Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains.

Tenacibaculosis is a bacterial ulcerative disease affecting marine fish and represents a major threat to aquaculture worldwide. Its aetiological agents, bacteria belonging to the genus Tenacibaculum, have been present in Norway since at least the late 1980's and lead to regular ulcerative outbreaks and high mortalities in production of farmed salmonids. Studies have shown the presence of several Tenacibaculum species in Norway and a lack of clonality in outbreak-related strains, thus preventing the development of an effective vaccine. Hence, a thorough examination of the bacterial diversity in farmed fish presenting ulcers and the geographical distribution of the pathogens should provide important insights needed to strengthen preventive actions. In this study, we investigated the diversity of Tenacibaculum strains isolated in 28 outbreaks that occurred in Norwegian fish farms in the period 2017-2020. We found that 95% of the 66 strains isolated and characterized, using an existing MultiLocus Sequence Typing system, have not previously been identified, confirming the high diversity of this genus of bacteria in Norway. Several of these Tenacibaculum species seem to be present within restricted areas (e.g., Tenacibaculum dicentrarchi in western Norway), but phylogenetic analysis reveals that several of the strains responsible of ulcerative outbreaks were isolated from different localities (e.g., ST- 172 isolated from northern to southern parts of Norway) and/or from different hosts. Understanding their reservoirs and transmission pathways could help to address major challenges in connection with prophylactic measures and development of vaccines.

Introduction

Tenacibaculosis, caused by members of the genus Tenacibaculum, is an ulcerative skin disease of many economically important marine farmed fish species worldwide. This disease is of major concern for global fish production and is currently considered one of the most threatening bacterial diseases impacting mariculture [1]. Tenacibaculosis is associated with characteristic clinical signs such as ulcerative skin lesions, mouth erosion, frayed fins, and tail. The best-known pathogen in the genus is the type species Tenacibaculum maritimum which has been reported from disease outbreaks in farmed fish since the 1970s [2-7]. However, in recent years an increasing number of novel Tenacibaculum species have been isolated and associated with ulcerative skin disease in farmed marine fishes [8-14]. Tenacibaculosis has been reported from marine wild and farmed fish in Europe, Asia, North and South America and Australia [2, 4, 5, 9, 15–18].

The first time a Tenacibaculum species was associated with fish disease in Norway was in 1989 with the novel bacterium Tenacibaculum ovolyticum. This bacterium was isolated from the adherent epiflora of halibut eggs and was shown to be an opportunistic pathogen for halibut egg and larvae [9, 19]. Since then, an increasing number of novel Tenacibaculum spp. have been associated with ulcerative skin disease in, or shown to be pathogenic to, farmed marine fish in Norway [11, 14, 20–24]. In the recent years, the increased use of sea salts-containing Marine Agar (MA) for bacterial isolation has improved the recovery rate of Tenacibaculum spp. [19-21]. Thus, Tenacibaculum spp. have also been associated with the winter ulcer disease, previously attributed to the bacterium Moritella viscosa [25, 26]. From phylogenetic analyses it has been shown that there is a large Tenacibaculum spp. diversity in Norway [11, 14, 21, 23, 27, 28]. However, most Tenacibaculum isolates associated with ulcerative disease belong to Tenacibaculum finnmarkense genomovar finnmarkense, Tenacibaculum finnmarkense genomovar ulcerans and Tenacibaculum dicentrarchi [11, 23, 27].

Through a MultiLocus Sequence Analysis, the present study aimed at investigating the Tenacibaculum spp. diversity associated with outbreaks of ulcerative disease in Norwegian fish farms for the period 2017–2020 with an emphasis on the northern part, where the aquaculture industry suffers the most from tenacibaculosis. By expanding the analysis to Tenacibaculum spp. isolated all along the Norwegian coast, the presence of ubiquitous or multi-hosts Tenacibaculum strains that could be of importance in terms of future epidemic management of tenacibaculosis in salmon production was explored.

Material and methods

Bacteria isolation

Fish samples were collected from licensed Norwegian fish farms (Norwegian Directorate of Fisheries). The fish were treated by veterinarians and certified fish-health biologists according to the Norwegian Animal Welfare Act (01.01.2010) and the study strictly followed the regulations set by the Norwegian Food Safety Authority.

Farmed marine fishes (Atlantic herring (Clupea harengus), Atlantic halibut (Hippoglossus hippoglossus), Atlantic salmon (Salmo salar), lumpfish (Cyclopterus lumpus) and rainbow trout (Oncorhynchus mykiss)) suffering ulcerative skin disease in Norway were sampled in the period from 2017 to 2019. Fish were sampled either in the frame of a project surveying tenacibaculosis in farmed salmonids in Norway during ulcerative disease outbreaks (fish sampled by veterinarians or certified fish-health biologists on site during outbreaks) or in the frame of other research projects focusing neither on tenacibaculosis nor specifically on salmon (fish sampled by fish-health biologists from our laboratory). Bacteria were isolated from affected fresh tissues (i.e., gills, skin, mouth, fins and from internal organs) directly on site on Marine Agar (MA) (Difco 2216) as a routine medium used by fish veterinarians, or on blood marine agar (BAMA) and BAMA supplemented with kanamycin to a final concentration of 50 μg/ml in our laboratory. Although the isolation process was optimized for the detection of Tenacibaculum spp. (especially culture media), growth of other bacteria species that can potentially cause ulcers (e.g., Moritella viscosa) was monitored.

A total of 197 fish were examined and sampled. Bacteria isolated from field samples were cultured for a minimum of three days at 16 °C. Plates were visually checked for the presence of Tenacibaculum spp. by looking for characteristic colonies as described for Tenacibaculum spp. [7, 9, 19]. The colonies were further examined by using a light microscope to identify the long and slender rods typical of Tenacibaculum spp. cell morphology [19]. The β-haemolytic activity of the colonies was readily observed by looking at the plates through a light source. Colonies containing bacteria with Tenacibaculum spp. morphology were further sub-cultured onto BAMA and incubated for two days at 16 °C or 20 °C. Clonal Tenacibaculum spp. cultures were stored in 50:50 Biofreeze freezing medium (Biochrom™, Germany) and Marine broth (DIFCO 2216) in liquid nitrogen.

DNA extraction, 16S rRNA PCR and sequencing

Genomic DNA was extracted from the recovered Tenacibaculum isolates either by using a DNeasy Blood & Tissue kit (Qiagen) or by heating single colonies in nuclease-free water at 96 °C for 5 min. The heated colonies were subsequently centrifuged at 10,000 G for 5 min and the DNA containing supernatant transferred into new vials. All genomic DNA were stored at—20 °C.

PCR and sequencing of the 16S rRNA gene were performed on all isolates using the primers 27F and 1518R [29]. Amplification details are given in [17]. PCR products were deposited on a 1% agarose electrophoresis gel stained with GelRed™ (Biotium, USA). Obtained amplicons were purified using ExoCleanUp FAST (VWR) in a Veriti thermal cycler (Applied Biosystems) at 37 °C for 5 min and 80 °C for 10 min before being sequenced at the Sequencing Facility at the University of Bergen (http://www.uib.no/seqlab) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).

Consensus sequences from each sample were obtained using VectorNTI 9.0.0 software (Invitrogen) and a BLAST search [30] was performed for preliminary bacterial identification.

Genetic characterization

Bacteria confirmed to be Tenacibaculum spp. from 16S rRNA gene sequence analyses were further included in a MLST analysis using primers targeting seven housekeeping genes atpA (567 bp), dnaK (573 bp), glyA (558 bp), gyrB (597 bp), infB (564 bp), rlmN (549 bp) and tgt (486 bp) as previously described [28].

The established Tenacibaculum MLST website (https://pubmlst.org/organisms/tenacibaculum-spp/) assigned unique allele identifiers for the seven loci considered and defined corresponding allelic profiles referred to as sequence types (STs) (S1 Table). In order to determine the DNA relatedness among Tenacibaculum strains, a minimum-spanning tree based on the seven loci was generated in PHYLOViZ 2.0 using the goeBURST Full MST algorithm (https://online.phyloviz.net/index) [31, 32].

MultiLocus Sequence Analysis (MLSA) was conducted to compare the Tenacibaculum spp. strains included in this study with strains previously described in other studies. The analysis included sequences of the following additional Tenacibaculum spp.: Tenacibaculum finnmarkense genomovar finnmarkense strain HFJ [14], and 36 sequences obtained from GenBank: 15 Tenacibaculum spp. strains isolated in Norway (TNO) and 21 type strains in the genus Tenacibaculum (T. adriaticumT, T. aestuariiT, T. aiptasiaeT, T. amylolyticumT, T. crassostreaeT, T. discolorT, T. dicentrarchiT, T. finnmarkense genomovar finnmarkenseT, T. finnmarkense genomovar ulceransT, T. gallaicumT, T. geojenseT, T. jejuenseT, T. litopenaeiT, T. litoreumT, T. lutimarisT, T. maritimumT, T. mesophilumT, T. ovolyticumT, T. pisciumT, T. skagerrakenseT, T. soleaeT). In accordance with [28], Kordia algicida (Accession number NZ_DS544873.1) was used as an outgroup.

Sequence alignments were constructed for all seven loci separately using AlignX in the VectorNTI 9.0.0 software package (Invitrogen). The sequences were trimmed and adjusted to correct reading frames in GeneDoc [33]. Concatenation of the seven housekeeping genes was performed using Kakusan4 [34]. The substitution rate, codon position and best fit model for the individual loci were calculated with Kakusan4. The Bayesian phylogenetic analysis was performed in MrBayes 3.2 [35] with a proportional codonproportional data bloc and a Markov Chain Monte Carlo (MCMC) analysis. The run included 20,000,000 generations and trees were sampled every 1000 generations. The initial 10,000 trees were discarded as a conservative “burn-in” in TreeAnnotator and the final tree was visualized in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/). GenBank accession numbers of the sequences from this study are provided in S2 Table.

Results

Isolation of bacteria from diseased fish

In the present study, more than 300 Tenacibaculum spp. isolates were recovered from five fish species: Atlantic herring (Clupea harengus), Atlantic halibut (Hippoglossus hippoglossus), Atlantic salmon (Salmo salar), lumpfish (Cyclopterus lumpus) and rainbow trout (Oncorhynchus mykiss). Bacterial colony characteristics are described in S3 Table. Twenty-eight outbreaks in fish farms were investigated in eight counties along the Norwegian coast (Akershus, Finnmark, Hordaland, Møre og Romsdal, Nordland, Rogaland, Troms and Sogn og Fjordane). Sampling details and tenacibaculosis ulcer pictures are given in S4 Table and S1 Fig, respectively. We successfully amplified and sequenced seven MLST loci from 66 strains isolated from the 28 outbreaks (Table 1).

Table 1

Data associated with the 66 Tenacibaculum strains isolated during this study.

ID	Sampling number	Country	Region	Date of collection	Fish host	Fish number	Organ isolation	Haemolytic activity
LIM001	1	Norway	Sogn og F.*	Apr-2018	Salmo salar	#1	skin ulcer	yes
LIM002	1	Norway	Sogn og F.*	Apr-2018	Salmo salar	#2	skin ulcer	yes
LIM003	1	Norway	Sogn og F.*	Apr-2018	Salmo salar	#4	skin ulcer	no
LIM004	1	Norway	Sogn og F.*	Apr-2018	Salmo salar	#4	skin ulcer	no
LIM005	2	Norway	Troms	Apr-2018	Salmo salar	#1	skin ulcer	yes
LIM006	2	Norway	Troms	Apr-2018	Salmo salar	#1	skin ulcer	no
LIM007	2	Norway	Troms	Apr-2018	Salmo salar	#2	skin ulcer	yes
LIM008	2	Norway	Troms	Apr-2018	Salmo salar	#2	skin ulcer	yes
LIM009	2	Norway	Troms	Apr-2018	Salmo salar	#8	skin ulcer	yes
LIM010	3	Norway	Troms	Apr-2018	Salmo salar	#1	skin ulcer	no
LIM011	5	Norway	Troms	Sep-2019	Salmo salar	#1	skin ulcer	yes
LIM012	5	Norway	Troms	Sep-2019	Salmo salar	#2	skin ulcer	no
LIM013	6	Norway	Hordaland	Apr-2017	Salmo salar	#2	skin ulcer	yes
LIM014	6	Norway	Hordaland	Apr-2017	Salmo salar	#3	skin ulcer	yes
LIM016	2	Norway	Troms	Apr-2018	Salmo salar	#3	skin ulcer	no
LIM017	2	Norway	Troms	Apr-2018	Salmo salar	#7	skin ulcer	no
LIM018	3	Norway	Troms	Apr-2018	Salmo salar	#4	skin ulcer	yes
LIM020	4	Norway	Troms	Apr-2018	Salmo salar	#3	skin ulcer	yes
LIM023	7	Norway	Troms	Apr-2018	Salmo salar	#4	skin ulcer	yes
LIM024	8	Norway	Hordaland	Mar-2019	Salmo salar	#1	skin ulcer	yes
LIM025	8	Norway	Hordaland	Mar-2019	Salmo salar	#2	skin ulcer	yes
LIM026	9	Norway	Nordland	Mar-2019	Salmo salar	#1	skin ulcer	yes
LIM027	9	Norway	Nordland	Mar-2019	Salmo salar	#2	head ulcer	yes
LIM032	10	Norway	Akershus	Mar-2019	Salmo salar	#1	gill	yes
LIM033	10	Norway	Akershus	Mar-2019	Salmo salar	#2	gill	yes
LIM036	11	Norway	Hordaland	Apr-2019	Salmo salar	#1	mouth, skin ulcer	yes
LIM040	12	Norway	Nordland	Apr-2019	Salmo salar	#6	skin ulcer	yes
LIM042	13	Norway	Hordaland	Apr-2019	Salmo salar	#11	mouth, gill	yes
LIM043	14	Norway	Hordaland	Apr-2019	Salmo salar	#17	mouth, gill	yes
LIM044	15	Norway	Troms	Apr-2019	Salmo salar	#1	mouth ulcer	yes
LIM046	2	Norway	Troms	Apr-2018	Salmo salar	#1	kidney	yes
LIM047	2	Norway	Troms	Apr-2018	Salmo salar	#3	kidney	no
LIM048	2	Norway	Troms	Apr-2018	Salmo salar	#9	kidney	yes
LIM049	2	Norway	Troms	Apr-2018	Salmo salar	#3	skin ulcer	no
LIM050	2	Norway	Troms	Apr-2018	Salmo salar	#4	skin ulcer	yes
LIM051	2	Norway	Troms	Apr-2018	Salmo salar	#5	skin ulcer	yes
LIM052	2	Norway	Troms	Apr-2018	Salmo salar	#6	skin ulcer	yes
LIM053	2	Norway	Troms	Apr-2018	Salmo salar	#9	skin ulcer	yes
LIM054	3	Norway	Troms	Apr-2018	Salmo salar	#2	skin ulcer	no
LIM055	3	Norway	Troms	Apr-2018	Salmo salar	#3	skin ulcer	no
LIM056	16	Norway	Hordaland	Oct-2017	Salmo salar	#1	mouth	no
LIM057	16	Norway	Hordaland	Oct-2017	Salmo salar	#1	mouth	no
LIM058	16	Norway	Hordaland	Oct-2017	Salmo salar	#3	mouth	yes
LIM059	7	Norway	Troms	Apr-2018	Salmo salar	#3	kidney	no
LIM060	7	Norway	Troms	Apr-2018	Salmo salar	#4	skin ulcer	no
LIM061	6	Norway	Hordaland	Apr-2017	Salmo salar	#1	mouth	yes
LIM062	11	Norway	Hordaland	Apr-2019	Salmo salar	#3	mouth, gill	yes
LIM063	17	Norway	Hordaland	Nov-2019	Salmo salar	#1	skin ulcer	yes
LIM064	18	Norway	Rogaland	Mar-2019	Hippoglossus hippoglossus	#1	skin ulcer	yes
LIM065	19	Norway	Rogaland	Apr-2019	Hippoglossus hippoglossus	#1	skin ulcer	yes
LIM066	20	Norway	Hordaland	Apr-2019	Cyclopterus lumpus	#1	skin ulcer, eye	no
LIM067	21	Norway	Troms	Mar-2019	Salmo salar	#4	skin ulcer	no
LIM068	21	Norway	Troms	Mar-2019	Salmo salar	#1	skin ulcer	no
LIM069	14	Norway	Hordaland	Apr-2019	Salmo salar	#23	skin ulcer	no
LIM070	13	Norway	Hordaland	Apr-2019	Salmo salar	#26	mouth, gill	yes
LIM071	22	Norway	Hordaland	Mar-2019	Salmo salar	#24	mouth, gill	yes
LIM072	23	Norway	Sogn og F.*	Dec-2018	Cyclopterus lumpus	#1	kidney	no
LIM073	23	Norway	Sogn og F.*	Dec-2018	Cyclopterus lumpus	#1	skin ulcer	yes
LIM074	24	Norway	Rogaland	Feb-2019	Oncorhynchus mykiss	#1	skin ulcer	yes
LIM075	24	Norway	Rogaland	Feb-2019	Oncorhynchus mykiss	#2	skin ulcer	no
LIM076	25	Norway	Troms	May-2019	Salmo salar	#1	mouth	yes
LIM077	25	Norway	Troms	May-2019	Salmo salar	#3	mouth	yes
LIM078	26	Norway	Rogaland	Jul-2019	Hippoglossus hippoglossus	#1	gill	no
LIM079	27	Norway	Rogaland	Oct-2019	Hippoglossus hippoglossus	#1	gill	no
LIM080	28	Norway	Hordaland	Feb-2020	Clupea harengus	#1	skin ulcer	yes
LIM081	28	Norway	Hordaland	Feb-2020	Clupea harengus	#2	skin ulcer	yes

*: Sogn og Fjordane.

To note, several other bacteria were isolated (e.g., Vibrio spp., Pseudoalteromonas spp., Photobacterium spp.), always isolated colonies outcompeted by the growth of a large number of Tenacibaculum spp. colonies.

The Tenacibaculum MLST website assigned a total of 29 STs from the Tenacibaculum spp. isolates included in this study, with 27 being new STs to the database. Two STs were previously isolated from Atlantic salmon in 2010 (ST-52 and ST-53) and in 2011 (ST-52). In the present study, ST-53 was isolated from a diseased Atlantic salmon farmed in Troms in April 2018. ST-52 was isolated from two Atlantic salmon, in March 2019 in Akershus and in May 2019 in Troms (Fig 1).

Fig 1

Minimum spanning tree.

Minimum spanning tree of Tenacibaculum strains based on 7 loci (atpA, dnaK, glyA, gyrB, infB, rlmN and tgt) using the goeBURST Full MLST algorithm. The sequence types (STs) are identified by a number (ST-, see https://pubmlst.org/tenacibaculum/). The circle size reflects their abundance in the data set. Group founders are indicated by a yellow circle. The analysis includes 67 new isolates for the database (66 STs from this study and ST-174 assigned to HFJ strain), and 18 STs already published. The DNA relatedness is presented with emphasis on regions (A) or hosts (B).

Of the 27 new STs, 22 were isolated only once during 22 different outbreaks of tenacibaculosis. Five STs were isolated during more than one outbreak: ST-160, ST-162, ST-107, ST-152 and ST-172.

ST-160 and ST-162 were associated with outbreaks of ulcerative skin disease that occurred in April 2018 in Troms. Both were isolated from Atlantic salmon from the same farm during two samplings separated by seven days. ST-160 was isolated from both a skin ulcer and from the head kidney of the same fish. Two Atlantic salmon with skin ulcers were also infected with ST-160 in another farm in Troms during the same period (April 2018).

ST-107 was isolated from Atlantic salmon during tenacibaculosis outbreaks at a single site in Hordaland in March, April, and November 2019. ST-107 was also isolated from a single site rearing herring that presented with ulcerative disease in February 2020.

ST-152 was isolated from Atlantic salmon in Hordaland (April 2017, March, and April 2019), in Troms (April and September 2018) and in Akershus (March 2019).

The most common ST found in this dataset was ST-172. It was isolated from salmon in three different counties: Sogn og Fjordane (April 2018), Nordland (Mars and April 2019) and Troms (April 2018, April, and May 2019). ST-172 was also isolated from rainbow trout (February 2019) and lumpfish in Rogaland (April 2019). This ST was associated with severe tenacibaculosis.

MultiLocus Sequence Analysis (MLSA)

A phylogenetic tree was constructed using the 66 concatenated sequences (3894 bp) included in this study. In addition, the analysis included the strain HFJ [14], 15 Tenacibaculum spp. strains previously isolated in Norway [28], and 21 type strains in the Tenacibaculum genus (Fig 2).

Fig 2

Bayesian analysis.

Bayesian phylogenetic tree of 103 Tenacibaculum strains based on concatenated sequences of seven genes (atpA, dnaK, glyA, gyrB, infB, rlmN and tgt, total size: 3894 bp). The data set includes 66 Tenacibaculum strains isolated during this study (LIM strains), the HFJ strain previously isolated and 36 strains from GenBank (15 from [28] (TNO strains) and 21 Tenacibaculum Type Strains). Kordia algicida was used as an outgroup. Posterior probabilities are indicated at each node. Fish symbols indicate the host from which each strain has been isolated (LIM and TNO) and colours indicate the Norwegian administrative regions, accordingly to the map. Colour dots indicate if the strains show haemolytic activity on blood agar (red dot), not (blue dot) or variable (red/blue dot). Clades I-V represent the structure of the T. finnmarkense/T. dicentrarchi group of isolates. The Norwegian map was reprinted from https://doi.org/10.1371/journal.pone.0215478 under a CC BY license, with permission from Are Nylund, original copyright 2019. This map is similar but not identical to the original image and is therefore for illustrative purposes only.

Fifty-seven out of the 66 strains (83%) belong to a large and highly diversified T. dicentrarchi/T. finnmarkense group of isolates that could be further separated into five highly supported distinct clades (clades I, II, III, IV and V).

Clade I (the T. finnmarkense genomovar ulcerans clade) consists of 14 Norwegian Tenacibaculum strains including strains isolated from Atlantic salmon from 1996 to 2011 (n = 5) and Atlantic cod in 2010 (n = 3) [20, 23, 28]. The six strains from this study were isolated from Atlantic salmon (n = 5) and lumpfish (n = 1). None of these strains showed β-haemolytic activity when grown on BAMA.

Clade II (the T. dicentrarchi clade) consists of eight isolates and includes the T. dicentrarchi type strain. The six isolates from the present study were isolated from Atlantic salmon in March (LIM024 and LIM025), April (LIM036) and November 2019 (LIM063) and from Atlantic herring in February 2020 (LIM080 and LIM081). All six isolates presented β-haemolytic activity on blood agar and were associated with clinical signs of tenacibaculosis. The clade II isolates were only isolated in Hordaland and showed 100% identity based on the seven housekeeping genes (ST-107). The Tenacibaculum isolates TNO012 and TNO015 were recovered from Atlantic cod in 2009 and 2010 and are genetically distinct from each other and from all other LIM isolates in the current study.

Clade III (the T. finnmarkense genomovar finnmarkense clade) includes the HFJ strain, 44 isolates recovered during this study and seven isolates from Atlantic salmon sampled in 2010 and 2011 [28]. Forty-two isolates were collected from Atlantic salmon in Akershus, Finnmark, Hordaland, Nordland and Troms. Three isolates were found in rainbow trout (LIM074) and Atlantic halibut (LIM064 and LIM065) in Rogaland. All these isolates showed β-haemolytic activity on blood agar except for ten isolates (TNO002, LIM006-010-017-047-049-054-055-059-060-068).

Clade IV and clade V are both represented by a single isolate; T. piscium type strain (isolated from an Atlantic salmon in 1998) and LIM075 (isolated from a rainbow trout in Rogaland in February 2019), respectively. Like T. piscium [11], LIM075 did not present any β-haemolytic activity on blood agar.

LIM057, LIM067 and LIM072 form a monophyletic group. They were isolated in Hordaland (LIM057 in October 2017), Sogn og Fjordane (LIM072 in December 2018) and Troms (LIM067 in March 2019), from 2 different hosts: Atlantic salmon (LIM057 and LIM067) and lumpfish (LIM072). None of them showed any β-haemolytic activity on blood agar.

Three isolates closely related to T. ovolyticum. LIM058 and LIM070 were isolated from salmon in Hordaland in October 2017 and April 2019, respectively. LIM073 was isolated from a lumpfish in Sogn og Fjordane in December 2018. They all showed β-haemolytic activity on blood agar.

LIM069 was isolated from an Atlantic salmon skin ulcer in Hordaland in April 2019. It did not present any β-haemolytic activity on blood agar.

LIM078 and LIM079 are two distinct Tenacibaculum spp. strains isolated from farmed Atlantic halibut in Rogaland. Neither strain showed β-haemolytic activity on blood agar.

Discussion

The diagnosis of tenacibaculosis has usually been based on histology and isolation of Tenacibaculum spp. from sick fish followed by morphological, biochemical, and serological characterization of the bacteria and analysis of their antimicrobial susceptibility profiles [8]. MultiLocus Sequence Analysis (MLSA) has, however, been increasingly used for bacterial genotyping [17, 23, 28, 36, 37] and has become an important tool for diagnosis and studies of epizootics. The results from the current study confirm the large diversity of Tenacibaculum spp. strains present in fish farms along the Norwegian coast and their association with ulcerative disease outbreaks.

Although the isolation process was optimized for the detection of Tenacibaculum spp., the implication of other bacteria species likely to be the cause of the observed ulcers can be fairly dismissed.

High diversity of Norwegian Tenacibaculum spp. strains

This study presents the genotyping of 66 Tenacibaculum strains isolated from April 2017 to February 2020 in Norway. Phylogenetic analyses show that 57 strains belong to a major lineage structured into five different clades consisting of T. finnmarkense genomovar ulcerans Clade I, T. dicentrarchi Clade II, T. finnmarkense genomovar finnmarkense Clade III, Tenacibaculum piscium Clade IV, and a putative new species Clade V (Fig 2). This is in accordance with previous phylogenetic analyses of Tenacibaculum spp. isolated from farmed fishes in Norway [11, 23, 27]. Several strains are not closely related to any Tenacibaculum spp. type strains (LIM078 and LIM079 isolated from Atlantic halibut in Rogaland, LIM069 isolated from Atlantic salmon in Hordaland; LIM057, LIM067 and LIM072 isolated from Atlantic salmon and lumpfish in Troms, Hordaland, and Sogn og Fjordane). Nevertheless, further identification steps must be undertaken to conclude whether these new strains represent novel Tenacibaculum species.

Altogether, these results confirm the large Tenacibaculum diversity in Norway, and suggest that other strains have yet to be identified.

The phylogenetic analysis clearly showed that most of the isolates recovered from Atlantic salmon suffering tenacibaculosis belong to the species T. finnmarkense genomovar finnmarkense and have β-haemolytic activity (except for five STs clustering in a strongly supported clade). The T. dicentrarchi strain isolated in Hordaland (ST-107, β-haemolytic) was associated with tenacibaculosis and mortality in both Atlantic salmon and Atlantic herring. The pathogenicity of T. dicentrarchi strains has been already described in several fish species including European sea bass, wrasse, Atlantic cod, and Atlantic salmon from Chile [13, 28, 37]. Olsen et al. suggested that three of the four T. dicentrarchi strains isolated from asymptomatic salmon could have a higher pathogenicity towards non-salmonids species [23]. The severe symptoms associated with ST-107 (LIM 024-025-036-063-080-081) suggests pathogenicity towards Atlantic salmon and Atlantic herrings and indicate that strains pathogenic to salmon may also occur in Norway. The three strains closely related to T. ovolyticum were associated with ulcers and showed β-haemolytic activities when grown on blood agar plates. Even though T. ovolyticum is already known for its ability to dissolve halibut eggshell, which could lead to the death of the embryo [9, 38], it is to our knowledge the first report of pathogenicity of T. ovolyticum toward Atlantic salmon and lumpfish. Nevertheless, it is uncommon and therefore unlikely to be a major threat to aquaculture.

β-haemolytic activity is commonly associated with pathogenic bacteria [39]. Among the strains isolated in the context of this study, non-haemolytic Tenacibaculum strains seem to be less pathogenic and therefore may not lead to tenacibaculosis outbreaks. Indeed, when associated with outbreaks, Clade I strains were isolated together with other haemolytic strains (LIM003, LIM004, LIM012, LIM016). Some Clade I strains were sporadically associated with ulcer symptoms (LIM066 for instance). This reinforces the need for the use of blood-supplemented culture media in the field. Estimating as early as possible the virulence of a strain is necessary in order to implement rapid sanitary measures. Nevertheless, it is important to temper this assessment, as non-haemolytic strains can also prove to be pathogenic. For instance, the non-haemolytic ST-160 (Clade III) was also associated with tenacibaculosis. While presence of haemolytic activity is to be seriously considered, all strains should be monitored.

Presence of clinically relevant ubiquitous strains

Tenacibaculum finnmarkense isolates from clades I and III are present all along the Norwegian coast, from Akershus in the southeast to Finnmark in the north. It is interesting to note that all the STs from Clade III except one (LIM064, isolated in Rogaland) have been isolated at least once in Troms or Finnmark. To note, Tenacibaculum finnmarkense has also been isolated outside of Norway, from Chilean Atlantic salmon, coho salmon, and rainbow trout (Oncorhynchus kisutch) [27, 36, 40]. Tenacibaculum dicentrarchi strains and the strains closely related to T. ovolyticum were only isolated in the western part of Norway. The reason why T. dicentrarchi and T. ovolyticum seem to be mostly present in western Norway is unknown but may be linked to several factors such as higher seawater temperature in western Norway compared to northern Norway.

Ninety-three % of the STs described herein were new to the MLST database. Only two STs (ST-52 and ST-53) were previously isolated from Atlantic salmon in 2010 and 2011 [23]. The majority of these new STs (81%) were recovered only once and therefore each were associated with a single locality and a single host species. This is in accordance with previous results that showed that the overall lack of clonality and host specificity among the Norwegian Tenacibaculum isolates indicated that tenacibaculosis infections arise as local epidemics involving multiple strains [23]. However, several STs were isolated from different localities and/or from different hosts and are therefore of extreme importance for the aquaculture. ST-152 is associated with tenacibaculosis outbreaks in farmed Atlantic salmon in three different counties: Troms, Hordaland and Akershus. ST-172 is the strain with the widest distribution, from Troms in the north, to Rogaland in the south. This ST, always associated with severe tenacibaculosis, might have a wider tolerance to different seawater conditions (salinity and temperature) than T. dicentrarchi. More importantly, both STs were isolated throughout the sampling period, from April 2017 to May 2019. We isolated ST-172 again in March 2020 during an outbreak in an Atlantic salmon farm in Troms. ST-172 has been isolated from three different fish species: Atlantic salmon, rainbow trout and lumpfishes. Even though it is uncommon, the ability of some Tenacibaculum strains to infect several fish species has been shown already [23, 28], but they were limited to a restricted geographic area. Likewise, the T. dicentrarchi related ST-107 isolated from both Atlantic salmon and Atlantic herring, was found only in Hordaland. But ST-172 and ST-152 present the ability to spread within a broad geographic area. How they spread out along the Norwegian coast and how they maintain themselves in the environment between outbreaks remain unknown, but several hypotheses can be made. Avendaño-Herrera et al. suggested that seawater is not an important route of transmission for T. maritimum and hypothesised that bacteria need to be attached to particle or substrate such as sediments or fish mucus to be maintained until favourable conditions occur [41]. Levipan et al. showed that all the T. dicentrarchi strains in their study were able to adhere to and form biofilms on polystyrene surfaces [42]. Survival of T. maritimum in fish mucus [43] suggests that bacteria can colonize and spread together with its hosts. Fish movements at different life stages can therefore be a spreading route for their associated bacteria. Furthermore, the transport of pathogens by fish ectoparasites have already been reported. For instance, the sea lice Lepeophtheirus salmonis, a common salmonid associated ectoparasite, can carry several micro-organisms such as Aeromonas salmonicida [44] and T. maritimum [45]. Thus, studies assessing the transport of Tenacibaculum sp. need to be implemented.

Conclusion

MultiLocus Sequence Typing is a powerful tool for studies of epizootics caused by bacteria and other pathogens. It is highly efficient to get an overview of a bacterial community and to separate strains with putative different pathogenicity. Overall, the results from this study consolidate the conclusions of previous studies and confirm the high diversity of the Tenacibaculum spp. strains infecting farmed fishes in Norway. This high diversity of Tenacibaculum spp. strains and their broad geographical distribution is a major challenge for aquaculture, not only in Norway but also globally. Attempts to tackle some of these major outbreaks by focusing on vaccines have so far been unsuccessful, which increases the importance of knowledge about reservoirs, transmission routes and virulence differences.

Allelic profiles.

Sequence type (ST) for 67 Tenacibaculum strains and unique allelic identifiers for the seven loci considered in the MLST scheme assigned by the established Tenacibaculum MLST website (https://pubmlst.org/tenacibaculum/).

(DOCX)

Click here for additional data file.

GenBank accession numbers of the sequences produced in this study.

(DOCX)

Click here for additional data file.

Colony characteristics.

Growth characteristics on blood Marine Agar of the 29 sequence types isolated in the study.

(DOCX)

Click here for additional data file.

Sampling details.

(XLSX)

Click here for additional data file.

Details on tenacibaculosis ulcers.

A: sampling 1. B: sampling 9. C: sampling 11. D: sampling 12. E: sampling 14. F: sampling 15. G: sampling 21. H: sampling 27. I: sampling 28. Image credits: Photo by Erwan Lagadec (C, I), MarinHelse AS (B, F, G), Heidrun Nylund (A, D, E), Kjetil Solheim (H).

(TIF)

Click here for additional data file.

11 Aug 2021

PONE-D-21-21158

Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains

PLOS ONE

Dear Dr. Lagadec,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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PLOS ONE

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Additional Editor Comments:

Dear Dr. Lagadec,

Thankyou for submitting this interesting manuscript to PloSOne. As you can see from the reviewers' comments, they are all in favour of publishing your findings, as I am as well, but have reservations which would need to first be addressed. These are all experienced fish pathologists whose opinions I value highly and I was especially happy that they all consented to review your manuscript. That they did so is no doubt also a reflection of your own work and that of your group.

I very much agree with their suggestions and recommendations and am convinced that by addressing the points they have raised that this will improve your manuscript, and inevitably counter any likely queries which would have been raised by a critical readership.

The diversity in Tenacibaculum strains you and your colleagues have discovered in Norwegian waters is truly impressive and I have no doubt that groups in other parts of the world will be stimulated by your findings to explore their own regions in greater detail. This will be key to developing vaccines which can be widely applied to countering this threat to aquaculture and the ensuing consequences on the environment. In this context, the additional details and information sought by the reviewers will be invaluable for those planning such studies.

I am very much looking forward to your response to these suggestions, just as much as I enjoyed reading the manuscript you sent in.

Kind regards,

Lloyd Vaughan

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: To better understanding the pathogen diversity underlying fish diseases is an important prerequisite for the control of the disease. The submitted Ms aims to examine the diversity of Tenacibaculum spp. Bacteria in Norwegian aquaculture. These bacteria are opportunistic pathogens and can cause ulcerative diseases. While originally, focus was T. maritimum , more recent research unraveled that there is much more diversity of Tenacibaculum species in aquaculture. The present study further extends the knowledge on the diversity of this genus, and as such it is of relevance.

Methodologically, the authors collected bacteria from marine fish farms suffering outbreaks of ulcerative skin disease during 2017-2019. The bacteria were isolated from diseased tissues, were cultured and then the 16S rRNA was sequenced using MLST. Personally, I am not an expert on MLST and thus are not in a position to judge on the adequacy of the molecular methodology. However, I have several questions on the sampling methodology: how many fish were sampled per farm? Were only diseased or also healthy fish/tissues sampled? Were all isolates from one fish pooled, were isolates from different individuals pooled, or were all isolates treated separately? Were only Tenacibuculum bacteria analysed or was a broader microbial characterization of the diseased fish performed? In particular I would be interested to know what evidence can be provided by the authors that the Tenacibaculum bacteria are indeed causative to the ulcerative changes. The mere presence of a bacterial species on a diseased fish dos not necessarily mean that this species is the etiological agent.

Results:

The data based in figure 1 are based on the analysis of 7 loci – what criteria were used to select those 7 loci?

Line 163: Since sampling details are inadequately described (see above), it is difficult to follow the results. For instance, in line 165 its says that ST-160 was isolated form both the skin ulcer and the head kidney of the same fish. Was ST-160 found in other tissues of this fish as well, or were other tissues not analysed? And was this fish the only fish with ST-160 in the farm/the only fish with ulcer, or were more fish positive for ST-160, and did they all show skin ulcer? This is a lot of detail, but this detail is critical to understand the association of T. spp with ulcer.

In addition, while the Results provide an in-depth MLST analysis, I missed an "epidemiological" analysis: For instance, the authors investigated a range of fish species: were certain strains/ST associated with certain host species or was no pattern detectable? Was there an association between farm conditions, ulcer prevalence/severity and presence of specific strains? If this study should go beyond a mere analysis of genetic diversity but would like to promote understanding of the disease epidemiology, more emphasis on this type of questions is required. In the way as presented now, the ms unfortunately does not provide insight how the results of this study would support "future epidemic management" (line 64)

The concluding sentence of the abstract says: "Understanding their reservoirs and transmission pathways could help to address major challenges in connection with prophylactic measures and development of vaccines". I fully agree with this statement, but I do not see how this Ms as presented now would support these aims. Interestingly, the authors address these questions in the Discussion, but unfortunately they do not apply it to their own study.

Reviewer #2: Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains, by Lagadec et al.

In this manuscript, the authors provide useful information about the diversity of Tenacibaculum spp. in the Norwegian fish farms. Bacteria belonging to the Tenacibaculum genus have become important opportunistic pathogens for many different marine fishes and lately have been associated with ulcerative diseases in farmed salmon. The authors have collected several strains from different regions in Norway and have used MLST analysis to map their genetic diversity.

Although the manuscript is well-written, and the information provided is useful there are some important points that should be considered. It seems to me that the strains used in the study have been collected by fish vets during routine diagnostic analyses and are not part of a specific research survey of the team. This if fully acceptable and understandable, however if this is true, it should be specifically mentioned in the manuscript. I am not very sure if the media used in the isolation would favor the growth of all possible Tenacibaculum spp. These bacteria are difficult to isolate, and in many cases, there is overgrowth by other faster growing opportunistic bacteria. The use of general isolation media may have hindered the growth of other species like Tenacibaculum maritimum.

Furthermore, I am not convinced that the heamolytic activity of Tenacibaculum strains is a valid indicator of pathogenicity. There are several non-haemolytic bacterial pathogens which are highly virulent. Although the authors state that the occurrence of beta haemolysis is an indicator of pathogenicity (supported by a relevant citation), the non-occurrence does not mean that the strains are less virulent. Haemolytic activity has been used extensively by the authors, however how it was assessed is not described in the M&M. More care should be given in the use of haemolysis as virulence factor for Tenacibaculum in order to be convincing.

Specific comments to the authors:

L53: move (MA) after Marine Agar

L53: Explain how the use of MA associates Tenacibaculum with winter ulcers

L68: Mention how many samples were used

L75-78: Are these the best media for isolating Tenacibaculum spp? What about FMA or TMA? It seems that these media are mostly used for routine microbiology by the fish vets. If this is the case, it should be stated either in the M&M or in the introduction, since this is a limitation of the study that may have biased the results. If this is not the case, then you should provide references supporting the use of these media for the specific isolation of Tenacibaculum spp.

L:78 It is not clear later in the results how many of the isolates were Tenacibaculum. If there were other bacteria present, which were these? What is the prevalence of Tenacibaculum during the outbreaks?

L80: What are the characteristics of the colonies? are these the same in all media? were they described also in Blood Agar? In the papers cited the authors have used different media.

L82: Describe the morphology

L143: Table legend: You talk about haemolytic activity but how this was assessed is not in the M&M. Since haemolytic capacity of the bacteria plays an important role in your paper, the method used to assess it should be described

L190-191: How do you define “variable”? Have you used replicates in the heamolytic activity assay?

L266: What are the severe symptoms? Where were they described?

L276: How did you come to this conclusion that the non-haemolytic strains are less pathogenic? If the conclusion is based on the field data (eg morbidity/mortality), these data should be provided. Even then, the conclusion is really based on circumstantial evidence, and it is indicative if not speculative. Unless there are comparative data from challenge tests (if yes, then you should provide these data).

L281: The association of hemolysis to virulence or to be more accurate, the non-hemolysis to non- or reduced virulence is not supported by evidence and it is speculative.

Reviewer #3: This manuscript provides additional information on the epidemiology of Tenacibaculosis and should be published. The information provided is sound and useful but not a significantly novel contribution to fish bacteriology, but should be published as it provides good information on the distribution and epidemiology of the bacterial isolates of concern.

Reviewer #4: The manuscript submitted by Lagadec et al (PONE-D-21-21158) is a well presented and informative paper showing the diversity that exists amongst Tenacibaculum spp. strains in Norwegian aquaculture.

I have a few minor points for the authors to address.

Line 39 - bacterial diseases

Line 44 - Tenacibaculosis has been reported in marine wild and farmed fish in Europe, Asia,

Line 52 - Due to the increased use of Marine Agar (MA) for bacterial isolation in recent years, Tenacibaculum spp. have also been associated with the winter ulcer disease, previously attributed to the bacterium Moritella viscosa [25, 26].

Line 89 - 10,000 rpm – state xg rather than rpm

Lines 139/194/213/294 - Numbers at the start of sentences should be written in words rather than as a number.

Line 151- March 2019

Line 161 - Of the 27 new STs, 22 were isolated during a single outbreak of tenacibaculosis. Was the outbreak at a single site - mention the location of this outbreak?

Line 278 - Overall, non-haemolytic Tenacibaculum strains seem to be less pathogenic and may not lead to tenacibaculosis outbreaks. Can you state the evidence for this e.g. a reference or findings from your study?

**********

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Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

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Submitted filename: PONE-D-21-21158.docx

Click here for additional data file.

30 Sep 2021

PONE-D-21-21158

Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high

bacterial diversity and suggest circulation of ubiquitous virulent strains

PLOS ONE

Dear Dr. Lagadec,

Thank you for submitting your manuscript to PLOS ONE. After careful

consideration, we feel that it has merit but does not fully meet PLOS ONE's

publication criteria as it currently stands. Therefore, we invite you to

submit a revised version of the manuscript that addresses the points raised

during the review process.

Please see my comments below.

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[1].

We look forward to receiving your revised manuscript.

Kind regards,

Lloyd Vaughan, PhD, BSc (Hons)

Academic Editor

PLOS ONE

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The authors: We apologize for the use of this map in Figure 2. We have modified figure 2 by integrating a map already published in PLOS ONE (https://doi.org/10.1371/journal.pone.0215478). This map has been modified and vectorized under Inkscape 0.91. The original holder of this map is Pr. Are Nylund, author of the present manuscript. However, he completed the Content Permission Form. This Content Permission Form is upload as an “Other” file within this new submission. We modified the caption accordingly by adding: “The Norwegian map was reprinted from https://doi.org/10.1371/journal.pone.0215478 under a CC BY license, with permission from Are Nylund, original copyright 2019. This map is similar but not identical to the original image and is therefore for illustrative purposes only " (see Lines 210-213).

Additional Editor Comments:

Dear Dr. Lagadec,

Thank you for submitting this interesting manuscript to PloSOne. As you can

see from the reviewers' comments, they are all in favour of publishing your

findings, as I am as well, but have reservations which would need to first

be addressed. These are all experienced fish pathologists whose opinions I

value highly and I was especially happy that they all consented to review

your manuscript. That they did so is no doubt also a reflection of your own

work and that of your group.

I very much agree with their suggestions and recommendations and am

convinced that by addressing the points they have raised that this will

improve your manuscript, and inevitably counter any likely queries which

would have been raised by a critical readership.

The diversity in Tenacibaculum strains you and your colleagues have

discovered in Norwegian waters is truly impressive and I have no doubt that

groups in other parts of the world will be stimulated by your findings to

explore their own regions in greater detail. This will be key to developing

vaccines which can be widely applied to countering this threat to

aquaculture and the ensuing consequences on the environment. In this

context, the additional details and information sought by the reviewers

will be invaluable for those planning such studies.

I am very much looking forward to your response to these suggestions, just

as much as I enjoyed reading the manuscript you sent in.

Kind regards,

Lloyd Vaughan

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

COMMENTS TO THE AUTHOR

1. Is the manuscript technically sound, and do the data support the

conclusions?

The manuscript must describe a technically sound piece of scientific

research with data that supports the conclusions. Experiments must have

been conducted rigorously, with appropriate controls, replication, and

sample sizes. The conclusions must be drawn appropriately based on the data

presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: Yes

Reviewer #4: Yes

-------------------------

2. Has the statistical analysis been performed appropriately and

rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

Reviewer #4: Yes

-------------------------

3. Have the authors made all data underlying the findings in their

manuscript fully available?

The PLOS Data policy [2] requires authors to make all data underlying the

findings described in their manuscript fully available without restriction,

with rare exception (please refer to the Data Availability Statement in the

manuscript PDF file). The data should be provided as part of the manuscript

or its supporting information, or deposited to a public repository. For

example, in addition to summary statistics, the data points behind means,

medians and variance measures should be available. If there are

restrictions on publicly sharing data--e.g. participant privacy or use of

data from a third party--those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

-------------------------

4. Is the manuscript presented in an intelligible fashion and written in

standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in

submitted articles must be clear, correct, and unambiguous. Any

typographical or grammatical errors should be corrected at revision, so

please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

-------------------------

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions

above. You may also include additional comments for the author, including

concerns about dual publication, research ethics, or publication ethics.

(Please upload your review as an attachment if it exceeds 20,000

characters)

Reviewer #1: To better understanding the pathogen diversity underlying fish

diseases is an important prerequisite for the control of the disease. The

submitted Ms aims to examine the diversity of Tenacibaculum spp. Bacteria

in Norwegian aquaculture. These bacteria are opportunistic pathogens and

can cause ulcerative diseases. While originally, focus was T. maritimum ,

more recent research unraveled that there is much more diversity of

Tenacibaculum species in aquaculture. The present study further extends the

knowledge on the diversity of this genus, and as such it is of relevance.

Methodologically, the authors collected bacteria from marine fish farms

suffering outbreaks of ulcerative skin disease during 2017-2019. The

bacteria were isolated from diseased tissues, were cultured and then the

16S rRNA was sequenced using MLST. Personally, I am not an expert on MLST

and thus are not in a position to judge on the adequacy of the molecular

methodology. However, I have several questions on the sampling methodology:

how many fish were sampled per farm? Were only diseased or also healthy

fish/tissues sampled?

The authors: We thank the reviewer#1 for his very constructive comments.

Indeed, the paper in its current form lacks details and does not allow to understand what the limits of our study were. We would first clarify the sampling methodology carried out in this study: fish presenting ulcers were sampled either in the frame of a project surveying tenacibaculosis in farmed salmonids in Norway during ulcerative disease outbreaks (fish sampled by veterinarians or fish-health biologists on site during outbreaks; culturing plates and/or tissue samples were sent to our laboratory) or in the frame of other surveying projects focusing neither on tenacibaculosis nor specifically on salmon (fish sampled by fish-health biologists from our laboratory). This sampling resulted in heterogeneity in the samples (sampling during an outbreak or not, different quantity of fish sampled per sites, sampling including or not tissue/organs) but made it possible to obtain the maximum number of bacterial strains from different hosts and different localities. We clarified that point in the M&M section (see lines 77-81) and added a new table providing details on the samplings (Supporting Information S4 Table). We have also modified Table 1.

Were all isolates from one fish pooled, were isolates

from different individuals pooled, or were all isolates treated separately?

The authors: All the bacterial strains included in this study were isolated and subsequently cloned on separate culturing plates. Each strain was therefore treated separately. It is also a prerequisite to be able to genotype with the MLST technique (MLST being a concatenation of several HK gene sequences, pure bacterial clones are needed to avoid concatenation of sequences from different strains).

Were only Tenacibuculum bacteria analysed or was a broader microbial

characterization of the diseased fish performed? In particular I would be

interested to know what evidence can be provided by the authors that the

Tenacibaculum bacteria are indeed causative to the ulcerative changes. The

mere presence of a bacterial species on a diseased fish dos not necessarily

mean that this species is the etiological agent.

The authors: We fully agree that a pathogen present on a sick fish does not necessarily mean that it is the etiological agent. Although our culture process (including media) was optimized for the detection of Tenacibaculum spp., we have monitored the growth of other bacteria, especially those known to cause ulcers on marine fish (e.g., Moritella viscosa). All the bacteria isolated were submitted to a 16S rRNA sequencing, the Tenacibaculum strains being subsequently included in the genotyping. All culture plates presented a dominant growth of Tenacibaculum spp. We isolated several other bacteria, such as Vibrio spp., Pseudoalteromonas spp., Photobacterium spp., but always small colonies in minority and outcompeted by a large number of Tenacibaculum spp. colonies.

Tenacibaculum spp. have been shown to be marine fish ulcerative pathogens in Norway for more than a decade. The dominant growth of bacteria species belonging to the genera Tenacibaculum on culture from ulcers seems to us a reasonable proof of its implication, especially because we did not detect other species likely to be the cause of the ulcers observed. However, we should clearly specify what the limits of our detection method were, and why we are fairly convinced of the implication of Tenacibaculum spp. in these ulcers (see in M&M lines 85-87, in Results lines 157-159 and in Discussion lines 264-266).

85-87

Results:

The data based in figure 1 are based on the analysis of 7 loci - what

criteria were used to select those 7 loci?

The authors: These 7 loci are part of a MultiLocus Sequence Typing scheme developed by Habib et al. (2014). They are located within protein coding genes conserved across the family Flavobacteriacea. This scheme has been since used by other teams, thus making it possible to enrich the official MLST database (https://pubmlst.org/) and to determine the DNA relatedness of the strains isolated in this study with those present in this open-access database.

To note, each of these loci are commonly used in bacterial molecular typing and had already been used in previous studies of other bacterial species and genera.

Line 163: Since sampling details are inadequately described (see above), it

is difficult to follow the results. For instance, in line 165 its says that

ST-160 was isolated form both the skin ulcer and the head kidney of the

same fish. Was ST-160 found in other tissues of this fish as well, or were

other tissues not analysed? And was this fish the only fish with ST-160 in

the farm/the only fish with ulcer, or were more fish positive for ST-160,

and did they all show skin ulcer? This is a lot of detail, but this detail

is critical to understand the association of T. spp with ulcer.

The authors: Indeed, the current version of this manuscript lacks some details. Besides the new S4 Table with sampling details, we added pictures from the ulcers observed on the fish (Supporting Information S5 Figure).

In addition, while the Results provide an in-depth MLST analysis, I missed

an "epidemiological" analysis: For instance, the authors investigated a

range of fish species: were certain strains/ST associated with certain host

species or was no pattern detectable? Was there an association between farm

conditions, ulcer prevalence/severity and presence of specific strains? If

this study should go beyond a mere analysis of genetic diversity but would

like to promote understanding of the disease epidemiology, more emphasis on

this type of questions is required. In the way as presented now, the ms

unfortunately does not provide insight how the results of this study would

support "future epidemic management" (line 64)

The concluding sentence of the abstract says: "Understanding their

reservoirs and transmission pathways could help to address major challenges

in connection with prophylactic measures and development of vaccines". I

fully agree with this statement, but I do not see how this Ms as presented

now would support these aims. Interestingly, the authors address these

questions in the Discussion, but unfortunately they do not apply it to

their own study.

The authors: Unfortunately, this unbalanced sampling distribution does not allow to answer some critical questions that would allow a global understanding of the disease (e.g., association host species and ST). Indeed, numbers are not sufficient to provide sufficiently robust statistical results.

In addition to continuing to build knowledge about the genetic diversity of Tenacibaculum spp. in Norway, the main result of this study is the presence of STs associated with different hosts and different localities. This partly contradicts some previous studies showing that tenacibaculosis infection arises as local epidemics and is an important result in terms of sanitary control strategy. For instance, ST-172, regularly isolated from different host species and associated with ulcers and epidemics, deserves to be considered carefully (challenge experiment, vaccine development, etc.). Not to be able to address some of the main issues of this disease of saltwater fish is a pity, and we hope that other research programmes will address them with more appropriate sampling methods.

Reviewer #2: Phylogenetic analyses of Norwegian Tenacibaculum strains

confirm high bacterial diversity and suggest circulation of ubiquitous

virulent strains, by Lagadec et al.

In this manuscript, the authors provide useful information about the

diversity of Tenacibaculum spp. in the Norwegian fish farms. Bacteria

belonging to the Tenacibaculum genus have become important opportunistic

pathogens for many different marine fishes and lately have been associated

with ulcerative diseases in farmed salmon. The authors have collected

several strains from different regions in Norway and have used MLST

analysis to map their genetic diversity.

Although the manuscript is well-written, and the information provided is

useful there are some important points that should be considered. It seems

to me that the strains used in the study have been collected by fish vets

during routine diagnostic analyses and are not part of a specific research

survey of the team. This if fully acceptable and understandable, however if

this is true, it should be specifically mentioned in the manuscript.

I am not very sure if the media used in the isolation would favor the growth of

all possible Tenacibaculum spp. These bacteria are difficult to isolate,

and in many cases, there is overgrowth by other faster growing

opportunistic bacteria. The use of general isolation media may have

hindered the growth of other species like Tenacibaculum maritimum.

Furthermore, I am not convinced that the heamolytic activity of

Tenacibaculum strains is a valid indicator of pathogenicity. There are

several non-haemolytic bacterial pathogens which are highly virulent.

Although the authors state that the occurrence of beta haemolysis is an

indicator of pathogenicity (supported by a relevant citation), the

non-occurrence does not mean that the strains are less virulent. Haemolytic activity has been used extensively by the authors, however how it was

assessed is not described in the M&M. More care should be given in the use

of haemolysis as virulence factor for Tenacibaculum in order to be

convincing.

The authors: We deeply thank the reviewer#2 for his pertinent remarks.

To clarify a first point, it is important to specify that the fish were sampled either in the frame of a project surveying tenacibaculosis in farmed salmonids in Norway (fish sampled by veterinarians or fish-health biologists on site during outbreaks) or during other projects (fish sampled by certified fish-health biologists from our laboratory). We have clarified this point in the M&M section (see lines 77-81).

We answered the other questions in the specific comments below.

Specific comments to the authors:

L53: move (MA) after Marine Agar

The authors: corrected (see line 53).

L53: Explain how the use of MA associates Tenacibaculum with winter ulcers

The authors: Tenacibaculum spp. are indeed difficult to isolate. Originally, blood agar containing 1.5-2.0 % NaCl (BAS) was used as the medium for investigation of bacterial diseases in saltwater salmon life stages. The use of Marine Agar (MA) which includes sea salts, has improved the isolation of Tenacibaculum bacteria. Several papers mention that some Tenacibaculum spp. only grow in the presence of sea salts [1-3]. We have added references in the Introduction and modified the sentence accordingly (see lines 52-54).

L68: Mention how many samples were used

The authors: We added this information in the M&M. The sentence “A total of 197 fish […]” was added line 88. The number of fish per sampling is now available in a new table (Supporting Information S4 Table).

L75-78: Are these the best media for isolating Tenacibaculum spp? What

about FMA or TMA? It seems that these media are mostly used for routine

microbiology by the fish vets. If this is the case, it should be stated

either in the M&M or in the introduction, since this is a limitation of the

study that may have biased the results. If this is not the case, then you

should provide references supporting the use of these media for the

specific isolation of Tenacibaculum spp.

The authors: Marine Agar (MA) is indeed used routinely by fish veterinarians. We specified this point in the M&M, line 83.

To prevent the outgrowth by faster growing bacteria, we also used a blood marine agar supplemented with kanamycin to a final concentration of 50 µg ml-1 (KABAMA). Based on our experience, this medium has drastically improved the recovery of Tenacibaculum spp.

L:78 It is not clear later in the results how many of the isolates were

Tenacibaculum. If there were other bacteria present, which were these? What

is the prevalence of Tenacibaculum during the outbreaks?

The authors: All the isolates specified in this paper are referring to Tenacibaculum spp. isolates.

This point was also raised by reviewer#1. We answered to his comment:

“Although our culture process (including media) was optimized for the detection of Tenacibaculum spp., we have monitored the growth of other bacteria, especially those known to cause ulcers on marine fish (e.g., Moritella viscosa). All the bacteria isolated were submitted to a 16S rRNA sequencing, the Tenacibaculum strains being subsequently included in the genotyping. All culture plates presented a dominant growth of Tenacibaculum spp. We isolated several other bacteria, such as Vibrio spp., Pseudoalteromonas spp., Photobacterium spp., but always small colonies in minority and outcompeted by a large number of Tenacibaculum spp. colonies.

Tenacibaculum spp. has been showed to be a marine fish ulcerative pathogens in Norway for more than a decade. The dominant growth of bacteria species belonging to the genera Tenacibaculum on culture from ulcers seems to us a reasonable proof of its implication, especially because we did not detect other species likely to be at the origin of the ulcers observed. However, we should clearly specify what the limits of our detection method were, and why we are fairly convinced of the implication of Tenacibaculum spp. in these ulcers (see in M&M lines 85-87, in Results lines 157-159 and in Discussion lines 264-266).”

L80: What are the characteristics of the colonies? are these the same in

all media? were they described also in Blood Agar? In the papers cited the

authors have used different media.

The authors: Colonies characteristics are provided in a new S3 Table. These colonies were described after growth on BAMA medium.

L82: Describe the morphology

The authors: We modified the sentence, which now reads: “The colonies were further examined by using a light microscope to identify the long and slender rods typical of Tenacibaculum spp. cell morphology” (lines 91-92).

L143: Table legend: You talk about haemolytic activity but how this was

assessed is not in the M&M. Since haemolytic capacity of the bacteria plays

an important role in your paper, the method used to assess it should be

described

The authors: This is an important point. We added a sentence in the M&M to detail how we assessed the haemolytic activity of the different strains: “The β-haemolytic activity of the colonies was readily observed by looking at the plates through a light source.” (see lines 92-94).

L190-191: How do you define "variable"? Have you used replicates in the

heamolytic activity assay?

The authors: The only “variable” strain in this study is the newly described T. finnmarkense genomovar finnmarkense [4]. Its haemolytic activity has been qualified as “variable”. They tested haemolysin production on Marine Agar with 5% bovine blood and 50 µg ml−1 kanamycin and recorded within 7 days. They performed phenotypic tests in parallel.

We assessed the haemolytic properties of our strains in duplicates and did not notice any variable haemolytic activity of the other strains.

L266: What are the severe symptoms? Where were they described?

The authors: Outbreaks characteristics and sampling details are described in S4 table. We also added a new S5 Figure with pictures of the ulcers for 9 different samplings (including Sampling 28).

L276: How did you come to this conclusion that the non-haemolytic strains

are less pathogenic? If the conclusion is based on the field data (eg

morbidity/mortality), these data should be provided. Even then, the

conclusion is really based on circumstantial evidence, and it is indicative

if not speculative. Unless there are comparative data from challenge tests

(if yes, then you should provide these data).

L281: The association of hemolysis to virulence or to be more accurate, the

non-hemolysis to non- or reduced virulence is not supported by evidence and

it is speculative.

The authors: We agree that precautions should be taken when discussing the link between haemolytic activity and pathogenicity, especially concerning the virulence of the non- haemolytic bacteria. We have modified the paragraph accordingly (see lines 307-311).

Reviewer #3: This manuscript provides additional information on the

epidemiology of Tenacibaculosis and should be published. The information

provided is sound and useful but not a significantly novel contribution to

fish bacteriology, but should be published as it provides good information

on the distribution and epidemiology of the bacterial isolates of concern.

The authors: We deeply thank the Reviewer #3. It is true that this paper does not add significant new insights to fish bacteriology, but we think these new findings are relevant for a better understanding of Tenacibaculum spp. diversity and tenacibaculosis.

Reviewer #4: The manuscript submitted by Lagadec et al (PONE-D-21-21158) is

a well presented and informative paper showing the diversity that exists

amongst Tenacibaculum spp. strains in Norwegian aquaculture.

I have a few minor points for the authors to address.

The authors: We thank Reviewer#4 for his comments. Please find our answers to each specific points below.

Line 39 - bacterial diseases

The authors: corrected (see line 39).

Line 44 - Tenacibaculosis has been reported in marine wild and farmed fish

in Europe, Asia,

The authors: corrected (see line 44).

Line 52 - Due to the increased use of Marine Agar (MA) for bacterial

isolation in recent years, Tenacibaculum spp. have also been associated

with the winter ulcer disease, previously attributed to the bacterium

Moritella viscosa [25, 26].

The authors: corrected (see lines 52-54).

Line 89 - 10,000 rpm - state xg rather than rpm

The authors: That was a mistake. It was corrected (see line 101).

Lines 139/194/213/294 - Numbers at the start of sentences should be written

in words rather than as a number.

The authors: We apologize for these mistakes. We have corrected these sentences accordingly (see lines 151, 215, 234, 323).

Line 151- March 2019

The authors: corrected (see line 168).

Line 161 - Of the 27 new STs, 22 were isolated during a single outbreak of

tenacibaculosis. Was the outbreak at a single site - mention the location

of this outbreak?

The authors: Our sentence was confusing. Twenty-two new STs were isolated only once, but during 22 different outbreaks. We have modified this sentence, which now reads “Of the 27 new STs, 22 were isolated only once during 22 different outbreaks of tenacibaculosis” (line 178). We have also modified a sentence in the Discussion (see lines 324-326).

Line 278 - Overall, non-haemolytic Tenacibaculum strains seem to be less

pathogenic and may not lead to tenacibaculosis outbreaks. Can you state the

evidence for this e.g. a reference or findings from your study?

The authors: This point has been raised by Reviewer#2. We should have taken more precautious in writing this paragraph. Indeed, even if the non-haemolytic strains in our study seem to be less virulent, some STs (e.g. ST-160) were associated with tenacibaculosis. We have modified the paragraph accordingly (see lines 307-311).

-------------------------

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1. Suzuki M, Nakagawa Y, Harayama S, Yamamoto S. Phylogenetic analysis and taxonomic study of marine Cytophaga-like bacteria: proposal for Tenacibaculum gen. nov. with Tenacibaculum maritimum comb. nov. and Tenacibaculum ovolyticum comb. nov., and description of Tenacibaculum mesophilum sp. nov. and Tenacibaculum amylolyticum sp. nov. Int J Syst Evol Microbiol. 2001;51(Pt 5):1639-52. Epub 2001/10/12. doi: 10.1099/00207713-51-5-1639. PubMed PMID: 11594591.

2. Olsen AB, Nilsen H, Sandlund N, Mikkelsen H, Sorum H, Colquhoun DJ. Tenacibaculum sp. associated with winter ulcers in sea-reared Atlantic salmon Salmo salar. Dis Aquat Organ. 2011;94(3):189-99. Epub 2011/07/28. doi: 10.3354/dao02324. PubMed PMID: 21790066.

3. Småge SB, Brevik OJ, Duesund H, Ottem KF, Watanabe K, Nylund A. Tenacibaculum finnmarkense sp. nov., a fish pathogenic bacterium of the family Flavobacteriaceae isolated from Atlantic salmon. Antonie Van Leeuwenhoek. 2016;109(2):273-85. Epub 2015/12/15. doi: 10.1007/s10482-015-0630-0. PubMed PMID: 26662517; PubMed Central PMCID: PMCPMC4751178.

4. Olsen AB, Spilsberg B, Nilsen HK, Lagesen K, Gulla S, Avendaño-Herrera R, et al. Tenacibaculum piscium sp. nov., isolated from skin ulcers of sea-farmed fish, and description of Tenacibaculum finnmarkense sp. nov. with subdivision into genomovars finnmarkense and ulcerans. Int J Syst Evol Microbiol. 2020. doi: https://doi.org/10.1099/ijsem.0.004501.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

15 Oct 2021

Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains

PONE-D-21-21158R1

Dear Dr. Lagadec,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

19 Oct 2021

PONE-D-21-21158R1

Phylogenetic analyses of Norwegian Tenacibaculum strains confirm high bacterial diversity and suggest circulation of ubiquitous virulent strains.

Dear Dr. Lagadec:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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