aquaculture [3]. However, there is very little genome-wide data on resistance determinants in S. algae from shellfish. Here we report the whole genome sequence of carbapenemresistant S. algae strain AC that was isolated from small abalone (Haliotis diversicolor). The isolate was preliminarily identified as S. algae by 16S rRNA gene sequencing. Antimicrobial susceptibility testing and interpretation were conducted using an automated VITEK12 system (bioMérieux) according to the manufacturer’s instructions. Strain AC was found to be resistant to colistin [minimum inhibitory concentration (MIC) = 64 mg/mL] and
https://doi.org/10.1016/j.jgar.2018.06.005 2213-7165/© 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
66
Y.-T. Huang et al. / Journal of Global Antimicrobial Resistance 14 (2018) 65–67
imipenem (MIC 16 mg/mL) but susceptible to amikacin (MIC 2 mg/mL), cefepime (MIC 1 mg/mL), piperacillin/tazobactam (MIC 4 mg/mL) and ciprofloxacin (MIC 0.25 mg/mL). Shewanella algae strain AC was cultured in tryptic soy broth (Becton Dickinson, Franklin Lakes, NJ) at 37 C overnight and its genomic DNA was extracted using a QIAGEN Genomic-tip 100/G kit and Genomic DNA Buffer Set (QIAGEN, Paisley, UK) following the manufacturer’s recommendations. A total of 2 mg of each DNA sample was used to build indexed PCR-free libraries with a multiplexed high-throughput sequencing TruSeq DNA Sample Preparation Kit (Illumina Inc., San Diego, CA) according to the manufacturer’s protocols with minor modifications. Sequencing was performed on an Illumina MiSeq platform (Illumina Inc.). Whole-genome sequencing was performed with a read length of 250-bp paired-end reads on an Illumina MiSeq sequencing platform and generated 7 233 426 reads. The total read depth was 458-fold coverage and the mean read length was 301 bp. The reads were filtered using DUK (http://duk.sourceforge.net/ ). After quality trimming using the FASTQ trimmer module of the FASTX toolkit (https://github.com/agordon/fastx_toolkit), filtered reads were assembled using Velvet v.1.2.07. The resulted contigs were then scaffolded using ALLPATHS v.R46652. Genome annotation was performed using the National Center for Biotechnology Information (NCBI) Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). Functional classification of these annotated genes was carried out by RPSBLAST v.2.2.15 in
conjunction with COGs (Clusters of Orthologous Groups of proteins) databases (e-value <0.001). The final assembled genome consisted of 28 scaffolds (>2 kbp) with a total size equal to 4 751156 bp and with a mean G + C content of 53.09%. The maximum contig size was equal to 1162 543 bp, and the N50 size was equal to 413 405 bp. Gene annotation included 4164 protein-coding sequences, 7 rRNAs (5S, 16S and 23S), 85 tRNAs and 5 non-coding RNAs. No extrachromosomal elements were detected in S. algae strain AC. The average nucleotide identity values (ANI) were determined using the modified ANI algorithm. Shewanella algae strains show high levels of reciprocal relatedness (98.24–98.27%). All other Shewanella spp. compared demonstrated lower ANI values (Fig. 1). The resistome of S. algae strain AC was annotated using the Resistance Gene Identifier from the Comprehensive Antibiotic Resistance Database (CARD), along with the Integrated Microbial Genomes & Microbiomes system. BLAST was used to detect functional homologues with the antimicrobial resistance genes. The results indicated that S. algae strain AC harbours genes encoding OXA-55 and OCH-6 β-lactamases. The results are in accordance with previous reports on the detection of the chromosomally-encoded oxacillinase gene in clinical isolates of Shewanella [1]. Oxacillinases belongs to Ambler class D βlactamases, which utilise a catalytically active serine residue to hydrolysis β-lactams. Previous studies demonstrated that OXA-55 is associated with carbapenem resistance in Shewanella [1,2].
Fig. 1. Heat map and phylogenetic tree based on average nucleotide identity (ANI) values (i.e. percentage) between each genome of Shewanella algae strains and related species.
Y.-T. Huang et al. / Journal of Global Antimicrobial Resistance 14 (2018) 65–67
The following resistance genes were detected: dfrA3 (trimethoprim resistance); tet(35) (tetracycline resistance); and qnrA3 (quinolone resistance). Shewanella algae strain AC also harbours the pmrCAB operon, which has been shown to mediate resistance to colistin [4]. The analysis further identified several efflux pump gene homologues (acrAB–tolC, mdtL and msbA) that may contribute to the development of carbapenem resistance in S. algae strain AC [5]. Two genomic islands, IS10 from family IS4 and IS200 from family IS200/IS605, were identified. Both are related to composite transposons. In summary, the presence of carbapenem-resistant S. algae in small abalone and the detection of multiple antibiotic resistance genes in its genomic sequence suggest an important role of S. algae as a foodborne zoonotic agent. These data also show that aquaculture may serve as a possible reservoir of these antimicrobial resistance genes [5]. Because small abalone is widely popular in Asian cooking and is often eaten partially cooked, transmission of its natural flora such as S. algae to humans is hard to avoid. Extensive surveillance and advanced studies are warranted to better characterise the evolution and spread of drug-resistant S. algae. Nucleotide sequence accession no. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. LVDH00000000. The version described in this paper is version LVDH00000000.
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Funding Y-TH was supported in part by a grant [106-2221-E-194-056MY3] from Taiwan’s Ministry of Science and Technology (MOST). PYL was supported by grants [TCVGH-1073901B and TCVGHNK1079003] from Taichung Veterans General Hospital (Taichung, Taiwan). Competing interests None declared. Ethical approval Not required. References [1] Yousfi K., Bekal S, Usongo V, Touati A. Current trends of human infections and antibiotic resistance of the genus Shewanella. Eur J Clin Microbiol Infect Dis 2017;36:1353–62. [2] Janda JM. Shewanella: a marine pathogen as an emerging cause of human disease. Clin Microbiol Newsl 2014;36:25–9. [3] Kang CH, Shin Y, Jeon H, Choi JH, Jeong S, So JS. Antibiotic resistance of Shewanella putrefaciens isolated from shellfish collected from the West Sea in Korea. Mar Pollut Bull 2013;76:85–8. [4] Telke AA, Rolain J-M. Functional genomics to discover antibiotic resistance genes: the paradigm of resistance to colistin mediated by ethanolamine phosphotransferase in Shewanella algae MARS 14. Int J Antimicrob Agents 2015;46:648–52. [5] Bush K. Carbapenemases: partners in crime. J Glob Antimicrob Resist 2013;1:7–16.
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Genome Note
Draft genome sequence of carbapenem-resistant Shewanella algae strain AC isolated from small abalone (Haliotis diversicolor) Yao-Ting Huanga , Jan-Fang Chengb , Shi-Yu Chena , Yu-Kai Honga , Zong-Yen Wub,c , Po-Yu Liud,e,f,* a
Department of Computer Science and Information Engineering, National Chung Cheng University, Chia-Yi, Taiwan Department of Energy, Joint Genome Institute, Walnut Creek, CA, USA Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan d Department of Nursing, Shu-Zen Junior College of Medicine and Management, Kaohsiung City, Taiwan e Rong Hsing Research Center for Translational Medicine, National Chung Hsing University, Taichung, Taiwan f Division of Infectious Diseases, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 30 March 2018 Received in revised form 17 May 2018 Accepted 11 June 2018 Available online 19 June 2018
Objectives: Shewanella algae is an environmental marine bacterium and an emerging opportunistic human pathogen. Moreover, there are increasing reports of strains showing multidrug resistance, particularly carbapenem-resistant isolates. Although S. algae has been found in bivalve shellfish aquaculture, there is very little genome-wide data on resistance determinants in S. algae from shellfish. The aim of this study was to determine the whole genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone in Taiwan. Methods: Bacterial genomic DNA was sequenced using an Illumina MiSeq platform with 250-bp pairedend reads. De novo genome assembly was performed using Velvet v.1.2.07. The whole genome was annotated and several candidate genes for antimicrobial resistance were identified. Results: The genome size was calculated at 4 751156 bp, with a mean G + C content of 53.09%. A total of 4164 protein-coding sequences, 7 rRNAs, 85 tRNAs and 5 non-coding RNAs were identified. The genome contains genes associated with resistance to β-lactams, trimethoprim, tetracycline, colistin and quinolones. Multiple efflux pump genes were also detected. Conclusion: Small abalone is a potential source of foodborne drug-resistant S. algae. The genome sequence of carbapenem-resistant S. algae strain AC isolated from small abalone will provide valuable information for further study of the dissemination of resistance genes at the human–animal interface. © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
Keywords: Shewanella algae Whole-genome sequencing Carbapenem resistance
Shewanella algae is a non-fermentative Gram-negative bacterium that is widely distributed in nature, in particular in the marine environment [1]. The organism is regarded as an emerging opportunistic human pathogen that is associated with rare but life-threatening cases of bacteraemia, soft tissue infection, bone and joint infection, lower respiratory tract infection and gastroenteritis [2]. Shewanella algae infection is associated with exposure to contaminated water [2]. Although this bacterial species is
* Corresponding author. Present address: Division of Infectious Diseases, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan. E-mail address: [email protected] (P.-Y. Liu).
generally susceptible to broad-spectrum cephalosporins, carbapenems and quinolones, there are reports of carbapenem-resistant S. algae in clinical isolates [1]. Antibiotic-resistant Shewanella have been found in bivalve shellfish aquaculture [3]. However, there is very little genome-wide data on resistance determinants in S. algae from shellfish. Here we report the whole genome sequence of carbapenemresistant S. algae strain AC that was isolated from small abalone (Haliotis diversicolor). The isolate was preliminarily identified as S. algae by 16S rRNA gene sequencing. Antimicrobial susceptibility testing and interpretation were conducted using an automated VITEK12 system (bioMérieux) according to the manufacturer’s instructions. Strain AC was found to be resistant to colistin [minimum inhibitory concentration (MIC) = 64 mg/mL] and
https://doi.org/10.1016/j.jgar.2018.06.005 2213-7165/© 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.
66
Y.-T. Huang et al. / Journal of Global Antimicrobial Resistance 14 (2018) 65–67
imipenem (MIC 16 mg/mL) but susceptible to amikacin (MIC 2 mg/mL), cefepime (MIC 1 mg/mL), piperacillin/tazobactam (MIC 4 mg/mL) and ciprofloxacin (MIC 0.25 mg/mL). Shewanella algae strain AC was cultured in tryptic soy broth (Becton Dickinson, Franklin Lakes, NJ) at 37 C overnight and its genomic DNA was extracted using a QIAGEN Genomic-tip 100/G kit and Genomic DNA Buffer Set (QIAGEN, Paisley, UK) following the manufacturer’s recommendations. A total of 2 mg of each DNA sample was used to build indexed PCR-free libraries with a multiplexed high-throughput sequencing TruSeq DNA Sample Preparation Kit (Illumina Inc., San Diego, CA) according to the manufacturer’s protocols with minor modifications. Sequencing was performed on an Illumina MiSeq platform (Illumina Inc.). Whole-genome sequencing was performed with a read length of 250-bp paired-end reads on an Illumina MiSeq sequencing platform and generated 7 233 426 reads. The total read depth was 458-fold coverage and the mean read length was 301 bp. The reads were filtered using DUK (http://duk.sourceforge.net/ ). After quality trimming using the FASTQ trimmer module of the FASTX toolkit (https://github.com/agordon/fastx_toolkit), filtered reads were assembled using Velvet v.1.2.07. The resulted contigs were then scaffolded using ALLPATHS v.R46652. Genome annotation was performed using the National Center for Biotechnology Information (NCBI) Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP). Functional classification of these annotated genes was carried out by RPSBLAST v.2.2.15 in
conjunction with COGs (Clusters of Orthologous Groups of proteins) databases (e-value <0.001). The final assembled genome consisted of 28 scaffolds (>2 kbp) with a total size equal to 4 751156 bp and with a mean G + C content of 53.09%. The maximum contig size was equal to 1162 543 bp, and the N50 size was equal to 413 405 bp. Gene annotation included 4164 protein-coding sequences, 7 rRNAs (5S, 16S and 23S), 85 tRNAs and 5 non-coding RNAs. No extrachromosomal elements were detected in S. algae strain AC. The average nucleotide identity values (ANI) were determined using the modified ANI algorithm. Shewanella algae strains show high levels of reciprocal relatedness (98.24–98.27%). All other Shewanella spp. compared demonstrated lower ANI values (Fig. 1). The resistome of S. algae strain AC was annotated using the Resistance Gene Identifier from the Comprehensive Antibiotic Resistance Database (CARD), along with the Integrated Microbial Genomes & Microbiomes system. BLAST was used to detect functional homologues with the antimicrobial resistance genes. The results indicated that S. algae strain AC harbours genes encoding OXA-55 and OCH-6 β-lactamases. The results are in accordance with previous reports on the detection of the chromosomally-encoded oxacillinase gene in clinical isolates of Shewanella [1]. Oxacillinases belongs to Ambler class D βlactamases, which utilise a catalytically active serine residue to hydrolysis β-lactams. Previous studies demonstrated that OXA-55 is associated with carbapenem resistance in Shewanella [1,2].
Fig. 1. Heat map and phylogenetic tree based on average nucleotide identity (ANI) values (i.e. percentage) between each genome of Shewanella algae strains and related species.
Y.-T. Huang et al. / Journal of Global Antimicrobial Resistance 14 (2018) 65–67
The following resistance genes were detected: dfrA3 (trimethoprim resistance); tet(35) (tetracycline resistance); and qnrA3 (quinolone resistance). Shewanella algae strain AC also harbours the pmrCAB operon, which has been shown to mediate resistance to colistin [4]. The analysis further identified several efflux pump gene homologues (acrAB–tolC, mdtL and msbA) that may contribute to the development of carbapenem resistance in S. algae strain AC [5]. Two genomic islands, IS10 from family IS4 and IS200 from family IS200/IS605, were identified. Both are related to composite transposons. In summary, the presence of carbapenem-resistant S. algae in small abalone and the detection of multiple antibiotic resistance genes in its genomic sequence suggest an important role of S. algae as a foodborne zoonotic agent. These data also show that aquaculture may serve as a possible reservoir of these antimicrobial resistance genes [5]. Because small abalone is widely popular in Asian cooking and is often eaten partially cooked, transmission of its natural flora such as S. algae to humans is hard to avoid. Extensive surveillance and advanced studies are warranted to better characterise the evolution and spread of drug-resistant S. algae. Nucleotide sequence accession no. This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. LVDH00000000. The version described in this paper is version LVDH00000000.
67
Funding Y-TH was supported in part by a grant [106-2221-E-194-056MY3] from Taiwan’s Ministry of Science and Technology (MOST). PYL was supported by grants [TCVGH-1073901B and TCVGHNK1079003] from Taichung Veterans General Hospital (Taichung, Taiwan). Competing interests None declared. Ethical approval Not required. References [1] Yousfi K., Bekal S, Usongo V, Touati A. Current trends of human infections and antibiotic resistance of the genus Shewanella. Eur J Clin Microbiol Infect Dis 2017;36:1353–62. [2] Janda JM. Shewanella: a marine pathogen as an emerging cause of human disease. Clin Microbiol Newsl 2014;36:25–9. [3] Kang CH, Shin Y, Jeon H, Choi JH, Jeong S, So JS. Antibiotic resistance of Shewanella putrefaciens isolated from shellfish collected from the West Sea in Korea. Mar Pollut Bull 2013;76:85–8. [4] Telke AA, Rolain J-M. Functional genomics to discover antibiotic resistance genes: the paradigm of resistance to colistin mediated by ethanolamine phosphotransferase in Shewanella algae MARS 14. Int J Antimicrob Agents 2015;46:648–52. [5] Bush K. Carbapenemases: partners in crime. J Glob Antimicrob Resist 2013;1:7–16.