- Email: [email protected]
Draft genome sequence of tetracycline-resistant Klebsiella oxytoca CCTCC M207023 producing 2,3-butanediol isolated from China
Journal of Global Antimicrobial Resistance 20 (2020) 160–162
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Genome Note
Draft genome sequence of tetracycline-resistant Klebsiella oxytoca CCTCC M207023 producing 2,3-butanediol isolated from China Kai-Feng Wang, Wei-Jian Wang, Xiao-Jun Ji* College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 August 2019 Received in revised form 22 November 2019 Accepted 9 December 2019 Available online 23 December 2019
Objectives: Recently, the Gram-negative bacterium Klebsiella oxytoca has been identified as an emerging pathogen. Here we report the draft genome of a 2,3-butanediol-producing strain, K. oxytoca CCTCC M207023, isolated from soil in Nanjing, China. The tetracycline-resistant phenotype and the high yield of 2,3-butanediol was demonstrated. Methods: The draft genome of K. oxytoca CCTCC M207023 was determined using an Illumina NovaSeqTM 6000 next-generation DNA sequencing platform. Clean sequencing data were subsequently assembled using SOAPdenovo. Results: The draft genome of K. oxytoca CCTCC M207023, comprising 5 658 144 bp and with a GC content of 56.50%, was assembled into 5262 open-reading frames (ORFs). Antimicrobial resistance genes were also annotated. Conclusions: The draft genome sequence of K. oxytoca CCTCC M207023 reported here will be a reference for comparative analysis with the antimicrobial resistance mechanisms for the safety of 2,3-butanediol industrial production. © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.
Keywords: Klebsiella oxytoca Whole-genome sequencing Genome analysis
A large majority of nosocomial infections are caused by Klebsiella spp., which is the second most common bacterium after Escherichia coli. The vast majority of Klebsiella isolated from the gastrointestinal tract of infected patients are Klebsiella pneumoniae. However, Klebsiella oxytoca, an opportunistic bacterium, has also been isolated as an emerging pathogen [1]. Klebsiella oxytoca is a Gram-negative rod-shaped bacterium that is capable of metabolising major sugars present in hemicellulose and cellulose hydrolysates to produce 2,3-butanediol [2]. Because of strain specificity, K. oxytoca isolated from patients can produce cytotoxin, whereas under normal circumstances it does not [3,4]. Importantly, K. oxytoca can produce β-lactamases that confer resistance to aminopenicillins and carboxypenicillins [5] leading to antibiotic-associated haemorrhagic colitis due to cytotoxin production following antibiotic consumption and a decrease in the microbial flora. Klebsiella oxytoca ME-303, a 2,3-butanediol-hyperproducing strain isolated from soil in Nanjing, China, was stored in the China
* Corresponding author. E-mail address: [email protected] (X.-J. Ji).
Center for Type Culture Collection (deposition no. CCTCC M207023). It has been shown to grow rapidly in simple media such as wheat and rice straw hydrolysates. It was also found that K. oxytoca CCTCC M207023 could grow on tetracycline-containing agar plates. To ensure the safety of K. oxytoca CCTCC M207023 in industrial production, more comprehensive information about the specific mechanisms of cytotoxin production and drug resistance should be investigated. Consequently, the sequenced genome of K. oxytoca CCTCC M207023 has been provided in this report. Genomic DNA of K. oxytoca CCTCC M207023 was extracted using a TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit v.3.0 (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The DNA was sequenced using an Illumina NovaSeqTM 6000 next-generation DNA sequencing platform (Illumina Inc., San Diego, CA, USA). Raw image data obtained by sequencing were converted into sequence data (10 959 622 raw reads) by base calling. Clean sequencing data were assembled using SOAPdenovo following filtration of the raw reads using Burrows–Wheeler Aligner (BWA). Then, MinHash and sort-merge were used to effectively reduce the noise between overlaps through a probabilistic algorithm. The coding sequence was predicted by Glimmer and the repeated sequence was estimated by RepeatMasker. The
http://dx.doi.org/10.1016/j.jgar.2019.12.011 2213-7165/© 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.
K.-F. Wang et al. / Journal of Global Antimicrobial Resistance 20 (2020) 160–162
open-reading frames (ORFs) of K. oxytoca CCTCC M207023 were annotated using Nr, SwissProt, Clusters of Orthologous Groups (COG), Gene Ontology (GO) and KEGG databases. The draft genome of K. oxytoca CCTCC M207023, comprising 5 658 144 bp and with a GC content of 56.50%, was assembled into 105 scaffolds. The number of bases N (Gap (N)) is 6382 and the maximum length of total scaffolds is 390 886. Protein-coding sequences were predicted by Glimmer, including 5262 genes with a maximum length of 9477 bp. The genome contains three 5S rRNA genes and 81 tRNA genes identified by rRNAmmer and tRNAscan. Moreover, ten transposable elements can be authenticated, six of them belonging to TY1_Copia type. The genome map was illustrated using Circos v.0.64 (Fig. 1). Many resistance genes can be found in the genome of K. oxytoca CCTCC M207023, which offers more information on virulence factors and developed antimicrobial resistance. Antimicrobial resistance genes conferring resistance to peptides (bacA), fosfomycin (fosA5 and uhpT) and polymyxins (arnA, arnC, arnD, arnE, arnF and arnT) were identified. BacA confers resistance to peptide antibiotics via molecular bypass. The fosA5 genes encodes an antibiotic inactivation enzyme related to fosfomycin resistance. In addition, many genes related to β-lactam resistance were identified, including blaOXY-1, nmcR, smeR and ampC. The blaOXY-1 gene encodes a class A β-lactamase that hydrolyses the antibiotic β-lactam ring and disables the molecule’s antibacterial properties. Both nmcR and smeR are transcriptional activators of carbapenem-hydrolysing βlactamases. Moreover, the aminoglycoside resistance gene aph(3ʹ)Ia (aminoglycoside O-phosphotransferase) was identifued, which modifies aminoglycosides by phosphorylation. The kdpE gene, encoding a two-component system response regulator modulating antibiotic efflux, is related to aminoglycoside resistance. Importantly, the tetracycline resistance gene tetA and the tetracycline
161
repressor protein TetRA were also identified, which may determine K. oxytoca CCTCC M207023 resistance to tetracycline. In addition, K. oxytoca CCTCC M207023 contains multidrug resistance proteins such as MdtE, MdtH, MdtK, MdtN, MexA and MexB. Moreover, there are many multidrug transport systems such as multidrug transporters EmrD, EmrE and PA4990, multidrug efflux pump subunits AcrA and AcrB, multidrug efflux pump accessory protein AcrZ, and other drug efflux system proteins including MdfA, MdtG, MdtI, MdtL, MdtM and MdtO. The ABC3 protein is related to multidrug resistance owing to protection from oxidative stress. The multidrug transporter ABC, including MdtA (membrane fusion protein) and the transmembrane MdtB–MdtC heteromultimer, constitutes a resistance–nodulation–cell division (RND) drug efflux system conferring resistance to bile salts but it does not work without the outer membrane subunit TolC. BaeR, a transcriptional regulator of the mdtABC operon, was also identified. Moreover, virulence genes including MsgA, VsdF, aerobactin (IutA) and yersiniabactin (Irp1, Irp2, YbtT and YbtE) as well as a major facilitator superfamily (MFS) transporter, related to two genes mfsd2b and mfsd3, were also observed in the genome. Ybt is a mixed iron carrier protein that is beneficial to the propagation of strains and can promote the formation of bacterial biofilms and enhance virulence. There are many genes in K. oxytoca CCTCC M207023 related to type 1 fimbriae (fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH and fimI) and type 3 fimbriae (mrkA, mrkB, mrkC, mrkD, mrkE and mrkF). Type 3 fimbriae contribute to biofilm formation, which may lead to increased antibiotic resistance. Consequently, this report provides a more convenient way for deeper investigation of the multidrug resistance mechanisms of K. oxytoca CCTCC M207023. In summary, the genome of K. oxytoca CCTCC M207023, with high 2,3-butanediol-producing capacity isolated from soil in Nanjing, China, is reported in this work. As presented in the
Fig. 1. Genome map of Klebsiella oxytoca CCTCC M207023. The circle, from outside to inside, is annotated as follows: positive and negative chain genes by Clusters of Orthologous Groups (COG) annotation, non-coding RNA (black indicates tRNA and red indicates rRNA), GC content (red indicates greater than mean and blue indicates less than mean), GC skew [GC deviation; GC skew = (GC)/(G + C); purple indicates greater than 0 and orange indicates less than 0].
162
K.-F. Wang et al. / Journal of Global Antimicrobial Resistance 20 (2020) 160–162
experimental data, K. oxytoca CCTCC M207023 is resistant to tetracycline, evidenced by the presence of the tetA gene and many tetracycline repressor proteins. The tetracycline resistance can offer more information on measures to inhibit the growth of K. oxytoca CCTCC M207023. Besides, it provides a possibility to use tetracycline resistance as a screening marker to knock-in or knockout genes in order to obtain dominant strains for industrial production. Moreover, many drug and multidrug resistance genes have also been annotated. Consequently, the genome of K. oxytoca CCTCC M207023 as well as annotation present an easy way to unveil the antimicrobial resistance mechanisms for the safety of 2,3-butanediol industrial production.
2018YFA0903700], the National Natural Science Foundation of China [No. 21776131], the Six Talent Peaks Project in Jiangsu Province of China [No. 2018-SWYY-047] and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture [No. XTD1814].
Nucleotide sequence accession no
References
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. VOJB00000000. The version described in this paper is version VOJB01000000. The BioProject ID in GenBank is PRJNA556821.
[1] Singh L, Cariappa MP, Kaur M. Klebsiella oxytoca: an emerging pathogen? Med J Armed Forces India 2016;72(Suppl. 1):S59–61. [2] Ji XJ, Huang H, Ouyang PK. Microbial 2,3-butanediol production: a state-of-theart review. Biotechnol Adv 2011;29:351–64. [3] Minami J, Saito S, Yoshida T, Uemura T, Okabe A. Biological activities and chemical composition of a cytotoxin of Klebsiella oxytoca. Microbiology 1992;138:1921–7. [4] Joainig MM, Gorkiewicz G, Leitner E, Weberhofer P, Zollner-Schwetz I, Lippe I, et al. Cytotoxic effects of Klebsiella oxytoca strains isolated from patients with antibiotic-associated hemorrhagic colitis or other diseases caused by infections and from healthy subjects. J Clin Microbiol 2010;48:817–24. [5] Livermore DM. β-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995;8:557–84.
Funding This work was financially supported by the National Science Fund for Excellent Young Scholars of China [No. 21922806], the National Key Research and Development Program of China [No.
Competing interests None declared. Ethical approval Not required.
Contents lists available at ScienceDirect
Journal of Global Antimicrobial Resistance journal homepage: www.elsevier.com/locate/jgar
Genome Note
Draft genome sequence of tetracycline-resistant Klebsiella oxytoca CCTCC M207023 producing 2,3-butanediol isolated from China Kai-Feng Wang, Wei-Jian Wang, Xiao-Jun Ji* College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, No. 30 South Puzhu Road, Nanjing 211816, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 4 August 2019 Received in revised form 22 November 2019 Accepted 9 December 2019 Available online 23 December 2019
Objectives: Recently, the Gram-negative bacterium Klebsiella oxytoca has been identified as an emerging pathogen. Here we report the draft genome of a 2,3-butanediol-producing strain, K. oxytoca CCTCC M207023, isolated from soil in Nanjing, China. The tetracycline-resistant phenotype and the high yield of 2,3-butanediol was demonstrated. Methods: The draft genome of K. oxytoca CCTCC M207023 was determined using an Illumina NovaSeqTM 6000 next-generation DNA sequencing platform. Clean sequencing data were subsequently assembled using SOAPdenovo. Results: The draft genome of K. oxytoca CCTCC M207023, comprising 5 658 144 bp and with a GC content of 56.50%, was assembled into 5262 open-reading frames (ORFs). Antimicrobial resistance genes were also annotated. Conclusions: The draft genome sequence of K. oxytoca CCTCC M207023 reported here will be a reference for comparative analysis with the antimicrobial resistance mechanisms for the safety of 2,3-butanediol industrial production. © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.
Keywords: Klebsiella oxytoca Whole-genome sequencing Genome analysis
A large majority of nosocomial infections are caused by Klebsiella spp., which is the second most common bacterium after Escherichia coli. The vast majority of Klebsiella isolated from the gastrointestinal tract of infected patients are Klebsiella pneumoniae. However, Klebsiella oxytoca, an opportunistic bacterium, has also been isolated as an emerging pathogen [1]. Klebsiella oxytoca is a Gram-negative rod-shaped bacterium that is capable of metabolising major sugars present in hemicellulose and cellulose hydrolysates to produce 2,3-butanediol [2]. Because of strain specificity, K. oxytoca isolated from patients can produce cytotoxin, whereas under normal circumstances it does not [3,4]. Importantly, K. oxytoca can produce β-lactamases that confer resistance to aminopenicillins and carboxypenicillins [5] leading to antibiotic-associated haemorrhagic colitis due to cytotoxin production following antibiotic consumption and a decrease in the microbial flora. Klebsiella oxytoca ME-303, a 2,3-butanediol-hyperproducing strain isolated from soil in Nanjing, China, was stored in the China
* Corresponding author. E-mail address: [email protected] (X.-J. Ji).
Center for Type Culture Collection (deposition no. CCTCC M207023). It has been shown to grow rapidly in simple media such as wheat and rice straw hydrolysates. It was also found that K. oxytoca CCTCC M207023 could grow on tetracycline-containing agar plates. To ensure the safety of K. oxytoca CCTCC M207023 in industrial production, more comprehensive information about the specific mechanisms of cytotoxin production and drug resistance should be investigated. Consequently, the sequenced genome of K. oxytoca CCTCC M207023 has been provided in this report. Genomic DNA of K. oxytoca CCTCC M207023 was extracted using a TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit v.3.0 (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The DNA was sequenced using an Illumina NovaSeqTM 6000 next-generation DNA sequencing platform (Illumina Inc., San Diego, CA, USA). Raw image data obtained by sequencing were converted into sequence data (10 959 622 raw reads) by base calling. Clean sequencing data were assembled using SOAPdenovo following filtration of the raw reads using Burrows–Wheeler Aligner (BWA). Then, MinHash and sort-merge were used to effectively reduce the noise between overlaps through a probabilistic algorithm. The coding sequence was predicted by Glimmer and the repeated sequence was estimated by RepeatMasker. The
http://dx.doi.org/10.1016/j.jgar.2019.12.011 2213-7165/© 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.
K.-F. Wang et al. / Journal of Global Antimicrobial Resistance 20 (2020) 160–162
open-reading frames (ORFs) of K. oxytoca CCTCC M207023 were annotated using Nr, SwissProt, Clusters of Orthologous Groups (COG), Gene Ontology (GO) and KEGG databases. The draft genome of K. oxytoca CCTCC M207023, comprising 5 658 144 bp and with a GC content of 56.50%, was assembled into 105 scaffolds. The number of bases N (Gap (N)) is 6382 and the maximum length of total scaffolds is 390 886. Protein-coding sequences were predicted by Glimmer, including 5262 genes with a maximum length of 9477 bp. The genome contains three 5S rRNA genes and 81 tRNA genes identified by rRNAmmer and tRNAscan. Moreover, ten transposable elements can be authenticated, six of them belonging to TY1_Copia type. The genome map was illustrated using Circos v.0.64 (Fig. 1). Many resistance genes can be found in the genome of K. oxytoca CCTCC M207023, which offers more information on virulence factors and developed antimicrobial resistance. Antimicrobial resistance genes conferring resistance to peptides (bacA), fosfomycin (fosA5 and uhpT) and polymyxins (arnA, arnC, arnD, arnE, arnF and arnT) were identified. BacA confers resistance to peptide antibiotics via molecular bypass. The fosA5 genes encodes an antibiotic inactivation enzyme related to fosfomycin resistance. In addition, many genes related to β-lactam resistance were identified, including blaOXY-1, nmcR, smeR and ampC. The blaOXY-1 gene encodes a class A β-lactamase that hydrolyses the antibiotic β-lactam ring and disables the molecule’s antibacterial properties. Both nmcR and smeR are transcriptional activators of carbapenem-hydrolysing βlactamases. Moreover, the aminoglycoside resistance gene aph(3ʹ)Ia (aminoglycoside O-phosphotransferase) was identifued, which modifies aminoglycosides by phosphorylation. The kdpE gene, encoding a two-component system response regulator modulating antibiotic efflux, is related to aminoglycoside resistance. Importantly, the tetracycline resistance gene tetA and the tetracycline
161
repressor protein TetRA were also identified, which may determine K. oxytoca CCTCC M207023 resistance to tetracycline. In addition, K. oxytoca CCTCC M207023 contains multidrug resistance proteins such as MdtE, MdtH, MdtK, MdtN, MexA and MexB. Moreover, there are many multidrug transport systems such as multidrug transporters EmrD, EmrE and PA4990, multidrug efflux pump subunits AcrA and AcrB, multidrug efflux pump accessory protein AcrZ, and other drug efflux system proteins including MdfA, MdtG, MdtI, MdtL, MdtM and MdtO. The ABC3 protein is related to multidrug resistance owing to protection from oxidative stress. The multidrug transporter ABC, including MdtA (membrane fusion protein) and the transmembrane MdtB–MdtC heteromultimer, constitutes a resistance–nodulation–cell division (RND) drug efflux system conferring resistance to bile salts but it does not work without the outer membrane subunit TolC. BaeR, a transcriptional regulator of the mdtABC operon, was also identified. Moreover, virulence genes including MsgA, VsdF, aerobactin (IutA) and yersiniabactin (Irp1, Irp2, YbtT and YbtE) as well as a major facilitator superfamily (MFS) transporter, related to two genes mfsd2b and mfsd3, were also observed in the genome. Ybt is a mixed iron carrier protein that is beneficial to the propagation of strains and can promote the formation of bacterial biofilms and enhance virulence. There are many genes in K. oxytoca CCTCC M207023 related to type 1 fimbriae (fimA, fimB, fimC, fimD, fimE, fimF, fimG, fimH and fimI) and type 3 fimbriae (mrkA, mrkB, mrkC, mrkD, mrkE and mrkF). Type 3 fimbriae contribute to biofilm formation, which may lead to increased antibiotic resistance. Consequently, this report provides a more convenient way for deeper investigation of the multidrug resistance mechanisms of K. oxytoca CCTCC M207023. In summary, the genome of K. oxytoca CCTCC M207023, with high 2,3-butanediol-producing capacity isolated from soil in Nanjing, China, is reported in this work. As presented in the
Fig. 1. Genome map of Klebsiella oxytoca CCTCC M207023. The circle, from outside to inside, is annotated as follows: positive and negative chain genes by Clusters of Orthologous Groups (COG) annotation, non-coding RNA (black indicates tRNA and red indicates rRNA), GC content (red indicates greater than mean and blue indicates less than mean), GC skew [GC deviation; GC skew = (GC)/(G + C); purple indicates greater than 0 and orange indicates less than 0].
162
K.-F. Wang et al. / Journal of Global Antimicrobial Resistance 20 (2020) 160–162
experimental data, K. oxytoca CCTCC M207023 is resistant to tetracycline, evidenced by the presence of the tetA gene and many tetracycline repressor proteins. The tetracycline resistance can offer more information on measures to inhibit the growth of K. oxytoca CCTCC M207023. Besides, it provides a possibility to use tetracycline resistance as a screening marker to knock-in or knockout genes in order to obtain dominant strains for industrial production. Moreover, many drug and multidrug resistance genes have also been annotated. Consequently, the genome of K. oxytoca CCTCC M207023 as well as annotation present an easy way to unveil the antimicrobial resistance mechanisms for the safety of 2,3-butanediol industrial production.
2018YFA0903700], the National Natural Science Foundation of China [No. 21776131], the Six Talent Peaks Project in Jiangsu Province of China [No. 2018-SWYY-047] and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture [No. XTD1814].
Nucleotide sequence accession no
References
This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession no. VOJB00000000. The version described in this paper is version VOJB01000000. The BioProject ID in GenBank is PRJNA556821.
[1] Singh L, Cariappa MP, Kaur M. Klebsiella oxytoca: an emerging pathogen? Med J Armed Forces India 2016;72(Suppl. 1):S59–61. [2] Ji XJ, Huang H, Ouyang PK. Microbial 2,3-butanediol production: a state-of-theart review. Biotechnol Adv 2011;29:351–64. [3] Minami J, Saito S, Yoshida T, Uemura T, Okabe A. Biological activities and chemical composition of a cytotoxin of Klebsiella oxytoca. Microbiology 1992;138:1921–7. [4] Joainig MM, Gorkiewicz G, Leitner E, Weberhofer P, Zollner-Schwetz I, Lippe I, et al. Cytotoxic effects of Klebsiella oxytoca strains isolated from patients with antibiotic-associated hemorrhagic colitis or other diseases caused by infections and from healthy subjects. J Clin Microbiol 2010;48:817–24. [5] Livermore DM. β-Lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995;8:557–84.
Funding This work was financially supported by the National Science Fund for Excellent Young Scholars of China [No. 21922806], the National Key Research and Development Program of China [No.
Competing interests None declared. Ethical approval Not required.