Genome Sequence of Colistin-Resistant Bacteremic Shewanella algae Carrying the Beta-Lactamase Gene bla OXA-55

Shewanella algae is an emerging pathogen widely distributed in aquatic environment. Bacteremia is a major manifestation of S. algae infections, and there are increasing reports of antibiotic-resistant strains. However, little is known about the genomic characteristics of human bacteremic S. algae. Here, we report the results of the whole-genome sequencing of colistin-resistant S. algae TYL, a blood isolate. Chromosome-encoded pmrC associated with colistin resistance and bla OXA-55 gene intrinsic to S. algae was identified. Continuous surveillance for the emergence of S. algae is needed.


Introduction
Shewanella algae is a nonfermenting Gram-negative bacterium and autochthonous inhabitant of aquatic environments [1]. e organism could tolerate a wide range of physiological conditions [2] and has been documented as an emerging zoonotic pathogen [3]. It has been reported to cause ulcerative disease in marine fish [4] and shellfish [5].
e clinical spectrum in human infection is broad, including bacteremia, biliary tract infection, pneumonia, and soft tissue infections [6]. Bacteremia is one of the most common presentations of S. algae infections, which is associated with considerable morbidity and mortality [7]. e emergence of colistin resistance has become a major public heath challenge. e major advances of sequencing technology in past decades have enabled improved understanding of the genomic background of many important pathogens and resistance determinants. High-quality genomic data have become critical for the study of pathogenesis and therapeutic intervention of infectious diseases.
Although there are increasing reports of colistinresistant S. algae bloodstream infections worldwide [8], little is known about the genomic characteristics of human bacteremic S. algae. Moreover, the widespread environmental nature of S. algae raises concern for its role as a resistance reservoir. In this study, we determined the wholegenome sequence of a colistin-resistant S. algae strain isolated from blood.

Materials and Methods
S. algae strain TYL was isolated on trypticase soy agar supplemented with 5% sheep blood (Becton Dickinson, San Jose, CA, USA) from the blood of a septic patient. e strain was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS, bio-Mérieux, Marcy-l'Etoile, France) and 16S ribosomal RNA gene sequencing. In brief, a PCR amplicon of the 16S ribosomal RNA gene was obtained with primers B27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and U1492R (5′-GGTTACCTTGTTACGACTT-3′) and subjected to Sanger sequencing. Sequences obtained were blasted against the bacterial 16S ribosomal RNA gene sequences in the Gen-Bank database (http://www.ncbi.nlm.nih.gov/) using the BLASTn (optimized for MegaBLAST) algorithm. Antibacterial susceptibility testing was performed by the Vitek 2 system (bioMérieux, Marcy-l′Etoile, France) according to the manufacturer's instructions. e reference strains Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality controls. e broth microdilution method was used to determine the minimum inhibitory concentration (MIC) of colistin.
Genomic DNA was extracted by using the QIAGEN Genomic-tip 100/G kit and the Genomic DNA Buffer Set (QIAGEN, Paisley, UK) then quantified by using the Qubit dsDNA HS Assay kit and the Qubit 2.0 fluorometer (Life 3 Technologies, Carlsbad, CA, USA). e indexed PCR-free library preparation was constructed using a multiplexed high-throughput sequencing TruSeq DNA Sample Preparation kit (Illumina, San Diego, CA, USA) following the standard protocol provided by the manufacturer with minor modification. Low-quality reads and bases were removed and trimmed using the Kmer-based tool DUK (http://duk. sourceforge.net/) and FASTQ Trimmer (https://github.com/ agordon/fastx_toolkit), respectively. e reads were assembled using Velvet version 1.2.07 [9]. Wgsim 0.3.0 (https://github.com/lh3/wgsim) was then used to generate 1-3 kb simulated paired-end reads.

Results and Discussion
Whole-genome sequencing was performed using the Illumina MiSeq platform which generated 4,270,654 reads (mean read length of 301 bp) and a gross amount of 1,285,466,854 bp. e assembly of the draft genome sequence consists of 100 scaffolds amounting to 4,821,720 bp (total read depth of 267-fold coverage), and the G + C content is 52.95%. An illustration of the genomic contents in the genome of TYL is shown in Figure 1. e maximum contig size was 234,074 bp, and the N50 size was 96,168 bp. Of the 4,304 genes predicted, 4,112 were protein-coding genes, 13 RNAs, 90 tRNAs, and 6 noncoding RNAs. e distribution of genes into COGs functional categories is presented in Table 1. is Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number: LVDB01000000. e version described in this paper is version LVDB01000000.
To measure the nucleotide-level genomic similarity between TYL and the other Shewanella genomes, the Average Nucleotide Identity was calculated using a modified algorithm [15]. Results from this analysis showed an Average Nucleotide Identity (ANI) value higher than 98% with both the C6G3 (NCBI accession number: NZ_JPMA00000000.1) and MARS 14 strains (NCBI accession number: NZ_CDQH00000000.1), confirming the identification at the species level ( Figure 2). e TYL strain is resistant to colistin (MIC � 32 μg/ml) and susceptible to imipenem Over the past decade, the emergence of multidrugresistant Gram-negative microorganisms increased the use of colistin as the remaining therapeutic option [17]. Nevertheless, the reports of colistin resistance globally are of great concern. e two most studied nonfermentative Gram-negative bacteria with acquired resistance to colistin are Acinetobacter baumannii and Pseudomonas aeruginosa [18]. Lipid A modification is a common mode of colistin resistance in clinical isolates belonging to these two species [19,20]. We identified the homolog of pmrC gene associated with colistin resistance [21]. Analysis of the upstream and downstream of pmrC gene was performed. We found pmrA and pmrE genes located downstream of pmrC gene. We noted a similar arrangement in colistin-resistant S. algae MARS 14 ( Figure S1). Functional genomic study on S. algae MARS 14 demonstrated that the colistin resistance in S. algae is associated with phosphoethanolamine transferase EptA-encoding pmrC gene [21]. EptA adds phosphoethanolamine to lipid A, resulting in structural modification of lipopolysaccharide and colistin resistance [21]. Studies of colistin resistance patterns of S. algae are limited to case series and case reports [22,23]. It was suggested that all S. algae are resistant to colistin [24]. However, there are also reports of colistin-susceptible strains [25]. Large-scale comparative genomics focus on pmr genes, and colistin resistance phenotype are needed to fully exploit mechanisms of colistin resistance in S. algae. e emergence of carbapenem-resistant S. algae is a growing concern worldwide. Carbapenemase producers are of major concern. In this study, we detected a chromosomeencoded bla OXA-55 ß-lactamase gene. OXA-type carbapenemases are members of class D ß-lactamases with carbapenem-hydrolysing activities.
e mechanisms of carbapenem resistance in S. algae are suggested to be caused by beta-lactamase gene bla OXA-55 [23]. Various bla OXA -type genes have been reported in different Shewanella species [26]. In addition, there are increasing numbers of studies on horizontal gene transfer in Shewanella that demonstrate the organism is a potential reservoir of antimicrobial resistance [27,28]. However, the OXA-55 ß-lactamase is also present in carbapenem-susceptible S. algae [29]. e resistance of carbapenems in S. algae may involve a combination of OXA-lance and further investigation are needed for the resistance emergence.
S. algae causes diseases in both humans and marine animals. e organism has been reported to cause various diseases in shrimp, marine fish, and shellfish [4,5,30]. In humans, S. algae infections have been associated with seafood ingestion and water exposure [6]. Severe bacteremic illness complicated with human shewanellosis is not uncommon [31]. Pathogenicity test showed the LD 50 of S. algae was 1.8 × 10 4 CFU/mL for 20-day-old abalone postlarvae [4]. Hemolysis was demonstrated in the pathogenic strains of S. algae [30].
Hemolytic activity has long been suspected as a virulence factor of S. algae [32]. e S. algae TYL genome carries genes encoding hemolysin A (hlyA) and hemolysin  III (hlyIII). Hemolysin A belongs to RTX pore-forming toxin α-hemolysin, which rearranges membrane permeability and causes cell lysis. Genomic analysis of S. algae TYL in this report supports the previous study, which demonstrated the hemolytic activity of environmental S. algae and thus suggested it as a possible virulence determinant [33]. Biliary tract infection is one of the major presentations and possible ports of entry in shewanellosis [34]. Bile salts possess antimicrobial activity, causing damage to lipids in the cell membrane and DNA. Hence, S. algae must be able to survive the deleterious action of bile salts. A previous study demonstrated bile resistance in S. algae [24]. In this study, we identified genes associated with bile tolerance (htpB, exbBD, and galU). e results support the earlier genomic study, suggesting a common mechanism of bile resistance in Shewanella [16].

Conclusions
In summary, multiple resistance and virulence determinants in S. algae TYL were identified by wholegenome sequencing. To the best of our knowledge, this report describes the first S. algae blood isolate. Our data provide the impetus for further research on the zoonotic potential of S. algae.

Data Availability
is Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the accession LVDB01000000. e version described in this paper is version LVDB01000000.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this article.