Résultats de caractérisations phénotypiques et génotypiques

Une partie des résultats de ce chapitre a fait l’objet d’une publication, présentée dans les pages suivantes, dans Letters in Applied Microbiology.

Dehaut A, Midelet-Bourdin G, Brisabois A & Duflos G (2014) Phenotypic and genotypic characterization of H2S-positive and H2S-negative strains of Shewanella baltica isolat-

ed from spoiled whiting (Merlangius merlangus). Letters in Applied Microbiology. 59 (5) : 542-548. http://dx.doi.org/10.1111/lam.12312

En outre, ce travail a également conduit au dépôt d’une souche de S. baltica auprès de la collection de l’Institut Pasteur (souche CIP 110804) et de séquences nucléotidiques (LK392312, LK392313, LK392314, LK392315, LK392308, LK392309, LK392310 et LK392311) sur EMBL.

Caractérisation de souches de S. baltica issues d’un poisson altéré

Phenotypic and genotypic characterization of H

S-positive and H

S-negative strains of

Shewanella baltica isolated from spoiled whiting (Merlangius merlangus).

Alexandre Dehaut, Graziella Midelet-Bourdin, Anne Brisabois, Guillaume Duflos*

Anses, Laboratoire de Sécurité des Aliments – Département des Produits de la Pêche et de l’Aquaculture, Boulevard du Bassin Napoléon, 62200 Boulogne-sur-Mer (France)

* Corresponding author: E-mail: guillaume.duflos[at]anses.fr Phone : +33.3.21.99.25.00 Fax : +33.3.21.99.17.25

Four strains were isolated from a spoiled whiting (Merlangius merlangus). All of them were able to grow aerobically from 4°C to 30°C and also able to develop anaerobically in presence of trimethylamine N-Oxide (TMAO) at 25°C. Biochemical characterization did not allow identification of the strains species but showed that one of the four strains was unable to produce H2S. Two strains synthetized an ornithine decarboxylase being potential putrescine

producers. Results of carbon sources use highlighted that the four strains were able to use citrate and D-sucrose, one strain was not able to use L-arabinose. Genotypic characterization of the strains thanks to 16S rRNA and gyrB partial gene sequencing led to their identification as members of Shewanella baltica species. These observations suggest that H2S production may

not be the most appropriate screening parameter for Shewanella species and further to monitor the development of spoilage flora.

Keywords: Shewanella baltica, fish spoilage, Taxonomy, H2S-negative strain, 16S rRNA, gyrB gene

1.Introduction

Fish is a highly perishable matrix subject to autolytic transformations (Huss 1995; Jiang 2000; Howgate 2006) and spoilage by specific microorganisms called specific spoilage organisms (SSO) (Dalgaard 1995). Among SSO, Shewanella putrefaciens has been de- scribed as the spoiling agent for fresh fish stored on melting ice (Gram and Dalgaard 2002), whereas fish conserved in modified atmosphere or vaccum packed is more likely spoiled by Photobacterium phospho-

reum (Dalgaard 1995).

S. putrefaciens previously named Alteromonas putre- faciens and Pseudomonas putrefaciens has been long

described as a unique florae. Actually, it was a heter- ogeneous species and S. putrefaciens was reclassified as a bacterial group composed of four subgroups based on DNA-DNA hybridization studies (Owen et al. 1978). S. putrefaciens group II and IV having been later reclassified as Shewanella baltica and S. alga species (Ziemke et al. 1998). It had been noticed that distinction between Shewanella species could not be achieved through biochemical characterization. Ge- nomic approaches using 16S rRNA sequencing and more confidently gyrB sequencing has been strongly encouraged to discriminate close species (Yamamoto and Harayama 1995; Satomi et al. 2006). A genomic diversity still remained inside Shewanella species, thus a recent multi locus sequence typing (MLST) study on S. baltica highlights nine clades into this sole species (Deng et al. 2014).

To date, numerous Shewanella species have been identified in fish, marine animals and sea water in- cluding Shewanella baltica, Shewanella hafniensis,

Shewanella morhuae, Shewanella frigidimarina, She- wanella livingstonensis, Shewanella marinintestina, Shewanella schlegeliana, Shewanella sairae (Ziemke

et al. 1998; Bozal et al. 2002; Satomi et al. 2003;

Satomi et al. 2006). S. baltica has been described as a potential spoilage organism and the most H2S produc

ing bacteria during storage of marine fish (Vogel et al. 2005). Most of the time isolation process of She-

wanella involved a screening of strains abilities to

produce H2S on iron agar plates (Vogel et al. 2005;

Tryfinopoulou et al. 2007).

This study describes the isolation of four strains of S.

baltica from a spoiled whiting (Merlangius merlan- gus) and their phenotypic and genotypic characteriza-

tion.

2.Results and discussion Growth abilities

A color screen selection was applied to colonies iso- lated from spoiled whiting and four colonies harbor- ing the target pink/purple pigment were recovered, called 14LSABSM1SHW,14LSABSM2SHW, 14LSABSM- 3SHW and 14LSABSM4SHW below. These four iso- lates were able to grow aerobically on a large spec- trum of temperatures ranging from 4°C to 30°C (Table 1).

This ability to grow on a wide range of temperature suggests that the four strains are psychrotolerant, this is consistent with observations previously de- scribed on Shewanella species (Jørgensen and Huss 1989; Bozal et al. 2002). However all isolates showed no growth at 37°C, as previously reported for She-

wanella baltica in the literature (Ziemke et al. 1998;

Satomi et al. 2006; Tryfinopoulou et al. 2007), where- as S. putrefaciens and the pathogenic Shewanella

algae are able to grow at such a temperature.

The four strains were also able to grow anaerobically on the trimethylamine N-Oxide (TMAO) containing agar medium (Table 1). Nonetheless it should be

Figure S1: Results obtained with growth on TSI medium tubes after 24H at 30°C, showing two types of phenotypes: H2S

negative strain on the left (14LSABSM1SHW) and H2S positive strain on the right (e.g. 14LSABSM2SHW, 14LSABSM3SHW

Caractérisation de souches de S. baltica issues d’un poisson altéré

mentioned that colonies were slightly thinner than in aerobic conditions and no characteristic pigmentation was observed. Moreover it was noticed that a trime- thylamine (TMA) characteristic odor, identical to the smell of pure TMA aqueous solution, was coming from plates placed in anaerobic conditions. This ob- servation is concordant with the existence of TMAO reductase in S. putrefaciens group species (Dos Santos et al. 1998).

Growth on TSI tubes showed two types of results (Figure S1). Three strains 14LSABSM2SHW, 14LSA- BSM3SHW and 14LSABSM4SHW displayed a clear ability to produce H2S with a deep black coloration

after 24H at 30°C. In contrast, 14LSABSM1SHW strain showed no black coloration indicating that no H2S

was produced.

Biochemical characterization of the strains

All the results obtained during the biochemical char- acterization of the strains are presented in Table 1. Only three strains, 14LSABSM2SHW, 14LSABSM3SHW and 14LSABSM4SHW were able to produce H2S. The

four strains showed no hydrolysis abilities against urea and o-nitrophenol-β-D-galactoside (ONPG); only

gelatin was catabolized by the whole strains. Con- cerning enzymes activities the four strains possess an oxidase activity and two of them, 14LSABSM1SHW and 14LSABSM4SHW, were able to decarboxylate ornithine being thus potential producers of a biogenic amine. The four strains were able to use few carbon sources, all of them used citrate as sole carbon source and D-sucrose even if the acidification of the solution was not clear. Only 14LSABSM2SHW strain was not able to use L-arabinose as carbon source. Regarding the biochemical tests, 14LSABSM1SHW, 14LSABSM- 2SHW, 14LSABSM3SHW and 14LSABSM4SHW strains were respectively identified as Burkholderia cepacia (87,3%ID – T=0.6) and members of S. putrefaciens group (14LSABSM2SHW strain: 99,5 %ID – T=0.58; 14LSABSM3SHW strain: 95,0%ID – T=0.3; 14LSABSM4SHW strain: 99,7%ID – T=0.4). Biochemi- cal pattern of the strains did not allow finding the species of the strains. Regarding studies of S. baltica strains (Ziemke et al. 1998; Vogel et al. 2005; Tryfinopoulou et al. 2007) it appeared that except one strain, all strains studied were able to produce H2S. Previous exposed results are in agreement with

those observed on three strains of S. putrefaciens Table 1: Results of phenotypic characterization of the four strains isolated from spoiled whiting obtained with plate cul- tures and API 20E system.

Characteristics Tests 14LSABSM1SHW 14LSABSM2SHW 14LSABSM3SHW 14LSABSM4SHW

Aerobic growth (n=2)* 4°C + † + + + 8°C + + + + 15°C + + + + 25°C + + + + 30°C + + + + 37°C - ‡ _{- - -} Anaerobic growth (n=2) 25°C + + + + Production (n=3) H2S production - + + + Hydrolysis (n=3) Urea - - - - Gelatin + + + + o-nitrophenol-β-D-galactoside - - - - Enzyme activity (n=3) Arginine dihydrolase - - - - Lysine decarboxylase - - - - Ornithine decarboxylase + - - + Tryptophane deaminase - - - - Tryptophanase - - - - Oxidase + + + + Use of (n=3)

carbon sources Citrate + + + +

Pyruvate - - - - D-glucose - - - - D-mannitol - - - - Inositol - - - - D-sorbitol - - - - L-rhamnose - - - - D-sucrose + ± § ± ± D-melobiose - - - - Amygdaline - - - - L-arabinose + - + +

* Numbers in parenthesis following “n” refers to the number of replicate of the test. † “+” signs refer, for “Growth temperatures”, to effec- tive growth at a given temperature and for the other tests to positive results on API 20E system. ‡ “-” signs refer, for “Growth tempera- tures”, to absence of growth at a given temperature and for the other tests to negative results on API 20E system. § “±” this sign means that results were not clear instead of yellow color, i.e acidification of the solution, only green color was observed, blue being the negative result.

Figure S2: Phylogenic tree based on partial 16S rRNA gene sequences comparison among Shewanella type strains and the four isolates, accession numbers are figured in parenthesis. Strains isolated in this study are tagged with  symbol. Phylogenetic tree was obtained using neighbor-joining method and Kimura’s 2 parameter model, the optimal tree with a sum of branch length equal to 0.61207491 was retained. The analysis involved 68 nucleotide sequences with a total of 718 positions in the final dataset. Numbers at nodes indicates occurrence of the node in 1000 bootstrapped trees; only values greater than 50% are displayed. Pseudoalteromonas haloplanktis subsp. tetraodonis 16S RNA sequence was used as outgroup. Bar indicates 0.01% sequence divergence

Caractérisation de souches de S. baltica issues d’un poisson altéré

group II concerning urea hydrolysis (Ziemke et al. 1998) and S. baltica NCTC 10735 regarding gelatin hydrolysis (Vogel et al. 2005), whereas S. baltica LMG2250 harbour an urease and is unable to lique- fied gelatin (Tryfinopoulou et al. 2007). By contrast, none of the isolated strains did show any arginine hydrolysis capacities as S. baltica LMG2250, whereas one strain of S. putrefaciens group II, S. baltica NCTC 10736, is able to produce ammonia from arginine (Ziemke et al. 1998; Tryfinopoulou et al. 2007). Use of carbon sources by the strains also lead to contradict- tory results. On the one hand conclusions concerning use of citrate as sole source of carbon and ability to use D-sucrose are consistent with previous observa- tion. On the other hand, conclusion concerning use of D-glucose, amygdaline and L-arabinose by the isolates are the exact opposite of previously reported data (Ziemke et al. 1998; Vogel et al. 2005; Tryfinopoulou

et al. 2007).

Phenotypic characterization of the four strains did not allow finding their species and showed that de- spite common growth and biochemical similarities,

specificities could be attribute for each strains; ab- sence of ornithine decarboxylase in 14LSABSM2SHW and 14LSABSM3SHW strains or incapacity to use L- arabinose as carbon source for 14LSABSM2SHW strain. More importantly, 14LSABSM1SHW strain showed inability to produce H2S. As encouraged by

previous studies (Satomi et al. 2003; Deng et al. 2014), a genotyping of these strains were achieved thanks to partial sequencing of 16S rRNA and gyrB gene.

Sequencing partial 16S rRNA sequences of the four isolated strains

The four partial 16S rRNA sequences of the isolates and 68 nucleotides sequences from GenBank, Pseu-

doalteromonas haloplanktis subsp. tetraodonis used

as outgroup, allowed building an initial phylogenic tree (Figure S2). The 22 closest sequences from the four isolates were retained to build a clearer subtree (Figure 1 a). The four stains were clustered together with S. baltica NCTC10735 type strain. There was an extremely low difference recorded between the 16S Figure 1: Phylogenic subtrees based on partial 16S rRNA gene sequences (a) and gyrB gene sequences (b). Trees com- pared the four isolates among Shewanella type strains (T) and non-type strains. Accession numbers are figured in paren- thesis and isolate of this study are tagged with ▲ symbols. Phylogenetic tree was obtained using neighbor-joining method and Kimura’s 2 parameter model. Concerning 16S rRNA the whole optimal tree with a sum of branch length equal to 0.61207491 was retained; analysis involved 68 nucleotides sequences with a total of 718 positions in the final dataset. Concerning gyrB the whole optimal tree with a sum of branch length equal to 3.79477621 was retained; analysis involved 54 nucleotides sequences with a total of 832 positions in the final dataset. Numbers at nodes indicates occur- rence of the node in 1000 bootstrapped trees; only values greater than 50% are displayed. Pseudoalteromonas halo-

rRNA sequences of this group. These strains clustered together 890 times in the 1000 bootstrapped replica- tions of phylogenic trees, meaning that this tree structure is highly likely. Regarding 16S rRNA se- quence, alignments in BLAST led to respectively 98.4%, 99.3%, 98.0% and 98.3% identity between 14LSABSM1SHW, 14LSABSM2SHW, 14LSABSM3SHW, 14LSABSM4SHW strains and S. baltica NCTC 10735 ty- pe strain (data not shown). Clustering of strains from different species based on 16S rRNA sequence led to confirm previous observations having concluded that this gene lacks of specificity to differentiate close relatives (Fox et al. 1992). To overcome the slow evolution rate of 16S rRNA, gyrB gene sequencing has previously been described to achieve taxonomic study for a lot of species including Shewanella species (Satomi et al. 2003). Thus, gyrB gene sequencing has been the second part of the genotyping approach of this study.

Sequencing of partial gyrB gene sequences of the four isolates

Fifty four gyrB gene sequences collected from Gen- Bank and the four partial gyrB gene sequences of isolated strains were gathered to build a phylogenic tree (Figure S3). Pseudoalteromonas haloplanktis subsp. tetraodonis was again used as outgroup refer- ence. For greater clarity, a new subtree was built (Figure 1 b) with a reduced number of sequences (17+4). As illustrated on Figure 1 b, 14LSABSM1SHW, 14LSABSM2SHW and 14LSABSM3SHW gyrB gene se- quences are closely related to S. baltica NCTC 10735

gyrB gene sequences. Alignments in BLAST led to

respectively 99.3 %, 99.0%, 98.9% and 99.1% identity between gyrB gene sequences of 14LSA-BSM1SHW, 14LSABSM2SHW, 14LSABSM3SHW, 14-LSABSM4SHW and S. baltica NCTC 10735 type strain gyrB gene se- quence (data not shown). gyrB gene sequencing con- firmed previous identifications thanks to 16S rRNA sequences, leading to the conclusion that the four strains belong to the S. baltica species.

Diversity among the S. baltica strains isolated from spoiled whiting.

Genotypic characterization of the strains isolated from spoiled whiting by both 16S rRNA and gyrB gene sequencing concluded that they belong to S. baltica species. As previously mentioned biochemical charac- terization of the strains was not efficient enough to identified strains species. Three strains were identi- fied at the bacterial group level and one, 14LSABSM1SHW, was misidentified as Burkholderia

cepacia. The misidentification of this strain seems to

solely result from its H2S negative phenotype as

above mentioned. Regarding biochemical patterns obtained hereby and in previous studies, it should be noted that S. baltica is a heterogeneous species re- garding enzymes activities and carbon sources use.

More knowledge is needed for the discrimination of the different strains isolated from spoiled fish and a MLST approach like the one realized on S. baltica coming from different environment (Deng et al. 2014) could interestingly be achieved. Based on 16S rRNA and gyrB gene partial sequencing, the four isolated strains are close to strains coming from three clades (H1, H2 and H3), defined in the MLST study and re- covered between 80 to 100 m depth. Surprisingly 14LSABSM1SHW strain grew anaerobically on TMAO- TSA-YE-NaCl plate, being able to respire TMAO whereas it was unable to respire thiosulfate. This phenotype seems thus to go against observation on strains of the sole clade, MLST-E, unable to respire thiosulfate that also could not respire TMAO (Deng et

al. 2014). More importantly, this study emphasized

that H2S production may not be the most appropriate

screening parameter to isolate Shewanella strains as previously achieved (Jørgensen and Huss 1989; Vogel

et al. 2005; Tryfinopoulou et al. 2007), S. baltica

14LSABSM1SHW being a H2S negative strain. None-

theless, more strains coming from more fish have to be characterized to confirm presented results and estimate the part of H2S negative isolates into spoil-

ing fish. Moreover, the spoiling potential of the strains has to be characterized, spiking sterile fish fillets for example and quantifying spoilage indicator as total volatile basic nitrogen (TVB-N).

Thus contrary to previously reported methods (Gram and Dalgaard 2002), TMAO reduction or pigment synthesis abilities seems to be a more appropriate detection method concerning Shewanella species. 3.Materials and Methods

Bacterial strains, growth conditions and biochemical characterization

Bacterial strains were isolated from a spoiled whiting bought at a local retailer (Boulogne-sur-Mer, France) and stored in its packaging for six days at + 8.0°C. Twenty five grams of flesh were added to 225 mL of peptone water (Oxoid, Dardilly, France) and were blended at 560 rpm with a Smasher lab homogenizer (AES Chemunex, Bruz, France). Two successive deci- mal dilutions of the homogenate were prepared in physiological water. For each dilution, two tryptone soya agar plus yeast extract with 1.5% (w/v) NaCl (TSA-YE-NaCl) (Oxoid, Dardilly, France) plates were inoculated and incubated at 25°C for 24H. After plate reading, colonies producing a purple/pink pigment were conserved, as this pigmentation was previously observed for growth of S. putrefaciens type strain (ATCC 8071) on TSA-YE-NaCl plates. Ability of these isolates to aerobically grow at 4°C (72H), 8°C (48H), 15°C (24H), 25°C (24H), 30°C (24H) and 37°C (24H) was screened on TSA-YE-NaCl plates. Ability of the isolates to grow on anaerobic conditions was tested on TSA-YE-NaCl supplemented with 40 mmol.L-1 of trimethylamine N-oxide (Sigma-Aldrich, St-Quentin-

Figure S3: Phylogenic tree based on partial gyrB gene sequences comparison among Shewanella type strains and the four isolates, accession numbers are figured in parenthesis. In some cases type strain sequences were not available this corresponds to genera not followed by “T”. Strains isolated in this study are tagged with  symbol. Phylogenetic tree was obtained using neighbor-joining method and Kimura’s 2 parameter model, the optimal tree with a sum of branch length equal to 3.79477621 was retained. The analysis involved 54 nucleotide sequences with a total of 832 positions in the final dataset. Numbers at nodes indicates occurrence of the node in 1000 bootstrapped trees; only values greater than 50% are displayed. Pseudoalteromonas haloplanktis subsp. tetraodonis gyrB gene sequence was used as outgroup. Bar indicates 0.02% sequence divergence.

Fallavier, France) (TMAO-TSA-YE-NaCl) for 24H at 25°C, anaerobic conditions being generated thanks to Genbag system (Biomérieux, Craponne, France). Con- firmation of H2S production was achieved by inocula-

tion of triple sugar iron (TSI) medium tubes (Oxoid, Dardilly, France) incubated at 30°C for 24H. Finally, biochemical characterization of these strains was achieved using API 20E system (Biomérieux, Crapon- ne, France) incubated at 30°C for 48H and depositing a 24H fresh bacterial colony on oxydase reagent (Biomérieux, Craponne, France).

DNA extractions & Sequencing

DNA extractions of the four strains was carried out on single colonies isolated from a fresh culture at 25°C for 24H on TSA-YE-NaCl plates, using Qiagen DNeasy Blood and Tissue Kit (Qiagen, Courtaboeuf, France). Extraction was performed using manufacturer in- structions except for the ultimate step: the final elu- tion was separated in two elutions with 50µL of TE buffer each. Quantity and quality of DNA (A260 nm/A280 nm and A260 nm/A230 nm ratio) were then controlled us-

ing a DS-11 Spectrophotometer (Denovix, Wilming- ton, NC, USA). A DNA extract was sent for each strain to the private genomics platform Genoscreen (Lille, France) that achieved PCR amplifications and se- quencing for the V1-V2-V3 and V3-V4-V5 regions of 16S rRNA (1200 bp) and partial sequence of gyrB gene (1200 bp).

Bioinformatics

Contig assembling of 16S rRNA and gyrB gene se- quences of the four strains were carried out using CAP3 algorithm (Perrière 1996; Huang and Madan 1999). Coding sequences of 16s rRNA and gyrB gene

of Shewanella genera were retrieved from GenBank using Shewanella type strain listed on list of prokary- otic names with standing in nomenclature (Euzéby 1997). Collected sequences of 16s rRNA and gyrB

gene and contigs of the four strains were aligned using MUSCLE algorithm (Edgar 2004). Phylogenic trees were traced using neighbor-joining method, genetic distance were computed using Kimura’s 2 parameter model (Kimura 1980). Percentage of repli- cate trees in which taxa clustered together were estimated computing 1000 bootstrapped trees (Felsenstein 1985). Sequences alignments and trees building was carried out using MEGA5 software (Tamura et al. 2011). Finally, sequences were BLAST checked using nucleotide BLAST (Altschul et al. 1990) with optimization of the program for highly similar sequences (megablast) and excluding models (XM/XP) and uncultured/environmental sample se- quences.

Accessions numbers and strain

The 16S rRNA and gyrB gene sequences of strains