Bridel et al. Vet Res (2020) 51:60 https://doi.org/10.1186/s13567-020-00782-0 RESEARCH ARTICLE Open Access Genetic diversity and population structure of Tenacibaculum maritimum, a serious bacterial pathogen of marine fsh: from genome comparisons to high throughput MALDI-TOF typing Sébastien Bridel1,2,3, Frédéric Bourgeon4, Arnaud Marie2, Denis Saulnier5, Sophie Pasek6, Pierre Nicolas7, Jean‑François Bernardet1 and Eric Duchaud1* Abstract Tenacibaculum maritimum is responsible for tenacibaculosis, a devastating marine fsh disease. This flamentous bacterium displays a very broad host range and a worldwide geographical distribution. We analyzed and compared the genomes of 25 T. maritimum strains, including 22 newly draft‑sequenced genomes from isolates selected based on available MLST data, geographical origin and host fsh. The genome size (~3.356 Mb in average) of all strains is very similar. The core genome is composed of 2116 protein‑coding genes accounting for ~75% of the genes in each 1 genome. These conserved regions harbor a moderate level of nucleotide diversity (~0.0071 bp− ) whose analysis reveals an important contribution of recombination (r/m 7) in the evolutionary process of this cohesive species that appears subdivided into several subgroups. Association trends≥ between these subgroups and specifc geographi‑ cal origin or ecological niche remains to be clarifed. We also evaluated the potential of MALDI‑TOF‑MS to assess the variability between T. maritimum isolates. Using genome sequence data, several detected mass peaks were assigned to ribosomal proteins. Additionally, variations corresponding to single or multiple amino acid changes in several ribo‑ somal proteins explaining the detected mass shifts were identifed. By combining nine polymorphic biomarker ions, we identifed combinations referred to as MALDI‑Types (MTs). By investigating 131 bacterial isolates retrieved from a variety of isolation sources, we identifed twenty MALDI‑Types as well as four MALDI‑Groups (MGs). We propose this MALDI‑TOF‑MS Multi Peak Shift Typing scheme as a cheap, fast and an accurate method for screening T. maritimum isolates for large‑scale epidemiological surveys. Introduction as a response to environmental changes has been docu- Te rapid development of intensive aquaculture has mented for some important viruses and bacteria [3, 4]. been associated with a dramatic increase in outbreaks of In this context, the success and sustainability of aqua- infectious diseases [1, 2]. Additionally, the international culture largely depend on the understanding of the evo- spread of pathogens through the trade of fsh and eggs or lution and epidemiology of pathogens [1]. Among those, several species of the genus Tenacibaculum (family Fla- vobacteriaceae, phylum Bacteroidetes) are responsible for *Correspondence: [email protected] 1 Université Paris‑Saclay, INRAE, UVSQ, VIM 78350 Jouy‑En‑Josas, France diseases collectively designated as tenacibaculosis, a very Full list of author information is available at the end of the article serious bacterial condition of many commercial marine © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bridel et al. Vet Res (2020) 51:60 Page 2 of 17 fsh species leading to considerable economic losses [5, species to draw a global picture of the genomic diversity 6]. Tenacibaculum maritimum (formerly Flexibacter of the species. In addition, we developed a genome-based maritimus) was the frst species to be characterized and MALDI-TOF MS scheme and typed 131 feld isolates. probably the best-known pathogen in the genus. More- While this technique is commonly used for species iden- over, T. maritimum can afect many feral, captive, and tifcation, bacterial characterization below the species cultured fsh species [5, 7] and has been repeatedly iden- level is much more challenging, requiring the identifca- tifed in many marine aquaculture systems worldwide. tion of subtle diferences between strains [22]. We also Diseased fsh usually exhibit a diversity of external symp- propose a dedicated website that allows both identifca- toms including corroded mouth, skin ulcers, fn necrosis, tion and typing of new Tenacibaculum isolates [23]. and rotted tail. Skin lesions are often colonized by oppor- tunistic pathogens such as Vibrio spp. So far, only one Materials and methods vaccine is commercially available, but it is restricted to Bacterial strains the protection of turbot. Hence, the control of T. mariti- Tenacibaclum maritimum strains were grown in marine mum outbreaks essentially relies on the use of antibiotics, 2216E broth (Difco, Becton, Dickinson and Co., Franklin sometimes combined with external disinfectants [8]. Lakes, New Jersey, USA) for 24 h at 28 °C and 170 rpm. Reliable methods for studying the relationships Stock cultures were preserved in marine 2216E broth between isolates of the same bacterial species (i.e., strain containing 20% (v/v) glycerol at −80 °C. Te 25 strains typing) are a key step for understanding the population used in this study are listed in Table 1 and the bacterial structure, the spreading and the epidemiology of patho- isolates subjected to MALDI-TOF MS analysis are listed gens. Diferent typing methods have been proposed for in Additional fle 1. epidemiological investigations of T. maritimum [9]. Tree serotypes displaying varying degrees of association with Genome sequencing, assembly and annotation host fsh species have been reported [10, 11] and difer- Following centrifugation of the liquid culture, genomic ent molecular technics have been used to determine the DNA was extracted from the pellet using the Wizard intraspecifc diversity of T. maritimum [12, 13]. Serologi- genomic DNA purifcation kit (Promega, Madison, Wis- cal data were compared with several PCR-based meth- consin, USA). Te genomes were sequenced using Illu- ods [14]. In 2014, we proposed a Multi Locus Sequence mina (HiSeq, 100 paired-end or MiSeq, 300 paired-end) Analysis (MLSA) scheme [15] that proved to be a pow- and genome assemblies were performed using Spades erful discriminating tool for isolate identifcation and and Velvet on the PATRIC website with default settings taxonomic afliation [16, 17]. MLSA revealed an unfore- [24]. Te resulting contigs (> 2000 bp) were integrated seen diversity including several, yet undescribed, patho- into the MicroScope platform [25]. genic species in Norway [18]. In addition, this 11-locus sequenced-based method revealed a high number of dis- Genome analysis and comparisons tinct genotypes for the species T. maritimum, suggest- Average Nucleotide Identity analyses were performed ing an endemic distribution of strains without signifcant using the ANIm method [26] with the Python module contribution of long-distance dissemination linked to Pyani [27] using proposed threshold of ≈ 95–96% for international fsh movements. More recently, whole-cell species delineation. Genome annotation, including man- matrix-assisted laser desorption ionization–time of fight ual curation, and comparisons, including pan and core mass spectrometry (MALDI-TOF MS) was used for the genome computation, were performed using the web diferentiation of several fsh-pathogenic Tenacibaculum interface MicroScope [28], which allows graphic visu- species [14, 19]. However, these studies did not reveal alization enhanced by a synchronized representation of any relationships between the proteomic profles and the synteny groups [29]. Persistent, shell and cloud genomes source of isolation of the strains for any of the Tenacib- were computed using the PPanGGOLiN 0.1.4 software aculum species analyzed, and no biomarker below the [30]. Considering the low levels of sequence divergence at species level (e.g., serotype-specifc peaks) was detected typical core genome loci previously reported for T. marit- for T. maritimum. Meanwhile, the complete genome imum [15], we chose a cutof of 80% identity and 80% on of the T. maritimum type strain [20] as well as the draft the minimal coverage of the length between the aligned genomes of the type strains and several feld isolates of portions of two proteins to determine whether two CDSs T. dicentrarchi and “T. fnnmarkense” have been recently were members of the same gene family. published [21], paving the way to comparative genomics. Te 25 genomes were aligned using Snippy [31] with In this study, we analyzed and compared 25 genomes the genome of strain NCIMB 2154T (the type strain of (including 22 newly draft-sequenced) of T. maritimum T. maritimum) [20] serving as a reference.