Received: 15 February 2020 | Revised: 20 May 2020 | Accepted: 27 May 2020 DOI: 10.1002/mbo3.1093 ORIGINAL ARTICLE Vibrios from the Norwegian marine environment: Characterization of associated antibiotic resistance and virulence genes Fredrik Håkonsholm1 | Bjørn Tore Lunestad1 | Jose Roberto Aguirre Sánchez2 | Jaime Martinez-Urtaza3 | Nachiket Prakash Marathe1 | Cecilie Smith Svanevik1 1 Institute of Marine Research, Bergen, Norway Abstract 2 Centro de Investigación en Alimentación y A total of 116 Vibrio isolates comprising V. alginolyticus (n = 53), V. metschnikovii Desarrollo (CIAD), Culiacán, Sinaloa, Mexico (n = 38), V. anguillarum (n = 21), V. antiquarius (n = 2), and V. fujianensis (n = 2) were 3 Department of Genetics and Microbiology, Universitat Autònoma de Barcelona (UAB), obtained from seawater, fish, or bivalve molluscs from temperate Oceanic and Polar Barcelona, Spain Oceanic area around Norway. Antibiotic sensitivity testing revealed resistance or re- Correspondence duced susceptibility to ampicillin (74%), oxolinic acid (33%), imipenem (21%), aztre- Cecilie Smith Svanevik, Institute of Marine onam (19%), and tobramycin (17%). Whole-genome sequence analysis of eighteen Research, P.O. Box 1870 Nordnes, NO-5817 Bergen, Norway. drug-resistant isolates revealed the presence of genes like β-lactamases, chloram- Email: cecilie.svanevik@hi.no phenicol-acetyltransferases, and genes conferring tetracycline and quinolone resist- ance. The strains also carried virulence genes like hlyA, tlh, rtxA to D and aceA, E and F. The genes for cholerae toxin (ctx), thermostable direct hemolysin (tdh), or zonula occludens toxin (zot) were not detected in any of the isolates. The present study shows low prevalence of multidrug resistance and absence of virulence genes of high global concern among environmental vibrios in Norway. However, in the light of cli- mate change, and projected rising sea surface temperatures, even in the cold tem- perate areas, there is a need for frequent monitoring of resistance and virulence in vibrios to be prepared for future public health challenges. KEYWORDS antimicrobial resistance, marine environment, Vibrio spp., virulence, whole-genome sequencing 1 | I NTRO D U C TI O N human pathogens, V. cholerae, V. parahaemolyticus, and V. vulnificus have been extensively studied (Baker-Austin et al., 2018; Stavric & Vibrio spp. have the sea and brackish water as their natural habi- Buchanan, 1997). tat and are among the most common bacteria found in surface V. cholerae has through history caused several pandemics waters worldwide (Vezzulli, Colwell, & Pruzzo, 2013). The genus and the main culprit being V. cholerae serotype O1/O139 encod- includes several fish and human pathogenic species. Among these ing cholerae toxin (CTX; Islam et al., 2013). However, non-O1/ This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. MicrobiologyOpen. 2020;9:e1093.  www.MicrobiologyOpen.com | 1 of 19 https://doi.org/10.1002/mbo3.1093 2 of 19 | HÅKONSHOLM et al. non-O139 V. cholerae can also cause infections. The virulence fac- the prevalence of different Vibrio spp. and associated resistance tors of non-O1 and non-O139 include a heat-stable enterotoxin, and virulence markers in the Norwegian marine environment. This repeat in toxin (rtx) and El Tor hemolysin (hlyA) (Kumar, Peter, & study aimed to examine the prevalence of different Vibrio spp. in the Thomas, 2010). In contrast, the pathogenicity of V. parahaemolyti- Norwegian marine environment and to characterize associated viru- cus is linked to their ability to produce a thermostable direct hemo- lence and antibiotic resistance genes among these. We here present lysin (TDH), or a TDH-related hemolysin (TRH), encoded by tdh and a detailed account of taxonomy, resistance, and virulence genes de- trh genes (Raghunath, 2015). For V. vulnificus, virulence is related to tected based on phenotypic culture-based methods and whole-ge- the production of a polysaccharide capsule and lipopolysaccharide nome sequence (WGS) analysis. (LPS), flagellum, hemolysin, and proteases (Roig et al., 2018). The genetic basis for human virulence is only partially known, although several studies suggest that all strains of V. vulnificus, regardless of 2 | E X PE R I M E NTA L PRO C E D U R E S their origin, may be able to cause infections in humans (Roig et al., 2018). Several other Vibrio spp., such as V. alginolyticus, V. fluvialis, 2.1 | Sampling V. mimicus, V. metschnikovii, V. furnissii, V. hollisae, and V. damsela, can occasionally cause infections in humans (Austin, 2010; Baker- Water samples were collected from four different locations (A–D) Austin et al., 2018). at the West coast of Norway (Oceanic temperate zones) at five dif- Vibrio infections in humans typically occur as a result of inges- ferent depths (0, 2, 5, 7, and 10 m) from each location during May tion of contaminated seafood, through the handling of raw seafood 2018, comprising 20 water samples. A total of 60 fish caught in the or by exposure of wounds to seawater during recreation (Iwamoto, North Sea, including 40 herring (Clupea harrengus) and 20 Atlantic Ayers, Mahon, & Swerdlow, 2010). The human pathogenic vibrios mackerel (Scomber scombrus), were sampled from May to November show strong seasonality and are more abundant when the water 2018. The fish were caught by commercial fishing vessels during the temperature exceeds 18°C and the salinity drops below 25 ‰ catch season for the respective species. The bivalve molluscs sam- (Vezzulli et al., 2013). In the last decades, an increase in infections ples were collected from 16 rearing locations along the Norwegian caused by Vibrio spp. has been reported, also in colder regions of coast (Oceanic and Polar Oceanic zones) in November 2018 South America and Northern Europe, including Norway, where (Figure 1), through the annual surveillance program on Escherichia this was previously rare (Baker-Austin et al., 2016). One of the coli in bivalves by the Norwegian Food Safety Authority (NFSA). This primary effects of climate change is increased sea surface tem- included 14 batch samples of blue mussels (Mytilus edulis), one batch peratures (SSTs), and this may facilitate the spread of seawater sample of flat oysters (Ostrea edulis), and one batch sample of scal- associated diseases (EEA, 2017). The temperature is predicted to lops (Pecten maximus), where each batch sample comprised batches increase further in northern temperate waters (EEA, 2017), and of 10–15 individual bivalve molluscs. All samples were further exam- new areas may become more favorable for the pathogenic vibrios. ined at the Institute of Marine Research (IMR). Several fish pathogenic vibrios have been identified and are a chal- lenge in aquaculture. The most common Vibrio species infecting farmed aquatic animals are V. parahaemolyticus, V. alginolyticus, 2.2 | Isolation of Vibrio spp. V. harveyi, V. owensii, V. campbellii, and V. anguillarum (Ina-Salwany et al., 2019). From each water sample, three aliquots of 100–250 ml were filtered The role of the marine environment in the development and dis- through 0.45 µm filters (Merck Millipore, Germany) using the EZ-fit semination of antimicrobial resistance is largely unknown. Vibrios Manifold 3-place system (Merck Millipore, Germany) connected to are indigenous to the sea (Banerjee & Farber, 2018), and in re- a vacuum pump. Each filter was transferred to thiosulfate-citrate- cent years, the occurrence of resistance genes in Vibrio spp. has bile-sucrose (TCBS) agar (Oxoid, UK) plates and incubated at 37°C been examined. Genes encoding resistance to β-lactams like penA, for 24–48 hrs. Also, an enrichment step was performed in duplicates blaTEM-1 (Letchumanan, Chan, & Lee, 2015), and blaVCC-1 (Hammerl on 500 ml water adding 50 ml concentrated (360 mg/ml) alkaline et al., 2017; Mangat et al., 2016), chloramphenicol resistance genes, peptone water (APW) with 2% sodium chloride (NaCl). The enrich- such as floR, catI, and catII, and several tet genes encoding resis- ment cultures were incubated at 42 ˚C for 18 hr. After incubation, tance to tetracycline (Letchumanan et al., 2015), have been de- 100 µl of the enrichment cultures was streaked on TCBS agar and tected in Vibrio spp. Clinically important mobile resistance genes incubated at 37 ˚C for 24–48 hr. Typical colonies were picked from like qnrVC and qnrS have originated in Vibrio spp. (Fonseca, Dos the plates and restreaked for obtaining pure cultures. Santos Freitas, Vieira, & Vicente, 2008). This makes Vibrio spp. a Isolation of Vibrio spp. from fish and bivalve molluscs followed good model organism for the studying antibiotic resistance in the a method based on NMKL method no. 156 (NMKL, 1997). The marine environment. method takes advantage of the vibrios alkaline and halophilic prop- Although V. parahaemolyticus, V. cholerae, and V. vulnificus erties (Vezzulli et al., 2013) and applies APW supplemented with have previously been isolated from Norway (Bauer, Ostensvik, 2% NaCl and 42°C as incubation temperature for selective enrich- Florvag, Ormen, & Rorvik, 2006), there is limited knowledge on ment of human pathogenic species (NMKL, 1997). For isolation of HÅKONSHOLM et al. | 3 of 19 F I G U R E 1 Map of Norway showing sampling locations for fish (herring and Atlantic mackerel) captured during commercial pelagic fisheries, seawater collected during the herring fisheries, and marine bivalves from harvesting areas included in the surveillance program of the Norwegian Food Safety Agency Vibrio spp., TCBS is a widely used medium. The alkaline pH (8.6), From bivalve molluscs, 100 g soft tissue and intravalvular fluid bile salts, and NaCl concentration in the agar inhibit the growth of from at least 10 individual bivalves were homogenized in sterile plas- Enterobacteriaceae and Gram-positive organisms (Donovan & van tic bags and 20 g was transferred to new sterile bags. Enrichment Netten, 1995). From herring collected in June 2018, samples were followed the same protocol as for fish samples. Additionally, from taken from the skin with muscle, gills, and intestine. From each tis- the homogenate tenfold dilution series were made using peptone sue type, 20 g was homogenized in 180 ml APW with 2% NaCl and water (bioMerièux, France). From dilutions and undiluted samples, APW with 2% NaCl supplemented with polymyxin B (250 IU/ml) for 100 µl was spread on TCBS and Vibrio ChromoSelect agar (VCS; 30 s. using a stomacher. The homogenate was incubated at 42 ± 1°C Sigma-Aldrich) and incubated at 37°C for 24–48 hrs followed by a for 18 ± 2 hrs. After incubation, 10 µl of the enrichment cultures selection of typical colonies. was streaked on TCBS agar and incubated at 37 ± 1°C for 24 ± 3 hrs. From mackerel collected in September, samples were taken from the skin with mussel following the same protocol as described 2.3 | Biochemical identification previously. Samples were also collected from gut content and ho- mogenized in phosphate-buffered saline (PBS) (Sigma-Aldrich), and Isolates were grown overnight on plate count agar (PCA) (Oxoid, UK) tenfold dilution series were made. From each sample, 100 µl was supplemented with 2% NaCl and characterized biochemical using the spread on TCBS and incubated at 37 ± 1°C for 24 ± 3 hrs. From her- Analytical Profile Index 20E (API 20E, bioMerièux, France) following ring collected in November, samples were collected from the skin the instructions of the manufacturer. Overnight cultures were used with muscle and prepared following the same method as described to prepare bacterial inoculums corresponding to 0.5 McFarland in previously. 2% sterile saline. 4 of 19 | HÅKONSHOLM et al. 2.4 | Identification by MALDI-TOF-MS called with Harvest Suit (Treangen, Ondov, Koren, & Phillippy, 2014). Phylogenetic inference by ML was performed on the All isolates were grown overnight on PCA supplemented with 2% core genome with RAxML v8.1 (Stamatakis, 2014) and the NaCl and sent to the Norwegian Veterinary Institute (NVI) in Bergen GTRGAMMA model (1,000 bootstrap replicates). The re- for identification by matrix-assisted laser desorption ionization time- sulting trees were visualized and edited using iTOL v4.3.3 (Letunic of-flight mass spectrometry (MALDI-TOF-MS) (Bruker, Germany). The et al., 2006). obtained peptide mass fingerprints (PMFs) were compared to spectra in the commercial MALDI-TOF-MS database (MALDI Biotyper, Bruker, Germany) and to spectra in an in-house generated database contain- 2.8 | Antimicrobial susceptibility testing ing spectra from Vibrio spp. known to be associated with marine fish. Antimicrobial susceptibility testing of isolated Vibrio spp. was con- ducted by disk diffusion according to the Clinical and Laboratory 2.5 | Whole-genome sequencing and Standards Institute (CLSI) method M42-A (CLSI, 2006). Each isolate sequence analysis was tested against 18 antibiotics belonging to 10 different classes commonly used for either human administration, agriculture, vet- Eighteen isolates were subjected to whole-genome sequencing erinary medicine, or aquaculture in Norway (NORM/NORM-VET, (WGS). DNA was extracted from isolates using the DNeasy Blood & 2018) using. These included mecillinam (10 µg), ampicillin (10 µg), Tissue kit (Qiagen, Germany). An additional lysis step was performed cefotaxime (5 µg), ceftazidime (10 µg), doxycycline (30 µg), tetra- by resuspending the samples in 180 µl lysis buffer and incubat- cycline (30 µg), ciprofloxacin (5 µg), oxolinic acid (2 µg), imipenem ing them at 37°C overnight. After incubation, DNA extraction was (10 µg), meropenem (10 µg), erythromycin (15 µg), azithromycin done as described by the manufacturer (Quiagen, 2006). The purity (15 µg), sulfamethoxazole/trimethoprim (25 µg), trimethoprim (260/280 and 260/230 ratios) and concentration in the DNA was (5 µg), gentamycin (10 µg), tobramycin (10 µg), florfenicol (30 µg), measured using Nanodrop ND-1000 (NanoDrop Technologies, USA) and aztreonam (30 µg). V. alginolyticus, V. metschnikovii, and V. an- and Qubit 2.0 broad range dsDNA kit (Invitrogen, USA). guillarum were incubated at 28°C. E. coli CCUG17620 was included Genomic libraries were prepared using Nextera DNA Flex as quality control in each setup. Inhibition zones were interpreted Tagmentation (Illumina, USA) and sequenced using the MiSeq (Illumina, according to breakpoints for Enterobacteriaceae from CLSI USA) platform to obtain 300 bp paired-end reads. The raw sequence method M100 (CLSI, 2017). For oxolinic acid, erythromycin and data were adapter and quality trimmed using BBDuk (https://jgi.doe. florfenicol breakpoints and epidemiological cutoff values (ECVs) gov/data-and-tools​/bbtoo​ls/bb-tools​-user-guide​/) and assembled for Aeromonas salmonicda from CLSI VET03/VET04 (CLSI, 2014) using SPAdes version 3.13.1 (Bankevich et al., 2012). Assembled were used. genomes were annotated using the NCBI Prokaryotic Genome For isolates showing reduced susceptibility for imipenem, MIC Annotation Pipeline (Tatusova et al., 2016) and the Rapid Annotations values were determined following CLSI method M42-A using MIC using Subsystems Technology (RAST) server (Aziz et al., 2008). evaluator strips (Oxoid, UK). Resistance genes were detected using the Comprehensive Antibiotic Resistance Database, CARD (Jia et al., 2017), and the Resistance Gene Identifier mode. Virulence genes were detected using virulence fac- 2.9 | CarbaNP test tors database (VFDB; Liu, Zheng, Jin, Chen, & Yang, 2019). Isolates showing reduced susceptibility to imipenem by the disk diffusion method were grown overnight on tryptic soy agar (TSA; 2.6 | Species identification of WGS Merck, Germany) at 37°C and examined for carbapenemase produc- tion by the CarbaNP test as described by Dortet, Poirel, Errera, and Raw forward and reverse reads in the FastQ format were uploaded Nordmann (2014). to The Microbial Genomes Atlas (MiGA) (Rodriguez et al., 2018) web server in the TypeMat mode. In this mode, the sequences are trimmed, assembled, and aligned to give the closest relatives found 2.10 | Hemolysis in the MiGA Reference database. V. alginolyticus and V. metschnikovii isolates were screened for hemo- lytic activity on TSA with 5% sheep blood (VWR, USA) or TSA with 2.7 | Phylogenetic inference 5% human blood. Agar plates containing human blood were pre- pared by using TSA (Merck, Germany) as a base and supplemented For each Vibrio species (V. metschnikovii, V. anguillarum, and with 5% EDTA blood. Isolates were cultivated on TSA and incubated V. alginolyticus), single nucleotide polymorphisms (SNPs) were at 37°C for 24 hrs. HÅKONSHOLM et al. | 5 of 19 F I G U R E 2 Physical parameters in seawater samples collected during herring fisheries, locations A, B, C, and D (Figure 1). (a) Measured temperature (°C). (b) Measured salinity (‰), note: missing measurement at 10 m from location B. (c) Number of colony-forming units (cfu)/100 ml water on TCBS plates incubated at 37°C for 24–48 hrs. two V fluvialis. The remaining isolates were identified as members 3 | R E S U LT S of Aeromonas, Pasteurella, Shewanella, and Proteus or yielded an “Unacceptable profile.” 3.1 | Physical parameters and bacteria plate count One hundred and fifteen (99%) of the 116 isolates were iden- tified as Vibrio spp. by matrix-assisted laser desorption ionization The highest measured temperatures in seawater samples were seen time-of-flight mass spectrometry (MALDI-TOF-MS), and one isolate close to the shore at location A (Figure 1), in samples collected in could not be identified. The MALDI-TOF-MS identified vibrios be- the surface and at 2 m depth, with temperatures of 16°C and 15°C, longing to the three species V. alginolyticus (n = 53), V. metschnikovii respectively (Figure 2a). All other samples had temperatures <15°C. (n = 38), and V. anguillarum (n = 24), respectively. In the seawater samples at location A (surface and 2 m), salinity of The Microbial Genomes Atlas (MiGA) run for 18 sequenced 12.4‰ and 16.9‰ was observed, which was the lowest of all sam- isolates identified seven V. alginolyticus, five V. anguillarum, ples (Figure 2b). The highest plate counts (cfu/100 ml) on thiosul- two V. metschnikovii, two V. antiquarius, and two V. fujianen- fate-citrate-bile-sucrose agar (TCBS) were observed in the samples sis. Incompliance between the identification by WGS MiGA and with the highest temperature and lowest salinity (Figure 2c). MALDI-TOF-MS was seen for the two isolates 1-2(7-a) and 11-4(1), identified as V. antiquarius and V. alginolyticus, the two isolates 3-2(1) and 2-2(8), identified as V. alginolyticus and V. anguillarum, 3.2 | Prevalence and identification of Vibrio spp and for the one isolate 1-1(7) identified as V. fujianensis and V. an- guillarum, respectively. Colonies were selected for further characterization based on mor- Among the species considered to be opportunistic human patho- phology on TCBS agar (NMKL, 1997) and color formation on Vibrio gens (Austin, 2010; Baker-Austin et al. 2018), V. alginolyticus was iso- ChromoSelect agar (VCS). Presumptive Vibrio spp. were detected lated from water, herring, and bivalves, while V. metschnikovii was in 50% of water samples, 33% of fish samples, and 31% of bivalve isolated from herring and water samples. On the other hand, species molluscs samples. In total, 60 isolates were recovered from water harboring virulence genes but not known to cause human disease, samples, 32 from fish and 24 from bivalves. Using Analytical Profile like V. antiquarius (Dahanayake, De Silva, Hossain, Shin, & Heo, 2018; Index (API) 20E, 54 (47%) of the 116 isolates were identified as Nur et al., 2015) and V. fujianensis (Fang et al., 2018), were iso- Vibrio spp., 49 of which were V. alginolyticus, three V. cholerae, and lated from water only. V. anguillarum, a well-known fish pathogen 6 of 19 | HÅKONSHOLM et al. (Ina-Salwany et al., 2019) rarely associated with serious human infec- numbers is presented in Table A2. Several genes related to viru- tion (Sinatra & Colby, 2018), was only isolated from bivalves. lence were detected in the examined genomes, including genes Global mapping of the sequenced isolates of V. alginolyticus and for mannose sensitive hemagglutinin (msh), adherence, type V. anguillarum (Figures A1 and A2) showed that Vibrio isolates from IV toxin-coregulated pilus (tcp), type IV pilus (pil), capsular an- Norway had high similarity to strains from other countries and con- tiphagocytosis polysaccharides (rml, vbf, cps, wec, wza, wzb, wzb), tinents, including the United States and China, indicating a global flagellar formation genes (che, fil, fla, flg, flh, fli, flr, mot, che), iron presence of these strains. uptake (irg, vct, viu, vib, vie), quorum sensing genes (eps), ESP se- cretion systems (esp, gsp), T3SS1 secretion systems, VAS effector proteins, endotoxin production, and immune evasion genes. None 3.3 | Hemolytic activity on blood agar of the isolates carried genes for cholerae toxin production (ctxA or ctxB), thermostable direct hemolysin (tdh), or zonula occludens None of the 53 V. alginolyticus isolates displayed hemolysis on blood toxin (zot). agar. All 38 V. metschnikovii isolates were hemolytic on both sheep The most prominent virulence genes detected in this study and human blood. On sheep blood, five V. metschnikovii isolates were were related to hemolysins. All Vibrio species examined had genes β-hemolytic, while the remaining isolates were α-hemolytic on both coding for the Aeromonas-related hemolysin type III (Hemolysin media. III). The V. cholerae cytolysin A (hlyA) was detected among V. al- ginolyticus, V. metschnikovii, and V. anguillarum, whereas the ther- molabile hemolysin gene (tlh) was present in all isolated species 3.4 | Characterization of virulence determinants except V. fujianensis. A variety of the repeats-in-toxin holotoxins in WGS genes (rtxA to D) was detected in V. alginolyticus, V. metschnikovii, V. anguillarum, and V. fujianensis. The accessory V. cholerae entero- Eighteen drug-resistant isolates were subjected to WGS. Detailed toxin genes ace (A, E, and F) were found in the two V. fujianensis overview of genome assembly statistics and GenBank accession isolates (Table 2). TA B L E 1 Antibiotic sensitivity pattern among the Vibrio isolates V. alginolyticus V. metschnikovii V. anguillarum V. antiquarius V. fujianensis (n = 53) (n = 38) (n = 21) (n = 2) (n = 2) R Agent S (%) I (%) R (%) S (%) I (%) R (%) S (%) I (%) R (%) S (%) I (%) R (%) S (%) I (%) (%) AMP 0 0 100 79 0 21 0 0 100 0 0 100 0 0 100 MEL 100 0 0 100 0 0 100 0 0 100 0 0 100 0 0 CTX 100 0 0 97 3 0 95 5 0 100 0 0 100 0 0 CAZ 100 0 0 97 3 0 100 0 0 100 0 0 100 0 0 TE 100 0 0 100 0 0 100 0 0 100 0 0 100 0 0 DO 100 0 0 100 0 0 100 0 0 100 0 0 100 0 0 IPM 96 4 0 100 0 0 0 0 100 100 0 0 50 50 0 MEM 100 0 0 100 0 0 100 0 0 100 0 0 100 0 0 E 98 0 2 100 0 0 71 0 29 100 0 0 100 0 0 SXT 100 0 0 100 0 0 100 0 0 100 0 0 100 0 0 W 98 2 0 100 0 0 100 0 0 50 50 0 100 0 0 OA 34 62 4 97 3 0 100 0 0 0 100 0 100 0 0 CIP 100 0 0 100 0 0 100 0 0 100 0 0 100 0 0 CN 100 0 0 82 18 0 100 0 0 100 0 0 100 0 0 TOB 100 0 0 47 39 13 100 0 0 100 0 0 100 0 0 FFC 100 0 0 100 0 0 100 0 0 100 0 0 100 0 0 AZM 100 0 0 100 0 0 100 0 0 100 0 0 100 0 0 ATM 98 2 0 100 0 0 5 86 10 100 0 0 50 50 0 Abbreviations: AMP: Ampicillin, MEL: Mecillinam, CTX: Cefotaxime, CAZ: Ceftazidime, TE: Tetracycline, DO: Doxycycline, CIP: Ciprofloxacin, OA: Oxolinic acid, IPM: Imipenem, MEM: Meropenem, E: Erythromycin, AZM: Azithromycin, SXT: Sulfamethoxazole/Trimethoprim, W: Trimethoprim, TOB: Tobramycin, CN: Gentamicin, FFC: Florfenicol, ATM: Aztreonam, S: Susceptible, I: intermediate, R: Resistant. TA B L E 2 Isolation source, identification, and list of resistance and virulence genes detected in whole-genome sequences of Vibrio spp. isolates Sample Isolate MiGA TypeMat p-value MALDI-TOF-MS Score API 20E ID % T-value Resistance genes Virulence genes Accession No. Bivalve B4-6 V. anguillarum 0.004 V. anguillarum 2.39 A. hydrophila/ 70.5 0.56 blaampC , tet(34) Hem III, hylA, tlh VHSL00000000 caviae/sobria2 rtxA, B, C, D Bivalve B7 V. anguillarum 0.004 V. anguillarum 2.48 A. hydrophila/ 70.5 0.56 catB-related, tet(34) Hem III, hylA, tlh, VHSN00000000 HÅKONSHOLM et al. caviae/sobria2 rtxA, B, C, D Bivalve B1−2 V. anguillarum 0.004 V. anguillarum 2.32 A. hydrophila/ 54.2 0.36 blaampC , varG, catB-related, Hem III, hylA, tlh, VHSK00000000 caviae/sobria1 tet(34) rtxA, B, C, D Bivalve B4−12 V. anguillarum 0.004 V. anguillarum 2.38 A. hydrophila/ 70.5 0.56 blaampC , catB-related, tet(34) Hem III, hylA, tlh, VHSM00000000 caviae/sobria2 rtxA, B, C, D Bivalve B8−1 V. anguillarum 0.004 V. anguillarum 2.38 A. hydrophila/ 70.5 0.56 blaampC , catB-related, tet(34) Hem III, hylA, tlh VHSO00000000 caviae/sobria2 Herring A8−1 V. metschnikovii 0.0094 V. metschnikovii 1.78 Identification - - adeF, blaCARB, catB-related, Hem III, hylA, tlh VHTC00000000 not valid Herring A11 V. metschnikovii 0.0094 V. metschnikovii 1.78 Unacceptable - - blaCARB, catB-related Hem III, hylA, tlh VHSI00000000 profile Seawater 2-1 (7) V. alginolyticus 0.0016 V. alginolyticus 2.2 V. alginolyticus 97.8 0.74 adeF, blaCARB, blaampC , catB- Hem III, tlh VHSR00000000 related, qnr, tet(34), tet(35) Seawater 2-2 (2) V. alginolyticus 0.0016 V. alginolyticus 2.09 V. alginolyticus 85.9 0.81 adeF, blaCARB, catB-related, Hem III, tlh VHSS00000000 qnr, tet(34), tet(35) Seawater 2-2 (7) V. alginolyticus 0.004 V. alginolyticus 2.21 V. alginolyticus 85.9 0.81 adeF, blaCARB, blaampC , qnr, Hem III, tlh VHST00000000 catB-related, tet(34), tet(35) Seawater 2-2 (9) V. alginolyticus 0.0016 V. alginolyticus 2.19 V. alginolyticus 97.8 0.74 adeF, blaCARB, blaampC , qnr, Hem III, tlh VHSV00000000 catB-related, tet(34), tet(35) Seawater 7-5 (1-a) V. alginolyticus 0.0016 V. alginolyticus 2.11 V. alginolyticus 85.9 0.81 adeF, blaCARB, qnr, catB- Hem III, tlh VHSX00000000 related, tet(34), tet(35) Seawater 3-2 (1) V. alginolyticus 0.0016 V. anguillarum 2.37 A. hydrophila/ 69.8 0.28 catB-related, tet(34) Hem III, hylA, tlh VHSW00000000 caviae/sobria2 rtxA, B, C, D Seawater 2-2 (8) V. alginolyticus 0.0016 V. anguillarum 2.37 Unacceptable - - Hem III, hylA, tlh VHSU00000000 profile rtxA, B, C, D Seawater 1-2 (7-a) V. antiquarius 0.0048 V. alginolyticus 2.21 V. alginolyticus 97.8 0.74 adeF, blaCARB, catB-related, Hem III, tlh VHSQ00000000 qnr, tet(34), tet(35) Seawater 11-4 (1) V. antiquarius 0.004 V. alginolyticus 2.13 V. alginolyticus 85.9 0.81 adeF, blaCARB, blaampC , catB- Hem III, tlh VHSY00000000 related, qnr, tet(34), tet(35) Seawater 1-1 (7) V. fujianensis 0.0008 V. anguillarum 2.32 A. hydrophila/ 37.3 0.33 blaampC , tet(34) Hem III, aceE, VHSP00000000 caviae/sobria1 aceF, rtxA Seawater 12-2(3a) V. fujianensis 0.0008 Id. not possible - Unacceptable - - Hem III, aceE, aceF VMQP00000000 profile | Abbreviations: CARB: Carbenicillin-hydrolyzation, catB-related o-acetyltransferase involved in chloramphenicol resistance, qnr family pentapeptide repeat protein involved in quinolone target protection, adeF is the membrane fusion protein of the multidrug efflux complex adeFGH, and tet(34) as well as tet(35) conferring resistance to tetracyclines. hlyA: V. cholerae cytolysin A, tlh: Thermolabile hemolysin, 7 of 19 Hem III: Aeromonas-related hemolysin type III, rtxA to D repeats-in-toxin holotoxins, aceA, E and F accessory cholerae enterotoxin genes. 8 of 19 | HÅKONSHOLM et al. 3.5 | Antimicrobial resistance 4.1 | Prevalence of Vibrio spp. in the Norwegian marine environment The phenotypic antimicrobial susceptibility testing of the 116 Vibrio spp. showed 74% to be resistant to ampicillin, 33% to oxolinic The highest plate count of aquatic bacteria was observed in the acid, 21% to imipenem, 19% to aztreonam, and 17% to tobramycin water samples collected closest to the shore, where the measured (Table 1). All isolates were susceptible to tetracycline, ciprofloxa- temperature was highest and the salinity lowest (Location A). A total cin, and trimethoprim-sulfamethoxazole, while most isolates were of 67% of isolated V. alginolyticus were isolated from these samples, susceptible to third-generation cephalosporins (98%) and amino- where the temperature was measured to above 15°C and the salin- glycosides (83%). For the isolates showing reduced susceptibility ity to ≤25 ‰, close to the preferred conditions for vibrios (Vezzulli (intermediate resistance) to imipenem, minimum inhibitory concen- et al., 2013; Vezzulli, Pezzati, Brettar, Höfle, & Pruzzo, 2015). V. algi- tration (MIC) for imipenem ranged from 2 to 8 µg/ml. Detailed over- nolyticus is usually the dominating species in Vibrio communities (Fu view of the individual inhibition zones obtained from disk diffusion et al., 2016), and our results are in accordance with this study. From test is included in Table A1. fish samples, V. metschnikovii was the dominating species, while V. an- guillarum was the species most frequently isolated from bivalves. Of the vibrios isolated from water samples, only four V. alginolyticus 3.6 | Examination of carbapenemase production isolates and one V. metschnikovii isolate were recovered from en- richment cultures, indicating a suboptimal enrichment method for Among the 116 Vibrio isolates examined, resistance to imipenem was water samples. V. vulnificus, V. cholerae, and V. parahaemolyticus all observed in all V. anguillarum isolates, while two V. alginolyticus iso- grow at 42°C (NMKL, 1997), and hence, 42°C is used as enrichment lates and one V. fujianensis isolate were intermediately susceptible to temperature for these species. Although such a high incubation tem- the agent. These imipenem-resistant isolates were also resistant to perature may affect the recovery of stressed cells (Huq et al., 2012), ampicillin but susceptible to meropenem. All but one V. anguillarum Bauer et al. (2006) showed that there was no difference in isolation isolate (B4-12) was susceptible to cefotaxime. CarbaNP test was rate of V. parahaemolyticus with enrichment at 37°C and 41.5°C for negative for all isolates, suggesting the absence of carbapenemase isolation of Vibrio spp. from bivalves. with high hydrolytic activity. In a previous study, three major pathogenic Vibrio spp. (V. vul- nificus, V. parahaemolyticus, and V. cholerae) were isolated from the Norwegian marine environment (Bauer et al., 2006). In summer of 3.7 | Genetic characterization of resistance 2018, several Vibrio infections were reported after bathing along the determinants Southeast coast of Norway (Naseer et al., 2019). However, none of these species was isolated in this study. Most of the samples that The sequenced genomes revealed the presence of β-lactamases were obtained for this study were from the west coast of Norway, like blaCARB genes in V. alginolyticus, V. metschnikovii, and V. an- where the seas are influenced by the North and Atlantic Ocean. As a tiquarius and ampC genes in V. alginolyticus, V. anguillarum, and result, the sea temperature in these areas is normally low and the sa- V. antiquarius (Table 2). One V. anguillarum isolate harbored varG linity is high. It is well known that the human pathogenic vibrios are metallo-β-lactamase first described in V. cholerae (Hong-Ting Victor most abundant at elevated sea temperatures, >18°C, and at lower et al., 2017). Genes encoding catB-related o-acetyltransferase, in- salinity levels, <25‰ (Vezzulli et al., 2013). This may explain the ab- volved in acetylation of chloramphenicol, were detected in isolated sence of the major human pathogenic Vibrio spp. in this study. The V. metschnikovii and V. anguillarum, while genes encoding tetracycline risk of increased numbers of vibrios due to elevated temperatures is resistance (tet34 and tet35) and multidrug membrane fusion protein greater in the east coast of Norway and closer toward the Baltic sea (adeF) were found in all examined sequences from V. alginolyticus. (Escobar et al., 2015) where the seas are less affected by the open V. alginolyticus also harbored genes encoding the qnr family penta- oceans. peptide repeat proteins conferring reduced susceptibility against quinolones (Marathe et al., 2019). 4.2 | Antimicrobial susceptibility 4 | D I S CU S S I O N For the treatment of infections caused by non-cholerae Vibrio spp., tetracyclines, fluoroquinolones, and third-generation cephalo- To the best of our knowledge, this study is the most comprehen- sporins are among the recommended agents (Elmahdi, DaSilva, & sive assessment of vibrios from the Norwegian marine environment Parveen, 2016; Wong, Brown, Luscombe, Wong, & Mendis, 2015). describing the prevalence of Vibrio spp. in Norwegian pelagic fish, Resistance to these agents has been reported within the genus bivalves, and seawater, and their characteristics concerning antimi- (Hernández-Robles et al., 2016; Lee, Ab Mutalib, Law, Wong, & crobial resistance and virulence. Letchumanan, 2018; Letchumanan et al., 2015). All Vibrio spp. HÅKONSHOLM et al. | 9 of 19 isolated during this study were phenotypically susceptible to tetra- our study. Pumps within these families are involved in the efflux of cycline, doxycycline, meropenem, sulfamethoxazole/trimethoprim, several classes of antibiotics, including aminoglycosides (Andersen ciprofloxacin, florfenicol, mecillinam, and azithromycin. et al., 2015; Garneau-Tsodikova & Labby, 2016; Krause, Serio, Kane, Consistent with previous reports, a high prevalence of resistance & Connolly, 2016). Phenotypic susceptibility testing and determina- to ampicillin was observed in all Vibrio spp. isolates in our study tion of MIC indicated the presence of resistance to imipenem in all (Banerjee & Farber, 2018; Chiou, Li, & Chen, 2015; Hernández- isolated V. anguillarum. Furthermore, two V. alginolyticus isolates and Robles et al., 2016; Li et al., 1999; Pan et al., 2013), and this resistance one V. fujianensis isolate were intermediately resistant to imipenem. is usually due to the presence of a blaCARB gene (Chiou et al., 2015; However, none of these isolates produced positive results in the car- Li et al., 2016). The blaCARB-like genes have been found in V. chol- baNP test indicating another resistance mechanism than the produc- erae predating the introduction of penicillins (Dorman et al., 2019). tion of a carbapenemase, or an imipenem hydrolyzing enzyme with In this study, the blaCARB genes were detected in V. alginolyticus, a slow turnover rate (Verma et al., 2011). The observed resistance V. metschnikovii, and V. antiquarius. Genes encoding ampC β-lact- is likely caused by an alteration in porins, the presence of low-affin- amase were found in V. alginolyticus, V. anguillarum, and V. fujianen- ity penicillin-binding proteins or overexpression of ampC (El Amin sis, which is conflicting to the results from phenotypic susceptibility et al., 2001; Nordmann, Dortet, & Poirel, 2012; Zapun, Contreras- testing as all these isolates were susceptible to cephalosporins. This Martel, & Vernet, 2008). One V. anguillarum isolate carried gene en- may indicate that the breakpoints used in this study are insufficient coding a VarG subclass B1-like lactamase, an enzyme with the ability for detection of these enzymes by a phenotypic method. This also to hydrolyze most β-lactam antibiotic, including cephalosporins and highlights the need for establishing breakpoints for environmental carbapenems (Lin et al., 2017). This isolate was, however, susceptible Vibrio species. However, differences between phenotype and geno- to both meropenem and cephalosporins. type may also be caused by a variable expression of genes in tested isolates (Sundsfjord et al., 2004). A study on the antimicrobial susceptibility of environmental V. al- 4.3 | Virulence ginolyticus isolated from oysters in Mexico reported a high preva- lence of resistance to tetracycline (Hernández-Robles et al., 2016). Members of the genus Vibrio are known to possess a range of viru- Although all isolates in our study were susceptible to both tetracy- lence factors connected to adherence (ACF, IlpA, MAM7, MSHA pili, cline and doxycycline, the tetracycline enzymatic inactivation gene OmpU, TCP, VpadF), pili production, motility by flagella, regulation tet34 (Akinbowale, Peng, & Barton, 2007) and efflux encoding gene (AI-2, CAI-1), iron uptake, secretion system (T3SS1, T3SS2, T6SS), tet35 were frequently detected within the examined genomes in the or toxin production (Ace, CT, MARTX, TDH, TRH, VCC, Zot, RTX), current study. often arranged in pathogenicity cassettes and islands (VPI, VPI-2) Resistance to oxolinic acid has been reported in V. alginolyticus (Pérez-Reytor, Jaña, Pavez, Navarrete, & García, 2018). (Scarano et al., 2014), and the prevalence of reduced susceptibility The lack of cholerae toxin (ctxA or ctxB) production, thermostable was quite high in this study. All examined isolates of V. alginolyticus direct hemolysin (tdh), or zonula occludens toxin (zot) indicates a low carried the qnr gene. It has been suggested that the marine bacteria level of virulence among the examined isolates. The most common may constitute the origin of plasmid-mediated quinolone resistance virulence genes among the isolates included in this study were the (PMQR) genes (Poirel, Cattoir, & Nordmann, 2012) and vibrios might Aeromonas-related hemolysin type III (Hemolysin III) (Goncalves Pessoa act as a reservoir for these genes (Poirel, Liard, Rodriguez-Martinez, et al., 2019). The V. cholerae cytolysin A gene (hlyA) was found among & Nordmann, 2005). V. alginolyticus, V. metschnikovii, and V. anguillarum, whereas the ther- Genes encoding chloramphenicol resistance are frequently molabile hemolysin gene (tlh) was present in all species except V. fujian- found in examined Vibrio spp. (Letchumanan et al., 2015), and in ensis. Different repeats-in-toxin holotoxins (rtxA to D) were detected in the current study, V. metschnikovii and V. anguillarum harbored the V. alginolyticus, V. metschnikovii, V. anguillarum, and V. fujianensis. catB-like acetyltransferase able to inactivate chloramphenicol. This The hemolysins produced by V. metschnikovii is known to lyse cells gene, however, does not give resistance to florfenicol (Schwarz, from several animals, including humans, sheep, and horse (Miyake, Kehrenberg, Doublet, & Cloeckaert, 2004), which was the only am- Honda, & Miwatani, 1988). All the V. metschnikovii isolates were α-he- phenicol tested in our study. molytic on tryptic soy agar (TSA) with 5% human blood and on TSA Reduced susceptibility to aminoglycoside has been reported with sheep blood, except five isolates that were β-hemolytic on TSA in clinical isolates of V. metschnikovii (Macarena Pariente, Elena with sheep blood. The results indicate that sheep erythrocytes are Escribano, Liria, & S. & María Dolores Crespo, S., 2008; Wallet, more susceptible to these hemolysins, even though a previous study Tachon, Nseir, Courcol, & Roussel-Delvallez, 2005). This was ob- showed the opposite, where human cells were more susceptible to the served quite frequently in our study; however, none of the acetyl- hemolysins produced by V. metschnikovii (Matté et al., 2007). transferases known to confer resistance to this class of agents was RTX is a pore-forming toxin found in several pathogenic Gram- detected in the isolates subjected to WGS. Several efflux pumps, negative bacteria (Lee, Choi, & Kim, 2008), while HlyA, also known including members of the RND, MATE, and ABC family, were found as V. cholerae cytolysin (VCC), is a hemolysin and cytolysin with in the isolates, but these have not been investigated in detail in activity against a range of eukaryotic cells (Ruenchit, Reamtong, 10 of 19 | HÅKONSHOLM et al. Siripanichgon, Chaicumpa, & Diraphat, 2017) and is found in both 5 | CO N C LU S I O N V. cholerae O1 and non-O1/non-O139. The cytotoxic activity has previously been described in V. metschnikovii isolated from a leg To the best of our knowledge, this study presents the most com- wound (Linde et al., 2004). Even though V. metschnikovii have caused prehensive assessment of vibrios from the Norwegian marine infections in humans, it is poorly described with regard to virulence environment, where potentially human pathogenic species like factors, and the presence of these genes may indicate a pathogenic V. alginolyticus and V. metschnikovii were detected. Although the potential. low frequency of multidrug-resistant isolates was observed, several Horizontal gene transfer can mediate transfer not only antibiotic clinically important resistance genes were detected in the Vibrio spp. resistance genes but also virulence factors. V. cholerae virulence en- isolates. These environmental vibrios could act as a reservoir of re- coding genes, for example, zonula occludens toxin (zot), are encoded sistance genes in the marine environment. by prophages, and it has been suggested that the transfer of zot en- coding phages occurs frequently in the Vibrio community (Castillo E T H I C S S TAT E M E N T et al., 2018). Similarly, fragments of V. cholerae pathogenicity islands None required. have been detected in V. alginolyticus, V. anguillarum, and V. met- schnikovii, indicating that important virulence genes can be present in AC K N OW L E D G E M E N T S environmental Vibrio spp. (Gennari, Ghidini, Caburlotto, & Lleo, 2012). We are grateful for samples provided for this study by the Norwegian Food Safety Agency and the research cruises monitoring pelagic fisheries organized by Dr Arne Levsen. We also thank Tone 4.4 | Species identification Galluzzi and Hui Shan Tung for help during the processing of sam- ples and analysis. We also want to acknowledge Hanne Nilsen at the Identification and discrimination of closely related Vibrio spp. can be Norwegian Veterinary Institute for help with identification of Vibrio difficult (Bauer & Rørvik, 2007; Cano-Gomez, Høj, Owens, Baillie, spp. by MALDI-TOF-MS. & Andreakis, 2015; Dieckmann, Strauch, & Alter, 2010; Moreno, Romero, & Espejo, 2002). In this study, several methods for iden- C O N FL I C T O F I N T E R E S T S tification of the isolates were applied. The API20E biochemical None declared. method was able to identify 47% of the isolates to the genus Vibrio. The API20E has a bias toward clinically relevant species (Viña-Feas, AU T H O R C O N T R I B U T I O N Lozano-Leon, de Novoa, Garcia-Martin, & Martinez-Urtaza, 2006) Fredrik Håkonsholm: Conceptualization (equal); Data cura- and does not include as many options for identification of en- tion (equal); Formal analysis (equal); Investigation (equal); vironmental species. A previous study showed that this system Methodology (equal); Project administration (equal); Software was able to correctly identify 63.9% of the Vibrio spp. included (equal); Visualization (equal); Writing-original draft (equal). Bjørn- in the database and performed best on the identification of V. al- Tore Lunestad: Conceptualization (equal); Data curation (equal); ginolyticus and V. parahaemolyticus (O'Hara, Sowers, Bopp, Duda, & Project administration (equal); Resources (equal); Writing-review Strockbine, 2003). MALDI-TOF-MS is primarily designed for clinical & editing (equal). Jose Roberto Aguirre-Sanchez: Software use, and thus, the library mainly contains clinically relevant species (equal); Writing-review & editing (equal). Jaime Martinez-Urtaza: (Santos, Hildenbrand, & Schug, 2016). By applying the Bruker stand- Software (equal); Writing-review & editing (equal). Nachiket P ard library and an external generated library consisting of marine Marathe: Data curation (equal); Investigation (equal); Resources bacteria, MALDI-TOF-MS determined 99% of the 116 isolates to (equal); Validation (equal); Writing-review & editing (equal). one of the three species of Vibrio. Although MALDI-TOF-MS can dif- Cecilie Smith Svanevik: Conceptualization (equal); Data cura- ferentiate between closely related Vibrio spp. (Eddabra, Prévost, & tion (equal); Methodology (equal); Project administration (equal); Scheftel, 2012), the performance of this method is dependent on Supervision (equal); Validation (equal); Writing-review & editing the strain catalogue in the reference library. For isolates identified (equal). by MiGA, a discrepancy with MALDI-TOF-MS was seen for five iso- lates. MiGA is based on average nucleotide identity (ANI) (Rodriguez DATA AVA I L A B I L I T Y S TAT E M E N T et al., 2018), a method where WGS data are used to calculate an The genomes sequences have been deposited to GenBank: https:// average similarity between homologues genomic regions shared www.ncbi.nlm.nih.gov/nuccore under the following accession between two genomes (Kim, Oh, Park, & Chun, 2014). MiGA can numbers: VHSL00000000, VHSN00000000, VHSK00000000, discriminate between closely related species (Rodriguez et al., 2018) VHSM00000000, VHSO00000000, VHTC00000000, and the reference database includes a large number of genomes, in- VHSI00000000, VHSR00000000, VHSS00000000, cluding the Vibrio spp. proposed by MALDI-TOF-MS (http://micro​ VHST00000000, VHSV00000000, VHSX00000000, bial-genom​es.org/proje​c ts/20). Hence, the results from identifica- VHSW00000000, VHSU00000000, VHSQ00000000, tion by MiGA should be considered most reliable. VHSY00000000, VHSP00000000, VMQP00000000. 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Genetic methods for detection of antimicrobial resistance. Apmis, 112, 815–837. APPENDIX 14 of 19 | TA B L E A 1 Measured inhibition zones (mm) from antimicrobial susceptibility testing of isolated Vibrio spp. by disk diffusion Antibacterial agent, Inhibition zone (mm) V. alginolyticus AMP MEL CTX CAZ TE DO CIP OA IPM MEM E AZM SXT W TOB CN FFC ATM 1-1 (4) 6 32 33 31 26 31 34 32 35 36 16/20 21 31 25 22 23 36 37 1-1 (4-a) 6 35 30 29 29 29 35 33 39 37 16/22 20 30 22 19 20 36 31 1-1 (8) 6 35 32 32 28/33 31 38 35 37 42 18 21 30 22 17 19 34 31 1-2 (7) 6 36 32 34 30 29 32 30 40 42 29 24 27 17 20 22 38 29 1-3 (1) 6 36 32 34 31 30 32 32 42 44 21 27 31 26 21 22 38 27 1-3 (1-a) 6 35 30 32 29 30 34 34 41 42 21 28 32 26 21 22 38 27 1-3 (2-a) 6 31 28 31 30 29 32 32 35 39 21 25 34 28 21 24 39 31 1-3 (3) 6 32 29 30 30 32 32 28 41 43 20 26 33 28 20 22 37 28 1-3 (4) 6 34 30 31 28 29 28 28 36 40 19 24 24 16 19 20 33 25 1-3 (4-a) 6 34 32 34 32 30 30 29 35 41 19 25 27 16 18 20 35 28 1-3 (4-b) 6 31 27 29 30 29 29 28 34 37 20 24 29 25 18 19 34 25 1-3 (5) 6 34 31 32 32 30 29 29 36 39 18 22 28 16 20 22 38 28 1-3 (6) 6 34 33 33 32 31 31 28 39 41 22 23 32 22 21 22 37 32 1-3 (6-a) 6 33 34 28 30 29 30 28 38 39 20 24 29 23 19 21 36 30 1-3 (7) 6 33 32 34 31 30 29 27 41 39 18/22 22 30 24 19 21 34 32 1-3 (10) 6 33 27 29 29 30 30 29 37 40 20 26 33 29 18 20 36 31 2-1 (2) 6 34 28 29 30 32 28 28 37 41 19 23 33 19 20 21 37 27 2-1 (5) 6 36 32 34 30 30 29 26 36 36 18 24 30 19 19 20 38 30 2-1 (6) 6 23 23 21 29 28 25 24 30 32 16 20 31 26 17 18 30 19 2-1 (6-a) 6 30 27 25 30 31 24 25 30 32 16 18 27 26 17 18 30 20 2-1 (7) 6 30 23 23 29 27 24 25 31 33 16 18 28 26 17 19 31 20 2-1 (7-a) 6 34 30 29 29 28 28 25 34 36 18 25 31 19 19 21 37 24 2-1 (9) 6 37 32 33 28 29 32 28 39 40 20 21 28 23 20 22 35 31 2-2 (2) 6 36 24/33 26/34 29/34 31/38 27 27 36 36 15/21 19/25 24/28 23 18 20 29/42 22/29 2-2 (2-a) 6 32 29 28 28 30 35 32 37 39 19 24 32 24 19 20 35 27 2-2 (3) 6 33 31 32 29 31 36 32 37 38 20 25 30 23 19 19 36 27 2-2 (3-a) 6 32 29 30 27 29 34 31 36 37 19 24 32 24 18 20 34 27 HÅKONSHOLM et al. (Continues) TA B L E A 1 (Continued) Antibacterial agent, Inhibition zone (mm) V. alginolyticus AMP MEL CTX CAZ TE DO CIP OA IPM MEM E AZM SXT W TOB CN FFC ATM 2-2 (7) 6 33 32 30 29 30 36 35 40 39 21 25 32 25 18 20 35 27 HÅKONSHOLM et al. 2-2 (8) 6 24 28 23 32 28 37 35 20 29 13 20 35 25 19 21 38 26 2-2 (9) 6 33 31 28 28 27 27 24 - 37 19 25 28 14 16 17 37 28 2-3 (1) 6 29 27 27 26 26 33 33 36 35 29 24 27 21 17 18 32 23 2-3 (5) 6 30 27 26 28 27 28 29 33 35 18/19 23 36 32 18 19 37 26 2-3 (5-a) 6 34 30 31 29/35 28/33 28 27 36 37 18/22 19/21 31 22 18 19 35 27 2-3 (6) 6 37 31 32 31 32 28 27 40 42 22 25 29 21 18 19 33 32 2-3 (6-a) 6 37 33 34 33 32 29 27 40 40 22 25 30 22 19 20 35 31 2-3 (9) 6 30 27 27 29 29 30 26 33 35 19 21/30 33 30 18 19 36 25 2-3 (9-a) 6 31 28 29 26/30 28 33 31 37 38 20 24 32 25 19 20 36 27 3-1 (1) 6 37 32 33 31/36 31 29 28 38 42 21 26/36 33 24 19 22 38 32 3-1 (1-a) 6 37 32 33 29/36 28/33 27 27 36 37 16/22 20/30 33 24 21 22 35 33 3-2 (1) 6 26 28 24 31 30 36 35 20 31 16 22 33 28 20 21 37 22 4-1 (2) 6 38 34 33 33 31 27 27 37 38 18/22 19 29 19 18 19 35 29 4-2 (2) 6 37 36 37 33 32 30 29 39 37 20 24 32 25 20 22 36 31 4-3 (1) 6 30 28 27 29 28 26 27 32 33 19 21 30 29 16 18 35 24 4-3 (2) 6 35 30 32 30 31 28 27 37 39 21 25 29 23 19 20 34 29 4-5 (1) 6 32 27 29 28 29 26 27 34 34 18 23 31 26 18 21 38 37 4-5 (1-a) 6 32 28 29 30 31 29 28 40 42 21 25 32 27 21 22/25 38 24 4-5 (2) 6 33 29 30 29 31 27 26 35 38 19 24 31 25 20 21 36 24 7-5 (1-a) 6 32 28 28 27 29 34 36 30 34 20 22/30 29 18 17 19 33 25 8-1 (1) 6 31 29 30 28 30 31 32 39 41 21 24 30 21 19 19 34 26 8-1 (1-a) 6 31 29 28 29 30 27 28 36 38 21 23 30 20 18 19 34 25 B9-1 6 32 31 30 31 29 31 29 35 36 20 23 29 23 20 20 35 26/38 B9-2 6 30 27 27 29 26 30 29 33 34 18 23 28 25 19 20 35 25 B9-3 6 33 28 28 29 29 31 30 35 37 20 24 28 24 19 19 34 24 V. metschnikovii 5-1 (4) 31 36 27 22 26 28 36 33 39 42 21/26 28 33 29 13 16 34 25 5-2 (1) 30 35 25 20 24 26 36 34 40 41 20 26 35 33 15 16 36 27 | 5-2 (2) 31 36 27 23 27 28 35 31 37 40 20/23 25 31 28 14 16 34 24 15 of 19 (Continues) TA B L E A 1 (Continued) 16 of 19 Antibacterial agent, Inhibition zone (mm) | V. alginolyticus AMP MEL CTX CAZ TE DO CIP OA IPM MEM E AZM SXT W TOB CN FFC ATM 5-2 (3) 31 37 31 23 29 30 35 33 40 42 21 25 33 30 14 15 35 25 6-2 (1) 29 35 26 21 28 27 40 35 41 44 23 27 33 30 14 15 34 22 7-5 (1) 32 36 32 25 28 32 36 30 36 40 21 24 30 27 14 14 33 23 A21 6 40 32 24 28 28 36 34 36 38 18 26 34 30 13 15 32 26 A2-2 8 38 30 26 32 30 40 40 42 44 22 26 32 28 26 17 34 28 A6 32 38 28 22 27 28 38 34 40 46 22 26 32 29 15 16 34 23 A7 30 38 28 22 28 26 40 32 42 44 20/26 26/32 34 32 13 14 36 27 A81 8 37 30 22 30 28 36 34 37 40 21 24 32 30 13 15 36 25 A82 9 36 29 26 30 30 42 42 44 44 22 26 36 32 15 16 38 28 A9 32 38 28 24 27 29 42 40 42 44 22 26 32 29 13 15 38 24 A10 32 38 30 26 31 32 41 33 41 44 20 23 29 27 11 13 32 22 A11 11 36 27 23 30 30 38 37 40 42 20 23 29 26 12 14 33 23 A12 31 36 28 22 27 28 38 36 44 44 22 24 36 30 13 15 34 27 A15 31 36 26 22 28 27 36 30 38 40 19 21 30 27 11 13 33 23 A17 29 35 26 21 27 26 33 29 37 40 19 22 31 28 12 13 34 23 TA 4-1 8 40 38 27 30 28 42 34 43 41 21 26/28 36 32 16 18 36 34 TA 4-2 27 40 42 44 40 29 42 36 44 43 26 28 38 35 16 18 40 36 TA 5 18 40 36 35 31 32 40 35 42 44 19 22 32 29 15 18 35 28 TA 13 30 37 30 24 26 28 38 36 46 45 28 28 34 30 15 17 36 27 TA 16 30 36 28 22 30 28 42 40 44 46 21 29 38 32 18 17 38 31 TS2 32 37 28 24 29 27 39 33 40 44 20 24 30 28 12 13 33 23 TS 4 31 38 30 22 26 26 40 36 44 45 21 25 37 35 15 15 39 30 TS 6 30 37 31 24 30 29 41 39 43 46 22 28 32 29 13 15 37 22 GA 5 27 38 27 22 27 27 36 32 38 39 23 24 33 31 13 15 35 27 GA 9 30 35 29 22 28 27 34 35 39 40 19 25 33 28 13 15 37 25 GA 10 32 38 33 26 30 32 37 33 40 43 19/24 25 32 29 - 15 34 24 GA 13 30 39 32 24 26 27 34 33 36 41 20/27 22/25 32 29 14 15 33 25 GA 14 32 38 32 24 30 30 36 34 37 40 23 25/34 33 30 17 18 33 25 GA 16 6 37 30 26 28 26 41 40 44 45 24 25 36 34 15 17 37 30 GA 20 32 40 36 26 32 31 34 33 37 42 20/27 24/32 35 31 15 16 33 26 GS 15 31 40 29 23 28 26 34 35 37 40 20/28 22/33 37 34 19 21 31 29 HÅKONSHOLM et al. (Continues) TA B L E A 1 (Continued) Antibacterial agent, Inhibition zone (mm) V. alginolyticus AMP MEL CTX CAZ TE DO CIP OA IPM MEM E AZM SXT W TOB CN FFC ATM A12 (T.2) 12 39 30 26 29 30 39 36 38 41 23 28 34 29 15 16 35 26 HÅKONSHOLM et al. A16 (T.2) 32 40 35 28 30 28 36 32 36 43 20/28 24/33 32 31 15 15 39 25 A17 (T.2) 32 40 34 26 29 30 41 37 36 38 24 29 32 29 14 18 33 24 T7 u.f (T.2) 40 41 37 29 34 32 37 38 40 44 26 27 34 31 16 18 38 26 V. anguillarum B1-2 6 25 27 23 33 31 37 31 18 27 13 20 32 26 20 20 35 19 B1-4 6 24 26 23 33 32 37 34 17 29 12 22 40 26 19 21 38 21 B4-1 6 26 28 23 32 30 39 35 18 28 12 18 35 26 18 20 36 18 B4-3 6 24 28 22 32 30 42 39 19 30 16 21 33 26 19 20 35 18 B4-4 6 25 27 23 32 31 41 36 18 29 14 16 35 27 19 20 38 20 B4-5 6 26 27 24 32 30 41 37 19 30 14 20 33 26 19 20 36 20 B4-6 6 24 27 23 31 29 39 35 18 28 13 19 32 24 18 19 35 17 B4-7 6 25 28 23 32 31 42 37 17 29 14 21 35 25 19 20 38 19 B4-8 6 25 27 22 31 31 41 36 19 30 16 20 32 25 19 19 38 18 B4-9 6 26 29 23 36 32 40 36 18 29 13 20 31 24 19 20 35 17 B4-10 6 25 27 23 33 30 39 35 19 29 14 20 30 23 19 19 35 18 B4-11 6 25 29 24 32 30 40 36 19 29 13 20 31 24 18 19 37 18 B4-12 6 27 25 22 34 32 42 38 19 30 16 21 33 25 19 20 35 18 B4-13 6 27 28 24 36 34 40 36 18 29 15 21 36 27 20 21 37 20 B4-14 6 25 27 23 32 31 38 36 18 29 14 20 33 25 19 20 37 18 B4-15 6 27 29 25 34 31 41 36 18 30 16 21 34 25 20 21 36 18 B4-16 6 27 29 25 36 34 43 38 19 31 15 23 36 28 20 21 37 20 B4 6 25 28 24 32 30 43 37 17 30 15 22 33 24 19 20 36 19 B7 6 24 27 24 32 31 42 37 18 29 15 23 34 24 18 19 36 18 B8-1 6 27 28 24 34 32 42 38 18 30 15 22 34 25 19 20 39 19 B8-2 6 25 29 22 34 32 39 35 18 29 16 22 35 26 20 21 38 20 V. antiquarius 1-2 (7-a) 6 30 28 27 27 28 30 28 37 39 20 25 26 15 15 17 34 25 11-4 (1) 6 32 30 29 29 30 29 29 39 40 21 24 30 20 19 19 33 26 V. fujianensis 1-1 (7) 6 26 26 22 32 31 36 32 21 29 18 17 33 29 19 19 35 19 | 12-2 (3-a) 12 25 27 25 34 33 42 39 23 31 17 24 39 34 21 21 39 24 Abbreviations: AMP, Ampicillin; ATM, Aztreonam; AZM, Azithromycin; CAZ, Ceftazidime; CIP, Ciprofloxacin; CN, Gentamicin; CTX, Cefotaxime; DO, Doxycycline; E, Erythromycin; FFC, Florfenicol; IPM, Imipenem; MEL, Mecillinam; MEM, Meropenem; OA, Oxolinic acid; SXT, Sulfamethoxazole/Trimethoprim; TE, Tetracycline; TOB, Tobramycin; W, Trimethoprim. 17 of 19 18 of 19 | HÅKONSHOLM et al. TA B L E A 2 Assembly statistics for whole-genome sequenced Vibrio spp Total CDSs Isolate Species Accession no. Coverage length No. Contigs GC (%) N50 (Total) B4-6 V. anguillarum VHSL00000000 54.8X 3,901,483 69 44.51 300,519 3,560 B7 V. anguillarum VHSN00000000 37.1X 3,954,657 40 44.64 283,309 3,541 B1-2 V. anguillarum VHSK00000000 52.4X 3,987,976 45 44.54 242,254 3,677 B4-12 V. anguillarum VHSM00000000 37.3X 3,965,239 48 44.65 283,309 3,555 B8-1 V. anguillarum VHSO00000000 60.9X 3,954,672 41 44.64 283,302 3,540 A8-1 V. metschnikovii VHTC00000000 30.2X 3,761,458 133 44.31 98,018 3,543 A11 V. metschnikovii VHSI00000000 55.8X 3,803,682 81 44.14 234,690 3,464 2-1 (7) V. alginolyticus VHSR00000000 49.2X 5,228,382 24 44.61 1,096,303 4,869 2-2 (2) V. alginolyticus VHSS00000000 38.1X 5,175,814 36 44.63 604,436 4,806 2-2 (7) V. alginolyticus VHST00000000 31.1X 5,176,990 39 44.58 501,798 4,812 2-2 (9) V. alginolyticus VHSV00000000 5.61X 5,444,598 416 44.53 112,490 5,262 7-5 (1-a) V. alginolyticus VHSX00000000 67.5X 5,176,459 36 44.63 1,221,862 4,806 3-2(1) V. alginolyticus VHSW00000000 79.8X 4,110,525 66 44.37 423,004 3,731 2-2(8) V. alginolyticus VHSU00000000 38.1X 3,980,405 55 44.53 368,657 3,677 1-2 (7-a) V. antiquarius VHSQ00000000 57.4X 5,204,341 32 44.77 494,432 4,786 11-4 (1) V. antiquarius VHSY00000000 31.7X 5,251,311 67 44.79 442,253 4,937 1-1 (7) V. fujianensis VHSP00000000 67.8X 3,651,083 37 43.48 834,663 3,560 12-2 (3-a) V. fujianensis VMQP00000000 40.8X 3,650,909 41 43.38 834,684 3,248 Abbreviation: CDSs, Coding sequences. F I G U R E A 1 ML phylogenetic inference of Vibrio anguillarum strains included in this study. Genome used as reference is red shaded, while the genomes from this study are in green. Blue dots show nodes with bootstrap values above 85% HÅKONSHOLM et al. | 19 of 19 F I G U R E A 2 ML phylogenetic inference of Vibrio alginolyticus strains included in this study. Genome used as reference is red shaded, while the genomes from this study are in green. Blue dots show nodes with bootstrap values above 85%