|Year : 2020 | Volume
| Issue : 2 | Page : 162-166
Genomic investigation of emerging zoonotic pathogen Shewanella xiamenensis
Jui-Hsing Wang1, Shu-Ying Tseng2, Kwong-Chung Tung2
1 Division of Infectious Disease, Department of Internal Medicine, Taichung Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Taichung; Department of Internal Medicine, School of Medicine, Tzu Chi University, Hualien, Taiwan
2 Department of Veterinary Medicine, National Chung Hsing University, Taichung, Taiwan
|Date of Submission||16-Mar-2019|
|Date of Decision||19-Mar-2019|
|Date of Acceptance||28-Mar-2019|
|Date of Web Publication||13-Jun-2019|
Prof. Kwong-Chung Tung
Department of Veterinary Medicine, National Chung Hsing University, Taichung, 145, Xingda Road, South District, Taichung
Source of Support: None, Conflict of Interest: None
Objective: Shewanella xiamenensis is an emerging zoonotic pathogen commonly found in aquatic ecosystem. Clustered regularly interspaced short palindromic repeats (CRISPR) and (CRISPR)-associated gene systems act as adaptive immune system of prokaryotes. Recently, growing evidence suggested their role in bacterial virulence and resistance. Despite its medical importance, little is known about the genomic characteristics of S. xiamenensis. Materials and Methods: Strain ZYW6 was isolated from Epinephelus awoara. We sequenced the 16S rRNA gene and blast against the GenBank bacterial database. Antibiotic susceptibility tests and interpretation were performed by automatic VITEK 2 system. We extracted the genomic DNA with QIAGEN Genomic-tip 100/G kit and QIAGEN Genomic DNA Buffer Set. Whole-genome shotgun sequencing was performed using the Illumina MiSeq sequencer. To identify the CRISPR-Cas System in the genome of S. xiamenensis ZYW6, the Integrated Microbial Genomes and Microbiomes and CRISPRFinder were used. Results: We characterized the genome of a S. xiamenensis strain. The genome is 4,765,190 bp in length and encodes 4262 open-reading frames. Type I CRISPR-Cas system and serine biosynthesis genes were identified. Conclusion: Our results demonstrate the genetic structure of CRISPR-Cas system, l-serine synthesis, and oxacillinase in S. xiamenensis. The report of antibiotics resistance genes in the study might indicate a possible reservoir of antimicrobial drug resistance determinants in food animal, resulting in potential infection source. The findings provide insights into the structure and composition of CRISPR-Cas system in S. xiamenensis and foundation for future biological validation.
Keywords: Clustered regularly interspaced short palindromic repeats-Cas system, Serine, Shewanella xiamenensis, Whole-genome sequencing
|How to cite this article:|
Wang JH, Tseng SY, Tung KC. Genomic investigation of emerging zoonotic pathogen Shewanella xiamenensis. Tzu Chi Med J 2020;32:162-6
| Introduction|| |
Emerging infection is a severe threat to global health. Most emerging infections are zoonoses or have zoonotic origins that warrant novel diagnostic and control strategies. Shewanella xiamenensis is an emerging zoonotic pathogen first reported in the coastal sea sediment in Xiamen, China. The organism has been reported from aquatic ecosystem worldwide, including wastewater,, freshwater, and seawater. It was also found to be the causative organism of human intra-abdominal infection and intestinal colonization. Identification of S. xiamenensis is difficult, as illustrated by Zong, because 16S rRNA gene sequencing alone is not capable to differentiate S. xiamenensis from closely related species. Thus, the true infection rate may be underestimated. The pathogenesis determinants in S. xiamenensis are largely unknown.
The rapid advance in sequencing technology changes the study of bacterial infections and leads to the discovery of novel pathogenesis determinants. Genomic sequencing allows the implementation of study of a broad range of pathogenic organisms' characteristics and is applicable on a wide range of pathogens. In many studies of prokaryotes, such as bacterial and archaeal genomes, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are applied. Early studies demonstrated that the CRISPR-Cas system is an adaptive immune system in prokaryotes. At present, the CRISPR-Cas systems are classified into three distinct types, type I, Type II, and Type III, based on the signature cas genes, sequence similarity, and the phylogeny of Cas1. Type I system consists of six major subtypes (I-A to I-F) which utilize a crRNP to identify targets. Recent studies further suggested its role in virulence and resistance. Earlier study demonstrated L-serine production is an important virulent feature in pathogenic bacteria. Although L-serine production was found in Shewanella,, little is known about the genetic background.
Despite its medical importance, little is known about the genomic characteristics of S. xiamenensis. To identify the CRISPR-Cas systems and genetic background of the L-serine synthesis, we sequenced and analyzed the genome of S. xiamenensis strain.
| Materials and Methods|| |
Strain isolation and antimicrobial susceptibility tests
Strain ZYW6 was isolated from Epinephelus awoara on trypticase soy agar with 5% sheep blood (Becton Dickinson, San Jose, CA, USA). The isolate was preliminary identified by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (bioMérieux, Marcy-l'Etoile, France) and by Sanger sequencing of its 16S rRNA gene sequencing. For amplification of the 16S rRNA gene, we used primers with B27F (5'-AGAGTTTGATCCTGGCTCAG-3') and U1492R (5'-GGTTACCTTGTTACGACTT-3'). Then, we sequenced the PCR product and BLAST against the GenBank bacterial database of the National Center for Biotechnology Information. To confirm the L-serine production in ZYW6, biochemical characterization was performed using API ID 32 GN strips inoculated and read according to the recommendations of the manufacturer (bioMérieux, Marcy-l'Etoile, France). Antibiotic susceptibility tests and interpretation were performed by the VITEK 2 system (bioMérieux, Marcy-l'Etoile, France) according to the manufacturer's instructions. Escherichia More Details coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality controls.
DNA extraction and library preparation
We extracted the genomic DNA with QIAGEN Genomic-tip 100/G kit and QIAGEN Genomic DNA Buffer Set (QIAGEN, Paisley, UK). The library preparation was conducted using a multiplexed high-throughput sequencing TruSeq DNA Sample Preparation Kit (Illumina, San Diego, CA, USA).
Genome sequencing and assembly
We performed sequencing by Illumina MiSeq sequencer using a read length of 250 bp. Consequently, 5,154,704 reads were obtained. There were 1,551,565,904 bp of sequence data. The average read length was 301 bp. The total read depth was 326-fold coverage. The genomic dataset quality was trimmed using duk (http://duk.sourceforge.net/) and the FASTQX-toolkit fastqTrimmer (https://github.com/agordon/fastx_toolkit). Sequencing data were first assembled using Velvet v. 1.2.07 and ALLPATHS v. R46652.
Whole-genome average nucleotide identity analysis
We conducted numerous average nucleotide identity analyses between genomes of ZYT6 and Shewanella strains. Pairwise average nucleotide identity between the genome of strains ZYW6 and Shewanella strains was calculated using the algorithm proposed by Goris et al.. A radial phylogram was constructed using distance matrix computations.
We performed the annotation of the strain ZYW6 according to the NCBI Prokaryotic Genomes Automatic Annotation Pipeline. Besides, functional classification was performed using the RPSBLAST version 2.2.15 and Clusters of Orthologous Groups of proteins databases. To identify the CRISPR-Cas system in the genome of S. xiamenensis ZYW6, the integrated microbial genomes and microbiomes and CRISPRFinder were used. All the coding sequences of the genomes were subjected to BLASTn analysis.
We predicated the antibiotic resistance gene (ARG) in the genome with a multiple database-based approach. The comprehensive antibiotic resistance database, ResFinder 3.0, and ARG-ANNOT. BLASTn analysis against the JGIs Integrated Microbial Genomes database was used to validate the results. When multiple results are overlapped at the same locus in the genome, the best-aligned virulent factor gene was retained. The candidate virulent genes were predicted using the virulence factor database. The protein sequences of annotated genes were aligned against virulence factor database protein sequences, using BLASTX. All BLASTX results were manually curated based on the consistency of annotations among different databases.
This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number LVDQ00000000. The version described in this paper is version LVDQ00000000.
| Results|| |
General genome features
The final assembled genome consisted of forty scaffolds with a total size of 4,765,190 bp (GC content of 46.20%). The maximum contig size was equal to 429,948 bp, and the N50 size was 250,841 bp. Among the 4,262 identified open-reading frames, 4,137 contained protein-coding genes. In addition, 15 rRNA genes (7 5S, 2 16S and 6 23S), 78 tRNA genes, and 5 noncoding RNAs were identified.
Whole-genome average nucleotide identity analysis
The pairwise average nucleotide value between ZYW6 and Shewanella strains was calculated. The average nucleotide identity values were 97.6% for ZYW6 and S. xiamenensis BC01, 72.3% for Shewanella algae YHL, 72.1% for Shewanella amazonensis SB2B, 79.9% for Shewanella baltica BA175, and 72.5% for Shewanella loihica PV-4. As shown in [Figure 1], the radial phylogram from alignment fraction analysis revealed an obvious relationship between strains ZYW6 and BC01, which supported the results from the average nucleotide identity analyses.
|Figure 1: Radial phylogram based on the genomic data from Shewanella strains|
Click here to view
The clustered regularly interspaced short palindromic repeats-Cas system and serABC operon
We identified a Type I-E CRISPR-Cas system, located at position 56,931–65,565 of the genome of S. algae ZYW6, containing cas2 (ygbF), cas1 (ygbT), casE (cse3, ygcH), casD (cas5e, ygcI), casC (cse4, ygcJ), casB (cse2, ygcK), casA (cse1, ygcL), and cas3 (ygcB) [Figure 2]. The S. xiamenensis ZYW6 assimilated l-serine. We detected serABC operon which are required for L-serine synthesis from 3-phosphoglycerate.
|Figure 2: Genetic structure of the clustered regularly interspaced short palindromic repeats-cas locus in Shewanella xiamenensis|
Click here to view
Resistance profile and related antibiotic resistance gene
The S. xiamenensis ZYW6 was susceptible to ampicillin/sulbactam, piperacillin/tazobactam, imipenem, third- and fourth-generation cephalosporin [Table 1]. In our study, blaOXA-48 was identified from the genome of S. xiamenensis ZYW6.
|Table 1: Antimicrobial susceptibility pro?les of the Shewanella xiamenensis ZYW6|
Click here to view
| Discussion|| |
There are increasing reports of S. xiamenensis worldwide, raising concern of its pathogenic potential. The emergence of the organism poses threat to both human and animals. Better understanding of the key genomic features is essential for the development of strategies for diagnostic and control measures of this zoonotic infection,. In the study, we use a genomic approach to characterize the possible genetic background of important antimicrobial resistance and virulence-associated traits.
The CRISPR immune system in prokaryotic organisms is a key defense element in neutralizing invading viruses and plasmids. Current evidence revealed that a number of important pathogens harbor Type II CRISPR-Cas system, including Streptococcus pyogenes, Neisseria More Details meningitidis, and Haemophilus influenzae. CRISPR-Cas system-mediated sialylation of the cell envelope was suggested to be one of the underlying mechanisms. The S. xiamenensis ZYW6 CRISPR-Cas Type I-E system consists of a gene cluster including cas2, cas1, the Cascade genes (casABCDE), and cas3. Complete functionally essential cascade complex is encoded in the system. Type I-E CRISPR-Cas system can be found in many pathogenic bacteria and is associated with their virulence. In enterohemorrhagic E. coli, polymorphisms in Type I-E CRISPR-Cas system are correlated with the presence of key virulence genes encoding Shiga toxin. Animal study of Salmonella More Details and Campylobacter also demonstrated that Type I-E CRISPR-Cas system is involved in the pathogenesis. The discovery of Type I-E CRISPR-Cas system in S. xiamenensis warrants further studies to elucidate its role in fitness and pathogenesis.
We identified the essential genes of de novo L-serine biosynthetic pathway. The biosynthesis of L-serine from D-3-phosphoglycerate includes three reactions, which is catalyzed by D-3-phosphoglycerate dehydrogenase, D-3-phosphoserine aminotransferase, and phosphoserine phosphatase. These are encoded by serA, serC, and serB, respectively. In the study, we identified genes involved in the L-serine biosynthesis pathway, serA, serC, and serB, which is consisted with the biochemical testing results. Overexpressing serABC results in improvement of cell growth. Large-scale genomic study has demonstrated the difference of amino acid biosynthesis capabilities between Staphylococcus aureus strains and suggested the association with the virulence. Further large-scale pangenomic study is needed to build genome-scale models of S. xiamenensis.
Chromosome-encoded carbapenem-hydrolyzing β-lactamase OXA-48 gene was detected in ZYW6. Genes encoding Ambler Class D β-lactamase have been detected in various Shewanella species regardless of phenotypic resistance pattern. Our data further suggest that aquaculture-associated S. xiamenensis could be a potential source of Class D β-lactamase gene. Ambler Class D β-Lactamase OXA-48 has been found in human intestinal carriage S. xiamenensis. The variants of blaOXA detected in S. xiamenensis also included blaOXA-181, blaOXA-416, blaOXA-204, blaOXA-514, blaOXA-252, and blaOXA-199.
| Conclusion|| |
Our results demonstrate the genetic structure of CRISPR-Cas system, l-serine synthesis, and oxacillinase in S. xiamenensis. The work also highlights the need to conduct large-scale genomic study to fully understand the zoonotic potential and evolutionary changes in S. xiamenensis.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Petersen E, Petrosillo N, Koopmans M; ESCMID Emerging Infections Task Force Expert Panel. Emerging infections-an increasingly important topic: Review by the Emerging Infections Task Force. Clin Microbiol Infect 2018;24:369-75.
van Doorn HR. Emerging infectious diseases. Medicine (Abingdon) 2014;42:60-3.
Huang J, Sun B, Zhang X. Shewanella xiamenensis
sp. nov. isolated from coastal sea sediment. Int J Syst Evol Microbiol 2010;60:1585-9.
Grouzdev DS, Safonov AV, Babich TL, Tourova TP, Krutkina MS, Nazina TN. Draft genome sequence of a dissimilatory U (VI)-reducing bacterium, Shewanella xiamenensis
strain DCB2-1, isolated from nitrate- and radionuclide-contaminated groundwater in Russia. Genome Announc 2018;6. pii: e00555-18.
Yousfi K, Touati A, Bekal S. Complete genome sequence of an extensively drug-resistant Shewanella xiamenensis
strain isolated from Algerian hospital effluents. Genome Announc 2016;4. pii: e01236-16.
Tafoukt R, Leangapichart T, Hadjadj L, Bakour S, Diene SM, Rolain JM, et al
. Characterisation of blaOXA-538, a new variant of blaOXA-48, in Shewanella xiamenensis
isolated from river water in Algeria. J Glob Antimicrob Resist 2018;13:70-3.
Ng IS, Chen T, Lin R, Zhang X, Ni C, Sun D. Decolorization of textile azo dye and Congo red by an isolated strain of the dissimilatory manganese-reducing bacterium Shewanella xiamenensis
BC01. Appl Microbiol Biotechnol 2014;98:2297-308.
Zong Z. Nosocomial peripancreatic infection associated with Shewanella xiamenensis
. J Med Microbiol 2011;60:1387-90.
Antonelli A, Di Palo DM, Galano A, Becciani S, Montagnani C, Pecile P, et al
. Intestinal carriage of Shewanella xiamenensis
simulating carriage of OXA-48-producing Enterobacteriaceae
. Diagn Microbiol Infect Dis 2015;82:1-3.
Wu ZY, Ho SP, Cheng JF, Tung KC, Hong YK, Chen SY, et al
. Whole-genome characterization of Shewanella algae
strain SYT3 isolated from seawater reveals insight into hemolysis. Future Microbiol 2018;13:1709-17.
Tseng SY, Tung KC, Cheng JF, Lee YH, Wu ZY, Hong YK, et al
. Genome characterization of bile-isolated Shewanella algae
ACCC. Gut Pathog 2018;10:38.
Koonin EV, Makarova KS, Zhang F. Diversity, classification and evolution of CRISPR-cas systems. Curr Opin Microbiol 2017;37:67-78.
Louwen R, Staals RH, Endtz HP, van Baarlen P, van der Oost J. The role of CRISPR-cas systems in virulence of pathogenic bacteria. Microbiol Mol Biol Rev 2014;78:74-88.
Hatoum-Aslan A, Marraffini LA. Impact of CRISPR immunity on the emergence and virulence of bacterial pathogens. Curr Opin Microbiol 2014;17:82-90.
Murch AL, Skipp PJ, Roach PL, Oyston PC. Whole genome transcriptomics reveals global effects including up-regulation of Francisella
pathogenicity island gene expression during active stringent response in the highly virulent Francisella tularensis
subsp. Tularensis SCHU S4. Microbiology 2017;163:1664-79.
Tseng SY, Liu PY, Lee YH, Wu ZY, Huang CC, Cheng CC, et al
. The pathogenicity of Shewanella algae
and ability to tolerate a wide range of temperatures and salinities. Can J Infect Dis Med Microbiol 2018;2018:6976897.
Jiang W, Xia B, Liu Z. A serine hydroxymethyltransferase from marine bacterium Shewanella algae
: Isolation, purification, characterization and l-serine production. Microbiol Res 2013;168:477-84.
Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 1991;173:697-703.
Huang YT, Cheng JF, Chen SY, Hong YK, Wu ZY, Liu PY. Draft genome sequence of carbapenem-resistant Shewanella algae
strain AC isolated from small abalone (Haliotis diversicolor
). J Glob Antimicrob Resist 2018;14:65-7.
Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al
. BLAST+: Architecture and applications. BMC Bioinformatics 2009;10:421.
Zerbino DR, Birney E. Velvet: Algorithms for de novo
short read assembly using de Bruijn graphs. Genome Res 2008;18:821-9.
Butler J, MacCallum I, Kleber M, Shlyakhter IA, Belmonte MK, Lander ES, et al
. ALLPATHS: De novo
assembly of whole-genome shotgun microreads. Genome Res 2008;18:810-20.
Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM, et al
. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 2007;57:81-91.
Huang YT, Chen JM, Ho BC, Wu ZY, Kuo RC, Liu PY. Genome sequencing and comparative analysis of Stenotrophomonas acidaminiphila
reveal evolutionary insights into sulfamethoxazole resistance. Front Microbiol 2018;9:1013.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-10.
Markowitz VM, Chen IM, Palaniappan K, Chu K, Szeto E, Grechkin Y, et al
. IMG: The integrated microbial genomes database and comparative analysis system. Nucleic Acids Res 2012;40:D115-22.
Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: A web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res 2007;35:W52-7.
Huang YT, Chuang WY, Ho BC, Wu ZY, Kuo RC, Ko M, et al
. Comparative genomics reveals diverse capsular polysaccharide synthesis gene clusters in emerging Raoultella planticola
. Mem Inst Oswaldo Cruz 2018;113:e180192.
McArthur AG, Waglechner N, Nizam F, Yan A, Azad MA, Baylay AJ, et al
. The comprehensive antibiotic resistance database. Antimicrob Agents Chemother 2013;57:3348-57.
Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S, Lund O, et al
. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 2012;67:2640-4.
Gupta SK, Padmanabhan BR, Diene SM, Lopez-Rojas R, Kempf M, Landraud L, et al
. ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes. Antimicrob Agents Chemother 2014;58:212-20.
Chen L, Zheng D, Liu B, Yang J, Jin Q. VFDB 2016: Hierarchical and refined dataset for big data analysis-10 years on. Nucleic Acids Res 2016;44:D694-7.
Wang JH, Chi CY, Lin KH, Ho MW, Kao CH. Tuberculous arthritis – Unexpected extrapulmonary tuberculosis detected by FDG PET/CT. Clin Nucl Med 2013;38:e93-4.
Wang JH, Lin PC, Chou CH, Ho CM, Lin KH, Tsai CT, et al
. Intraventricular antimicrobial therapy in postneurosurgical gram-negative bacillary meningitis or ventriculitis: A hospital-based retrospective study. J Microbiol Immunol Infect 2014;47:204-10.
Lee YH, Tung KC, Cheng JF, Wu ZY, Chen SY, Hong YK, et al.
Genomic characterization of carbapenem-resistant Shewanella algae
isolated from Asian hard clam (Meretrix lusoria
). Aquaculture 2019;500:300-4.
Wu ZY, Huang YT, Chao WC, Ho SP, Cheng JF, Liu PY. Reversal of carbapenem-resistance in Shewanella algae
by CRISPR/Cas9 genome editing. J Adv Res 2019;18:61-9.
Louwen R, Horst-Kreft D, de Boer AG, van der Graaf L, de Knegt G, Hamersma M, et al
. Anovel link between Campylobacter jejuni
bacteriophage defence, virulence and Guillain-Barré syndrome. Eur J Clin Microbiol Infect Dis 2013;32:207-26.
Delannoy S, Beutin L, Fach P. Use of clustered regularly interspaced short palindromic repeat sequence polymorphisms for specific detection of enterohemorrhagic Escherichia coli
strains of serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by real-time PCR. J Clin Microbiol 2012;50:4035-40.
Bosi E, Monk JM, Aziz RK, Fondi M, Nizet V, Palsson BØ. Comparative genome-scale modelling of Staphylococcus aureus
strains identifies strain-specific metabolic capabilities linked to pathogenicity. Proc Natl Acad Sci U S A 2016;113:E3801-9.
Ceccarelli D, van Essen-Zandbergen A, Veldman KT, Tafro N, Haenen O, Mevius DJ. Chromosome-based blaOXA-48-like variants in Shewanella
species isolates from food-producing animals, fish, and the aquatic environment. Antimicrob Agents Chemother 2017;61. pii: e01013-16.
Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: The phantom menace. J Antimicrob Chemother 2012;67:1597-606.
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.
[Figure 1], [Figure 2]