Annals of Microbiology (2019) 69:1–15 https://doi.org/10.1007/s13213-018-1357-8

ORIGINAL ARTICLE

Rapid genus identification of selected lactic acid isolated from Mugil cephalis and Oreochromis niloticus organs using MALDI-TOF

Rim El-Jeni1,2 & Karola Böhme3 & Monia El Bour1 & Pilar Calo-Mata 3 & Rym Kefi4 & Jorge Barros-Velázquez3 & Balkiss Bouhaouala-Zahar2,5

Received: 20 December 2017 /Accepted: 12 July 2018 /Published online: 21 July 2018 # Springer-Verlag GmbH Germany, part of Springer Nature and the University of Milan 2018

Abstract (LAB) are traditionally used in food and feed bio-industries as they have health-promoting probiotic effect for their host and currently considered safe for human and animal consumption. Recently, we isolated 177 strains from freshwater fishes of dams of Tunisia exhibiting antimicrobial activities against various food-borne pathogens and generated a laboratory biobank to be characterized. Herein, we investigated whether MALDI-TOF could assist rapid identification of LAB genus. First, a lactic microflora-selective Man Rogosa Sharpe medium was used. Isolates were further screened according to the antimicrobial activity, using well-difusion agar. In total, four major genera have been well identified and species frequency has been estimated (74% Enterococcus, 24% Leuconostoc,3%Lactococcus,and2%). Eighteen isolates was further analyzed using MALDI- TOF. Comparative analysis of spectral fingerprints to six referenced MALDI-TOF fingerprints was carried out from the recently developed USC library (www.spectrabank.org). The intra- and inter-specific phyloproteomic relationships among strains were compared to available phylogenetic data based on 16S rDNA genes. This study showed that MALDI-TOF MS is a potent and reliable rapid method for both discrimination and identification of LABs. This highlights the insight of proteomic approach into the screening of food-derived beneficial microorganisms.

Keywords Lactic acid bacteria . Fish . MALDI-TOF MS . 16S rDNA . Bacteriocin

Introduction efficacy benefit when ingested. Nowadays, LAB were safely applied in various foodstuffs fermentation processes (Stiles and Lactic acid bacteria (LAB) are known to have potential appli- Holzapfel 1997), in particular in industrial food sector, such as cation as beneficial bacteria in foods thanks to their high manufacturing cheese, fermented milk, some vegetables,

* Balkiss Bouhaouala-Zahar 1 Laboratory of Microbiology and Pathology of Aquatic Organisms, [email protected]; [email protected] Institut National des Sciences et Technologies de la Mer (INSTM), rue 2 mars 1934, 2025 Salammbô, Tunisia Rim El-Jeni [email protected] 2 Laboratory of Venoms and Therapeutic Molecules, Pasteur Institute Karola Böhme of Tunisia/University of Tunis El Manar, 13 Place Pasteur, BP-74, [email protected] 1002 Tunis, Tunisia Monia El Bour 3 Laboratory of Food Technology, LHICA, Department of Analytical [email protected] Chemistry, Nutrition and Food Science, University of Santiago de Compostela, Lugo, Spain Pilar Calo-Mata [email protected] 4 Biomedical and oncogenetic genomics laboratory, Pasteur Institute of Rym Kefi Tunisia/University of Tunis El Manar, 13 Place Pasteur, BP-74, [email protected] 1002 Tunis, Tunisia Jorge Barros-Velázquez 5 Medical School of Tunis, University of Tunis El Manar, 15 Rue [email protected] Djebel Lakhdhar. La Rabta, 1007 Tunis, Tunisia 2 Ann Microbiol (2019) 69:1–15 fermented meat products, and wines (Abee 1995; rapid method leads to the rapid differentiation of three clusters Vandamme et al. 1996; Hugenholtz and Kleerebezem related to Enterococcus sp. and two clusters related to 1999). The microbiota’s health is shown by the occur- Leuconostoc sp. Based on this MS data, phylogeny relation- rence of LAB in humans (Majamaa and Isolauri 1997; ships were compared to 16S rRNA phylogenetic analysis and Fooks et al. 1999) and animals (Bezkorovainy 2001;Ehrmann its application for early species detection and differentiation et al. 2002). was emphasized. Such bacteria are a major microbial population that mainly colonizes larvae and alevins’ intestines (Ringø and Birkbeck 1999). They were isolated from gills, skins, kidneys, and go- Material and methods nads of different fish species (Spanggaard et al. 2001; Mesalhy et al. 2008; Al-Harbi and Uddin 2005; Ringø and LAB biobank and pre-identification conditions Holzapfel 2000). Due to their significance for maintaining the microbial stabil- The northern fish-farming station BBechima^ and southern ity in the gut as well as inhibiting pathogenic microflora, through Tunisian dams were concerned (i.e., El Abid, Bir Mcherga production of antimicrobial substances and metabolites (i.e., lac- and Sidi Salem). A total of 12 individual Mugil tic acid, acetic acid, formic acid, hydrogen peroxide and bacteri- cephalus and 10 individual Oreochromis niloticus were ocin), they became frequently used and admitted as natural taken (Fig. 1). Microbial strains (from skin, gills, intes- probiotics for the benefit of both animals and humans (Soccol tine, and mucus organs) able to grow on MRS medium et al. 2010). Rapid identification of new species and genera were selected and stored at − 80 °C. This LAB lab from foodstuffs remains essential. Matrix-assisted laser biobank of isolates was screened against different fish desorption ionization-time of flight approach (MALDI- pathogen indicators causing food poisoning to humans TOF MS) has demonstrated its performance in enabling using the drop test (Shnit-Orland and Kushmaro 2008). very fast and cost-effective diagnosis of isolated colo- Bacterial isolation and morphological and biochemical char- nies (bacteria or fungi) on solid culture media (Claydon acterization were performed according to the previously de- et al. 1996; Carbonnelle et al. 1987, 2011). Later on, scribed method (El-Jeni et al. 2015). this strategy has been reported as useful for the charac- Briefly, the phenotypic bacterial characterization was terization of pathogenic and food-spoilage microorgan- carried out on solid medium (viscosity, color). Gram isms present in fish and seafood products (Böhme et al. staining, catalase, and peroxidase-specific tests were per- 2010). More recently, MALDI-TOF was used for rapid formed as described before (Prescott et al. 2003). Their identification of LAB strains (Amenan et al. 2014). pre-identification conditions and ability to grow on Since, the MS characteristics and overall spectral finger- MRS media were examined (Mathara et al. 2004). All print were considered as revolutionary in microbiology the selected strains were further incubated on Mueller– and especially for novel isolated species (Holland et al. Hinton broth medium without shaking at 37 °C, 48 h, and 1999;Fagerquistetal.2005; Pineda et al. 2003). stored at − 80 °C until used. In our recent study, we reported the in vitro probiotic po- tential of four Bbest in class^ cultured LABs selected from a Antimicrobial activity assay of the cell-free biobank of 177 LAB strains (Eljeni et al. 2015). In this study, supernatants we further investigated whether a MALDI-TOF MS approach could help in the rapid clustering of genus and species from Screening test was done according to the well-established this freshwater fish-associated LAB (El Jeni et al. 2015). In protocol with some modifications (El-Jeni et al. 2015). practice, strains were cultivated on Man Rogosa Sharpe Briefly, strains from the biobank were inoculated in MRS (MRS) medium and the 72 isolates exhibiting antimicrobial medium broth to grow and on MRS agar plates to check pu- activities against a set of human and fish pathogens were fur- rity. After 48 h at 20 °C incubation, bacterial cultures were ther phylogenetically characterized. A selection of 18 strains centrifuged, 12,000×g for 10 min. Each cell-free supernatant with bioactive supernatants displaying high antimicrobial ac- was filtered on 0.22-m pore diameter filters and then tested tivity was further investigated. MALDI-TOF MS of low- against 23 different fish pathogen indicators causing food poi- molecular-weight proteins extracted from intact bacterial cells soning to humans using the well diffusion test (i.e., were used to construct a library of specific mass spectral fin- Aeromonas hydrophila, Aureus salmonecida, Candida gerprints. We analyzed the spectral fingerprints using the web albicans, Escherichia coli O126B16 ATCC 14948, application SPECLUST based on clustering and according to Escherichia coli ATCC 25922, Escherichia coli ATCC a representative lists of peak mass range of 2000–10,000 Da. 8739, Enterococcus faecalis, Pseudomonas aeruginosa, The obtained phyloproteomic profile was compared to the Pseudomonas cephasia, Salmonella typhimurium, phylogenetic profile generated using MEGA software. This Staphylococcus ATCC 25923, Staphylococcus aureus ATCC Ann Microbiol (2019) 69:1–15 3

Fig. 1 The geographical repartition of both Oreochromis niloticus and Mugil cephalis species

6538, Streptococcus B, Vibrio alginolyticus, Vibrio Once dry, 1 mm3 wells are created into the inundated plate and anguillarum, Vibrio tapetis (INSTM collection, Tunisia), one drop of agar is put to avoid bacterial culture diffusing. Bacillus cereus ATCC 14893, Escherichia coli CECT 4076, Then, the supernatants of each LAB were applied into the Listeria innocua ATCC 33090, Listeria monocytogenes wells (centrifugation 5000 rpm, 10 min at 4 °C) and incubated CECT 4032, Staphylococcus aureus ATCC 35845 (LHICA at 20 °C during 24 h. Antimicrobial activity was qualitatively collection, spain)). Precisely, the Tryptic Soy Agar (TSA) monitored on the basis of transparency and the diameter of plate is inundated with indicator strain, already incubated in inhibition (Shnit-Orland and Kushmaro 2008). All the tests 5 ml Tryptic Soy Broth medium (TSB) (for 24 h at 20 °C). were performed in duplicates. 4 Ann Microbiol (2019) 69:1–15

Determination LAB inhibitory activities to proteolytic (Merck, Darmstadt, Germany) and 1% aqueous trifluoroacetic action acid (TFA; Acros Organics, NJ, USA) and mixed well by vortexing. After centrifugation at 8000 rpm for 10 min, the su- For further characterization of the proteolytic activity, 2.5 mg of pernatant was removed into a fresh tube and stored at − 20 °C Proteinase K (Sigma) was added to 1 ml cell-free supernatant until analysis. For the MS analysis, an aliquot of every sample from each bacteria displaying inhibitory activity. The mixed ex- extract (2 μl) was mixed with 10 μL of the matrix solution; tract was incubated at 37 °C during 3 h. Thereafter, the capacity saturated α-cyano-4-hydroxycinnamic acid (a-CHCA; Sigma- in inhibiting indicator microorganism growth was analyzed using Aldrich, St Louis, MO, USA) in 50% ACN and 2.5% aqueous well diffusion technique (Campos et al. 2006). A set of 18 strains TFA. Then, an aliquot of 1 μl of the sample/matrix solution was with active supernatants were selected for further investigation. manually deposited onto a target well on a stainless steel plate for MALDI-TOF analysis and allowed to dry at room temperature. Phylogenetic analysis based on the 16S rRNA gene Mass spectra were obtained using a Voyager DE STR MALDI- TOF Mass Spectrometer (Applied Biosystems, Foster City, CA, To prepare the extraction of genomic DNA, the bacterial USA) operating in linear mode, extracting positive ions with an strains were inoculated in MRS broth (Oxoid) and incubated accelerating voltage of 25,000 V and a delay time of 350 ms. at 37 °C for 24 h. After spinning 1 ml of cultures at 7500 rpm Spectra taken in the m/z range of 2000–10,000 were obtained in for 10 min, the supernatants were discarded and the bacterial ten different regions of the same sample spot, and each was the pellets resuspended in 180 μL of enzymatic lysis buffer result of the accumulation of at least 1000 laser shots. Two ex- (20 mg/ml lysozyme in 20 mM Tris–Cl, 2 mM EDTA, 1.2% tractions per sample were carried, out and each of the extracts Triton X-100; Sigma-Aldrich) and incubated for 30 min at was evaluated in duplicate, ultimately acquiring four spectra for 37 °C. Then, the DNA was extracted and purified using the each bacterial strain. The spectra were externally calibrated using DNeasy Tissue kit (Qiagen, Valencia, CA, USA) as described a mix of 1 pmol/μl oxidized B chain of insulin and 1 pmol/μl previously Hosseini et al. 2009). bovine insulin (Sigma-Aldrich). Analysis of the spectra and the A fragment of the 16S rRNA gene was amplified by PCR extraction of peak mass lists were carried out with Data Explorer using the universal primer pair P8FPL/P806R (McCabe et al. (Version 4.0.0.0) and the freely available, web-based application 1995). All PCR assays were carried out on a MyCycler SPECLUST (http://bioinfo.thep.lu.se/speclust.html)(Almetal. Thermal Cycler (BioRad Laboratories, Hercules, CA, USA). 2006;Böhmeetal.2010) compare peak lists by measuring a Prior to sequencing, the PCR products were purified with the distance between each pair of peak lists. To avoid small measure- ExoSAP-IT Kit (GE Healthcare, Uppsala, Sweden). Direct se- ment errors, a measure was defined to calculate match scores quencing was done with the BigDye Terminator v3. 1 Cycle which are then used to align the peak lists and to calculate a Sequencing Kit (Applied Biosystems). The same primers used distance between the lists. Similar methods have been used in for PCR were as well used for sequencing of both strands of the tandem mass spectrometry (MS/MS) database search algorithms. PCR products. The sequencing reactions were analyzed in an To find a maximum value, the Needleman-Wunsch algorithm automatic sequencing system (ABI 3730XL DNA Analyzer, was used, which was commonly in global sequence alignment Applied Biosystems) provided with the POP-7 system. The (Needleman and Wunsch 1970). DNA sequences were analyzed with Chromas (Griffith Thefinalpeakmasslistswereobtained,selectingrepresentative University, Queensland, Australia) and were aligned against peaks present in all four spectra acquired for each sample. Finally, themselves using CLUSTALW software (Thompson et al. all strain-specific mass lists were compared to each other using the 1994). The bacterial strains isolated from freshwater fishes were web application SPECLUSTand characteristic peak masses were identified by searching for sequence homologies among pub- determined. The clustering option, also available on the web inter- lished reference sequences with the BLAST tool (National face of SPECLUST, was applied to cluster the mass lists of all the Center for Biotechnology Information). Phylogenetic and mo- studied strains as described by Böhme et al. (2010). lecular evolutionary analyses were conducted with MEGA 4.0 software (Kumar et al. 2008), using the Neighbor-Joining meth- od (Saito and Nei 1987) and the Kimura two-parameter model Results with 1000 bootstrap replicates to construct distance-based trees. Identification of bacteriocinogenic LAB MALDI-TOF MS experiments The biobank of 177 strains isolated from mucus, skin, gills, and For MALDI-TOF MS analysis, the strains were grown on plate intestines of Mugil cephalus and Oreochromis niloticus fishes liv- count agar (PCA) (Oxoid, Hampshire, UK) for 24 h at 37 °C. ing in dams was previously constructed and stored at − 80 °C (Fig. One loopful biomass of each bacterial culture was taken and 1). Among them, only 72 isolates (41%) exhibit antimicrobial harvestedin100μl of a solution containing 50% ACN activity and 4 have been identified as displaying in vitro probiotic Ann Microbiol (2019) 69:1–15 5 characteristics (El Jeni et al. 2015). Different fish organs are con- Vibrio alginolyticus,Candidaalbicans,Pseudomonasaeruginosa cerned: 6 from skins, 22 from mucus, 7 strains from the intestine, INSTM, Escherichia coli ATCC 25922, Enterococcus faecalis, and 36 from gills. All isolates were Gram-positive, oxidase-nega- and Escherichia coli ATCC 8739. Their antimicrobial potential tive, catalase-negative, and non-mobile, indicating that isolate was inhibited by proteinase K, indicating the inhibitory activity forms were of various cocci or genus (data not shown). was of proteinaceous nature. As illustrated in Fig. 2a, no inhibition against Staphylococcus All the 72 isolates were identified through 16S rDNA gene aureus bacteria ATCC 25923, INSTM nor Bacillus cereus ATCC amplification and sequencing. According to the sequences, 14893, LHICA were induced by the LAB isolates. The Listeria the dominated genus belongs to Enterococcus sp. (more than innocua ATCC 33090 LHICA demonstrated a high sensitivity to 80%) with E. faecium (35%) and E. faecalis (38%) dominated bacteriocins generated by LAB, whereas only about half of select- species, whereas the rest of the genera belong to Leuconostoc ed strains inhibited Listeria monocytogenes CECT 4032 LHICA (16%), Lactococcus (3%), and Vagococcus species (1%) (Fig. growth. High inhibition was observed against 20 pathogenic spe- 2b). The 16S rDNA sequence multiple alignment showed a cies: Streptococcus B, Staphylococcus ATCC 25923 INSTM, very high homology (about 96%) compared to the other Enterococcus faecalis, Aureus salmonecida,andEscherichia coli GenBank strains (http://www.ncbi.nlm.nih.gov/pubmed/). CECT 4076 LHICA, Vibrio tapetis INSTM, Staphylococcus au- According to the fish origin, we noticed that 34 different reus ATCC 35845 LHICA, Escherichia coli O126B16 ATCC strains belonging to two different clusters were present in 14948 INSTM, Staphylococcus aureus ATCC 6538 INSTM, Oreochromis niloticus (Fig. 3): (i) cluster 1 consisting of three Pseudomonas fluorescens AH2 INSTM, Pseudomonas cephasia, subclusters including Enterococcus mundtii (sub-cluster 1.1) Aeromonas hydrophila INSTM, Salmonella Typhimurium C52, and Enterococcus faecium (sub-cluster 1.2) strain types with

Fig. 2 a Percentage of active isolates obtained from freshwater fishes against several fish and shellfish pathogens. Antibacterial activity was detected using the well diffusion technique. b Bacterial species and genus distribution of lactic acid bacteria 6 Ann Microbiol (2019) 69:1–15

Fig. 3 Phylogenetic tree of lactic acid bacteria isolated from Oreochromis niloticus,using MEGA software. Red tick matches reference strains.

Enterococcus durans species (HG937749) and (ii) cluster 2 (BECHIMA aquaculture in south of Tunisia), these environ- includes Leuconostoc mesenteroides isolates. Interestingly, the mental conditions (i.e. temperature parameter) could influence Enterococcus gallinarum sub-cluster (cluster 1) includes an natural evolution in favor of the dominated genus. Indeed, it is Enterococcus faecium particular strain (HG937762). Since well noticed that Enterococcus faecalis HG937755, both strains have been isolated from same warm-zone fish Leuconostoc citreum HG937743, and Leuconostoc Ann Microbiol (2019) 69:1–15 7 mesenteroides HG937751 strains were distinct from the previ- Likewise, 38 different isolates were present in Mugil ous cited clusters and may result from divergent species cephalus and belong to 4 distinct clusters (Fig. 4). Cluster 3 evolution. consists of three subclusters: (i) sub-cluster 3.1 includes

Fig. 4 Phylogenetic tree of lactic acid bacteria isolated from Mugil cephalis, using MEGA software. Red tick matches reference strains. 8 Ann Microbiol (2019) 69:1–15

Enterococcus faecium strains and an Enterococcus lactis strains with significant antimicrobial activities were chosen as HG937719 strain, (ii) sub-cluster 3.2 contains Enterococcus part of a more in-depth study: five LAB are isolated from the hirae type strains, and (iii) sub-cluster 3.3 consists of gills (HG937695, HG937696, HG937741, HG937758, and Enterococcus mundtii strains. Cluster 4 included HG937762), four from the mucosal skin (HG937754, Enterococcus faecalis strains. Cluster 5 contains two HG937697, HG937757, and HG937770), and two Lactococcus.sp strains. (HG937753 and HG937767) from the gut of Oreochromis Hence, the differential distribution can also be reported niloticus. Two isolates (HG937719, HG937715) were from depending upon which parameters are considered. gills and five strains (HG937699, HG937700, HG937716, It is noteworthy that lactic flora is most diverse within HG937753, HG937720, HG937724) from mucosal skin of fishes collected from two particular dams: Bir Mcherga and gray Mugil cephalis. El Abid dams. Interestingly, mucus and gill flora in both fishes As illustrated in Table 1, 14 strains belong to the were more dominated by LAB than intestinal and skin flora, Enterococcus genus. On the other hand, four strains belong with Enterococcus dominance. to the Leuconostoc genus.

Characteristics of the 18 strains with active cell-free MALDI-TOF MS profiles supernatants Using MALDI-TOF MS-described conditions, spectral profiles The characteristics of the 18 isolates displaying active cell-free in the mass range from 2000 to 10,000 Da were obtained. A supernatants were further considered based on their 16S rDNA library of MS data was compiled, comprising the mass spectra gene sequences and phylogenetic identification (Fig. 5). These profile obtained by MALDI-TOF MS. For the analysis of the

Fig. 5 Phylogenetic analysis of the nucleotide sequences of the 16S rDNA gene of 18 selected lactic acid bacteria. Numbers above and below branches indicate bootstrap values from neighbor-joining analysis. Blue tick matches reference strains Ann Microbiol (2019) 69:1–15 9

Table 1 List of lactic acid bacteria strains IC 16S rRNA gene ID Fish Origin Tissue Accession number

R.A4 Leuconostoc mesenteroides Oreochromis niloticus Skin HG937739 R.A5 Enterococcus faecium Oreochromis niloticus Gills HG937696 R.A11 Leuconostoc pseudomesenteroides Oreochromis niloticus Gills HG937742 R.A13 Leuconostoc mesenteroides Oreochromis niloticus Intestine HG937744 R.A18 Leuconostoc mesenteroides Oreochromis niloticus Gills HG937747 R.A73 Enterococcus faecium Oreochromis niloticus Mucus HG937697 R.A74 Enterococcus faecium Oreochromis niloticus Mucus HG937757 R.M4 Enterococcus lactis Mugil cephalis Gills HG937719 R.M05 Enterococcus faecalis Mugil cephalis Gills HG937737 R.M08 Enterococcus faecalis Mugil cephalis Skin HG937721 R.M18 Enterococcus mundtii Mugil cephalis Mucus HG937724 R.M07 Enterococcus faecalis Mugil cephalis Skin HG937726 R.M10 Enterococcus faecalis Mugil cephalis Skin HG937727 R.S8 Enterococcus mundtii Mugil cephalis Gills HG937711 R.S3 Enterococcus faecalis Mugil cephalis Gills HG937712 R.S9 Enterococcus mundtii Mugil cephalis Gills HG937715 R.S15 Enterococcus faecium Mugil cephalis Mucus HG937716 R.B21 Enterococcus mundtii Oreochromis niloticus Intestine HG937767

Accession numbers were from EMBL IC isolate code

18 spectra, arithmetic means for m/z values were calculated and was considered as an important biomarker for such genus. the corresponding mass variability was less than ± 5 Da in the Despite this, among the Enterococcus species, we showed mass range above 7000 Da and less than ± 3 Da in the lower different profiles that could allow the discrimination at the mass range. Final peak mass lists were generated, comprising species level. In details, 4 or 5 species-specific peak masses 10–35 reproducible and representative peak masses for each could be defined. Hence, E. faecium, exhibited 5 species- bacterial strain. By comparing the mass lists, we defined the specific peak masses of 3644 ± 1, 3881 ± 2, 6341 ± 2, 6510 similarities between different species and genera, as well as ± 2, and 7292 + 6 m/z, respectively, while E. mundtii exhibited unique masses for each particular strain. To facilitate the anal- two species-specific peak masses at m/z 3441 and 6480 + 3 ysis of spectra, symbols were added to highlight common and and 7782 ± 4, respectively. Interestingly, the spectral profiles specific peaks that were observed among strains of the same of Enterococcus faecalis species were divergent from those genus or for each species. described before and exhibited 4 species-specific peak masses Data analysis revealed that genus-specific peak masses (black at m/z 3429 + 1, 4409 + 2, 6222 + 4, and 6671 ± 2, respective- inverted triangle in Fig. 6a, b) were present in all spectra of the ly (Table 2). corresponding genus and did not appear in the spectra of other Leuconostoc spp. spectral profiles were analyzed by using genera considered in this work. In summation, we defined at least the same approach. A reproducible peak (m/z 5915 ± 2) was one species-specific peak mass for each studied species (white observed to be common to all Leuconostoc studied strains circle in Fig. 6a, b). Besides labeling of characteristic peak (i.e., 4 strains); therefore, it was considered as a genus-specif- masses in the spectral profiles, species- and genus-specific ic peak. The two species-specific peak masses of peak masses are indicated in Table 2. Leuconostoc mesenteroides were corresponding to m/z Subsequently, to obtain a characteristic and representative 5117 ± 1 and 5424 + 3, respectively. A single specific- profile of each studied strain, the whole spectral pattern has species mass (m/z 6222 + 7) was observed for Leuconostoc been examined individually. For instance, if a peak showed a pseudomesenteroides (Table 2). mass in common with one or more peak masses of other spe- cies, then this peak could not be considered as a species- Mass spectra hierarchical clustering specific peak but rather as a characteristic peak. Data showed an intense peak of m/z 4425 ± 2 mass, commonly observed Due to limited access to performant phyloproteomic analysis among 14 Enterococcus spp. spectra examined and therefore equipment, mass spectrum was generated using free 10 Ann Microbiol (2019) 69:1–15

Fig. 6 MALDI-TOF MS spectral of Leuconostoc (a)andEnterococcus (b) species. Species-specific peaks are indicated by (white circle) and genus- specific peaks by (black inverted triangle) Ann Microbiol (2019) 69:1–15 11

Table 2 Species-specific peak masses of every studied strain- Strains Species-specific peak masses Genus-specific and genus-specific peak masses peak masses

Enterococcus faecium CECT_4932 3645; 3883;6343; 6513; 7296 4427 Enterococcus faecium HG937697 3882; 6340; 6510; 7292 4427 Enterococcus faecium HG937719 3643; 3883; 6340; 6512; 7293 4427 Enterococcus faecium HG937716 3644; 3881; 6341; 6509; 7294 4427 Enterococcus faecium HG937696 3644; 3880; 6339; 6508; 7292 4427 Enterococcus faecium HG937757 3644; 3882; 6339; 6508; 7292 4427 Enterococcus faecalis CECT_4039 3429; 4411; 6225; 6673 4427 Enterococcus faecalis HG937765 3429; 4409; 6222; 6669 4427 Enterococcus faecalis HG937726 3429; 4411; 6226; 6673 4427 Enterococcus faecalis HG937712 3429; 4411; 6223; 6671 4427 Enterococcus faecalis HG937721 3429; 4409; 6226; 6671 4427 Enterococcus faecalis HG937763 3430; 4411; 6225; 6672 4427 Enteroccus mundtii CECT_972 3441; 6482; 7784 4427 Enteroccus mundtii HG937711 3441; 6478; 7758 4427 Enteroccus mundtii HG937715 3443; 7784 4427 Enteroccus mundtii HG937767 3443; 6483; 7786 4427 Enteroccus mundtii HG937724 6480; 7779 4427 Leuconostoc pseudomesenteroides LMG_11482 6229 5917 Leuconostoc pseudomesenteroides HG937742 6222 5917 Leuconostoc mesenteroides CECT_219 5118; 5427 5917 Leuconostoc mesenteroides HG937744 5117; 5424 5917 Leuconostoc mesenteroides HG937747 5116; 5424 5917 Leuconostoc mesenteroides HG937739 5117; 5424 5917

application SPECLUST (Alm et al. 2006), as an optional CECT_4039, presenting five subclusters. Cluster 4 (i.e., method available online for hierarchical clustering of mass HG937747, HG937739, HG937742, and HG937743) formed lists (http://bioinfo.thep.lu.se/speclust.html). For each a distinct cluster containing five branch nodes, close to one species, an available reference within the USC library was distinct cluster (i.e., cluster 5) containing the three referenced used. The files contained mass spectrum in a zip format strains (i.e., L. pseudomesenteroides LMG_11482, L. (corresponding to the 18 isolates and the 5 referenced mesenteroides LMG_6908, and L. mesenteroides strains) were first uploaded (i.e., input) and then used for CECT_219). cluster analysis using the software. Results were obtained using adequate parameter settings (i.e., correlation based select metric, average select linkage). Mass spectra were Discussions clustered according to the peptide masses and similar masses were grouped in the same cluster (output), as illustrated in the The objective of the study was to isolate, characterize, and dendrogram in Fig. 7. In total, five distinct clusters were rapidly identify lactic flora in freshwater fish from dams of retrieved, 3 clusters related to Enterococcus sp., and 2 Tunisia in order to select strains exhibiting antimicrobial clusters related to Leuconostoc sp. activities. Cluster 1 (i.e., HG937697, HG937757, HG937696, In this regard, a total of 177 bacterial strains from HG937716, and HG937719 isolates) is subdivided into five freshwater fish organs (intestine, gills, skin, and mucus) subclusters and constituted a common cluster nearby the E. were isolated and purified. Such a study has been reported faecium CECT_4932 reference strain. Four subclusters (i.e., by Olsson et al., (1992), Austin et al., (1995), and HG937715, HG937767, HG937711, HG937724 isolates) Robertson et al., (2000). LAB have been described to be formed the distinct cluster 2 with the E. mundti CECT_972 present in fish intestines (Gonzalez et al. 2000;. Bucio et reference strain. In cluster 3, five strains (i.e., HG937712, al. 2004;Bucioetal.2006;HagiandHoshino2009). HG937721, HG937737, HG937727, and HG937726, iso- They have also been reported to occur in gills, skin, kid- lates) formed a common cluster with E. faecalis neys, and gonads (Spanggaard et al. 2001;. Mesalhy et al. 12 Ann Microbiol (2019) 69:1–15

inhibitions levels. The Listeria innocua was most sensi- tive to Bacteriocin produced by selecting LAB. On the other hand, half of that collection was able to inhibit L. monocytogenes growth (Ahn and Stiles 1990). Significantly, Gram-negative bacterial inhibition is not typical because their external membranes block bacterio- cins. Consequently, few studies reported growth inhibition of Gram-negative bacteria due to bacteriocins from lactic strains (Messi et al. 2001; Todorov and Dicks 2004; Stevens et al. 1991). Inhibitory effect can be attributed to several antibacterial factors produced by LAB such as lactic acid, hydrogen peroxide, diacetyl, bacteriocin, or synergy between some of them. The proteinaceous nature wasaffirmedthroughtheirsensitivitytoproteinaseK.In the current study, the inhibitor-produced LAB can be identified as bacteriocins, and the other antibacterial fac- tors were excluded. On the laboratory scale, the bacterial identification needs rapid and more reliable techniques at reduced costs. Recently, Fig. 7 Phyloproteomic analysis of spectral fingerprints of 18 LAB, using a MALDI-TOF MS proteomic technique was developed to web-based application SPECLUST. E.FAECALIS_CECT_4039, analyze the molecular weight of soluble proteins extracted E.FAECIUM_CECT_4932, E.MUNDTII_CECT_972, from bacterial cells (Amenan et al. 2014). It permits the bac- L.MESENTEROIDES_LMG_219, L.MESENTEROIDES_LMG_6908,and terial identification within minutes with a high precision (com- L.PSEUDOMESENTEROIDES_LMG_11482 are MALDI-TOF finger- prints from USC library (www.spectrabank.org) E.FAECALIS_CECT_ pared to the molecular identification based on sequencing the 4039, E.FAECIUM_CECT_4932, E.MUNDTII_CECT_972, L. rDNA 16S gene). MESENTEROIDES_LMG_219, L.MESENTEROIDES_LMG_6908,and Within this context, the proteomics approach by mass L.PSEUDOMESENTEROIDES_LMG_11482 are known sequences in the spectrometry was validated (i.e., MALDI-TOF MS), in NCBI database order to identify and classify a set of 18 bioactive LAB (Fig. 7) belonging to different genera and species. It has 2008;. Al-Harbi and Uddin 2005; Ringo and Holzapfel also been used successfully to differentiate probiotic bac- 2000). teria from food and yoghurt (Tanigawa et al. 2010, Isolates were identified using a 16S rDNA sequencing Angelakis et al. 2011), resulting in subspecies identifica- molecular approach. Results elaborated in the present man- tion (Bifidobacterium animalis, L. delbrueckii, L. casei, uscript showed that the cultivatable lactic flora related to Lact. Lactis,andS. thermophilus). Similarly, 23 strains both freshwater fishes (Mugil cephalis and Oreochromis of LAB from fermented vegetables and meats (Doan et niloticus) was dominated by the Enterococcus genus. al. 2012, Nguyen et al. 2013) and fermented cereals They are pervasive and can be adapted to various environ- (Sorroza et al. 2012) were identified. ments (Manero and Blanch 1999; Migaw et al. 2013). Such The peaks mass lists were obtained using the freely avail- results have been found in other research (Dicentrarchus able, web-based application SPECLUST. If a consensus peak labrax and Sparus aurata) (Chahad et al. 2012). list (in the consensus output) contains several peaks Additional Leuconostoc genera were used as probiotics from one peak list, all peaks are included in the output, (Vendrell et al. 2008; Balcázar et al. 2007; Vázquez et al. semicolon separated. 2005). Recently, Sorroza et al. (2012) has isolated an iden- Various peaks were characterized as a species- or genus- tified strain of Vagococcus fluvialis from shellfish. It is specific peaks based on the spectral profiles, and considered to be a viable future probiotic for use in Leuconostoc spp. strains could easily be differentiated aquaculture. Slama et al. (2013) isolated powerful pro- from Enterococcus spp. spectra. A number of character- biotic Lactobacillus strains from Tunisian traditional istic peak masses were detected in the spectral profiles food and fermented fish preserved. Sica et al., (2010) of E. faecalis and E. faecium at: m/z 4409.23, 6222.4, and Jini et al. (2011) reported the presence of E. feacalis in and 6669.24 kDa and 3644.90, 3881.56, 6509.53, and freshwater fish. 7292.49 kDa, respectively. Based on a selection from 72 antimicrobial-producing Our data are in accordance with similar spectra obtained by isolates, many of them have an inhibitory activity against Andrés-Barrao et al. (2013). Interestingly, unique genus-specific Gram-positive and Gram-negative pathogens, at different peak mass at m/z 5912.92 was observed for Leuconostoc spp.A Ann Microbiol (2019) 69:1–15 13 distinctive peak mass at m/z 6225.18 and two peak masses (i.e., Compliance with ethical standards m/z 5117.01 and 5424 kDa) were specifically detected in the spectral profiles of L. pseudomesentroides and L. mesenteroides Conflict of interest The authors declare that they have no conflict of strains, respectively. interest. Using the same SPECLUST application, we elucidat- ed the intra-specific and inter-specific phyloproteomic References relationships among the studied strains and compared these data to the ones obtained using basic 16S rRNA Abee T (1995) Pore-forming bacteriocins of Gram+ bacteria and self- phylogenetic approach. protection mechanisms of producer organisms. FEMS Microbiol As the main result, we showed that the MALDI-TOF MS Lett 129:1–9 data analysis closely correlated with phylogenetic analysis Alm R, Johansson P, Hjerno K, Emanuelsson C, Ringnér M, Häkkinen J based on the 16S rRNA genes. Precisely, we showed more (2006) Detection and identification of protein isoforms using cluster analysis of MALDI-MS mass spectra. J Proteome Res 5:785–792 accurate diversity that could lead to better differentiation of Amenan A, Soro-Yao, Peter Schumann, Philippe Thonart, Koffi M. Djè, species in the particular case of the phyloproteomic tree. Rüdiger Pukall (2014) The Use of MALDI-TOF Mass Interestingly, the phylogenetic analysis revealed one cluster Spectrometry, Ribotyping and Phenotypic Tests to Identify Lactic of Leuconostoc spp. subdivided in two subclusters: in one Acid Bacteria from Fermented Cereal Foods in Abidjan (Côte ’ hand, L. pseudomesenteroides strains and in the other hand, d Ivoire). The Open Microbiology Journal 8:79 Andrés-Barrao C, Benagli C, Chappuis M, Ortega Pérez R, Tonolla M, L. mesenteroides strains. Barja F (2013) Rapid identification of acetic acid bacteria using However, with MS spectral profiles, a divergence was MALDI-TOF mass spectrometry fingerprinting. Syst Appl noticed since Leuconostoc species (cluster numbers 4 and Microbiol 36:75–81 5) between isolates and reference strain MS spectra, but Angelakis E, Million M, Henry M, Raoult D (2011) Rapid and accurate bacterial identification in probiotics and yoghurts by MALDI-TOF they are still close. Therefore, using MALDI-TOF MS, mass spectrometry. J Food Sci 76:568–571 accurate results were obtained when we used the web Al-Harbi AH, Uddin MN (2005) Microbiological quality changes in the application SPECLUST to cluster lists of peptide peak intestine of hybrid tilapia (Oreochromis niloticus Oreochromis au- masses and to identify spectra coming from similar pro- reus) in fresh and frozen storage condition. Lett Appl Microbiol 40: – teins. This comparative study was recently mentioned for 486 490 Ahn C, Stiles ME (1990) Antibacterial activity of lactic acid bactefia reliability of MALDI-TOF MS technique in bacterial isolated from vacuum-packaged meats. J Appl Bacteriol 69:302– identification (Emami et al. 2012), demonstrating the 310 good correlation between MALDI-TOF MS and 16S Austin B, Stuckey LF, Robertson PA, Efendi I, Griffith DRW (1995) A rDNA gene analysis for the characterization of 36 probiotic strain of Vibrio alginolyticus effective in reducing diseases isolates. cause by Aeromonas salmonicida, Vibrio anguillarum and Vibrio ordalii. J Fish Dis 18:93–96 We demonstrated that taxonomic resolution using MALDI- Balcàzar JL, de Blas I, Ruiz-Zarzuela I, Vendrell D, Calvo AC, Marquez TOF/MS was comparable or superior to the phylogenetic I, Girones O, Muzquiz JL (2007) Changes in intestinal microbiota analysis using 16S rDNA gene sequencing. Bacterial and humoral immune response following probiotic administration in species may be very well discriminated by proteomic brown trout (Salmo trutta). Br J Nutr 97:522–527 identification using MALDI-TOF MS of LAB with rel- Bezkorovainy A (2001) Probiotics: determinants of survival and growth in the gut. Am J Clin Nutr 73:399–405 evance in food and biomedical applications. This strat- Böhme K, Fernández-No I, Barros-Velázquez J, Gallardo JM, Calo-Mata egy allows significant time savings and needs only a P, Cañas B (2010) Species differentiation of seafood spoilage and little amount of material (i.e., drops of bacterial culture and pathogenic gram-negative bacteria by MALDI-TOF mass finger- – matrix solution) to successfully perform bacterial identifica- printing. J Proteome Res 9:3169 3183 tion in a few minutes and at moderate cost. Bucio A, Hartemink R, Schrama JW, Rombouts FM (2004) Screening of lactobacilli from fish intestines to select a probiotic for warm fresh- water fish. Bioscience Microflore 23:21–30 Acknowledgments The authors would like to thank Dr. Mohamed El Bucio A, Hartermink R, Schrama JW, Verreth J, Rombouts FM (2006) Ayeb, Director of Laboratoire des Venins et Molécules Thérapeutiques, Presence of lactobacilli in the intestinal content of freshwater fish Institut Pasteur de Tunis for his constant encouragements. Special thanks from a river and from a farm with a recirculation system. Food are addressed to Mr. Marcos Quintela Baluja (LHICA) for his technical Microbiol (5):476–482 assistance. Campos C, Rodrıguez O, Calo-Mata P, Prado M, Barros-Velazquez J (2006) Preliminary characterization of bacteriocins from Funding This work was initially co-funded by a grant from the Tunisia- Lactococcus lactis, Enterococcus faecium and Enterococcus mundtii – Spain bilateral AECI PROJECT A1/038311/11 and secondly sup- strains isolated from turbot (Psetta maxima). Food Res Int 39:356 ported by the project BIOVecQ P.S.1.3/08 from the National 364 Institute of Marine Sciences and Technology (INSTM) and the Chahad OB, El Bour M, Calo-Mata P, Boudabous A, Barros-Velazquez J Pasteur Institute of Tunis (IPT), Ministry of Higher Education (2012) Discovery of novel biopreservation agents with inhibitory and Scientific Research and Technology of Information and effects on growth of food-borne pathogens and their application to Communication, Tunisia. seafood products. Res Microbiol 163:44–54 14 Ann Microbiol (2019) 69:1–15

Carbonnelle B, Denis F, Marmonier A, Pinon G, Vargues R (1987) microorganisms of Kule naoto: the Maasai traditional fermented Bactériologie médicale Techniques Usuelles. SIMEP-MASSON milk in Kenya. Int J Food Microbiol 94:269–278 Paris 96 Mesalhy Aly S, Abdel-Galil Ahmed Y, Abdel-Aziz Ghareeb A, Mohamed Carbonnelle E, Mesquita C, Bille E, Day N, Dauphin B, Beretti JL, MF (2008) Studies on Bacillus subtilis and Lactobacillus acidophilus, Ferroni A, Gutmann L, Nassif X (2011) MALDI-TOF mass spec- as potential probiotics, on the immune response and resistance of trometry tools for bacterial identification in clinical microbiology Tilapia nilotica (Oreochromis niloticus) to challenge infections. Fsih laboratory. Clin Biochem 44:104–109 and Shellfish Immunology 25:128–136 Claydon MA, Davey SN, Edwards-Jones V,Gordon DB (1996) The rapid McCabe KM, Zhang YH, Khan G, Mason EO, McCabe ERB (1995) identification of intact microorganisms using mass spectrometry. Amplification of bacteria DNA using highly conserved sequences: Nat Biotechnol 14:1584–1586 automated analysis and potential for molecular triage of sepsis. Doan NTL, Van Hoorde K, Cnockaert M, De Brandt E, Aerts M, Le Pediatrics 95:165–169 Thanh B, Vandamme P (2012) Validation of MALDI-TOF MS for Messi P, Bondi M, Sabia C, Battini R, Manicardi G (2001) Detection and rapid classification and identification of lactic acid bacteria, with a preliminary characterization of a bacteriocin (plantaricin 35d) pro- focus on isolates from traditional fermented foods in northern duced by a Lactobacillus plantarum strain. Int J Food Microbiol 64: Vietnam. Lett Appl Microbiol 55:265–273 193–198 Ehrmann MA, Kurzak P, Bauer J, Vogel RF (2002) Characterization of Migaw S, Ghrairi T, Le Chevalier P, Brillet B, Fleury Y, Khaled H (2013) lactobacilli towards their use as probiotic adjuncts in poultry. J Appl Isolation and characterization of enterococci bacteriocinic strains Microbiol 92:966–975 from Tunisian fish viscera. Food Nutr Sci 4:701–708 El-Jeni R, El Bour M, Calo-Mata P, Böhme K, Fernández-No I,C, Barros- Needleman SB, Wunsch CDA (1970) General method applicable to the Velázquez J, Bouhaouala-Zahar B (2015) In vitro probiotic profiling search for similarities in the amino acid sequence of two proteins. J – of novel Enterococcus faecium and Leuconostoc mesenteroides MolBiol48:443 453 from Tunisian freshwater fishes. Can J Microbiol 62:60–71 Nguyen DTL, Van Hoorde K, Cnockaert M, De Brandt E, Aerts M, Le Emami K, Askari V, Ullrich M, Mohinudeen K, Anil AC, Khandeparker Binh T, Vandamme P (2013) A description of the lactic acid bacteria L, Burgess JG, Mesbahi E (2012) Characterization of bacteria in microbiota associated with the production of traditional fermented – ballast water using MALDI-TOF mass spectrometry. PLoS One 7: vegetables in Vietnam. Int J Food Microbiol 163:19 27 38515 Olsson JC, Westerdahl A, Conway PL, Kjelleberg S (1992) Intestinal Fooks LJ, Fuller R, Gibson GR (1999) Prebiotics, probiotics and human colonization potential of turbot (Scophthalmus rnaximus)- and dab gut microbiology. Int Dairy J 9:53–61 (Limanda limanda)-associated bacteria with inhibitory effects against Vibrio anguillarum. Appl Environ Microhiol 58:1–5 Fagerquist CK, Miller WG, Harden LA, Bates AH, Vensel WH, Wang G, Pineda FJ, Antoine MD, Demirev PA, Feldmann AB, Jackman J, Mandrell RE (2005) Genomic and proteomic identification of a Longenecker M, Lin JS (2003) Microorganism identification by DNA-binding protein used in the Bfingerprinting^ of campylobacter matrix-assisted laser/desorption ionization mass spectrometry and species and strains by MALDI-TOF-MS protein biomarkers analy- model-derived ribosomal protein biomarkers. Anal Chem 75: sis. Anal Chem 77:4897–4907 3817–3822 González CJ, Encinas JP, García-López ML, Otero A (2000) Prescott N, Wathes C, Jarvis J (2003) Light, vision and the welfare of Characterization and identification of lactic acid bacteria from fresh- poultry. Anim Welf 12:269–288 water fishes. Food Microbiol 17:383–391 Ringø E, Holzapfel W (2000) Identification and characterization of Hagi T, Hoshino T (2009) Screening and characterization of potential carnobacteria associated with the gills of Atlantic salmon (Salmo probiotic lactic acid bacteria from cultured common carp intestine. salar L.). Syst Appl Microbiol 23:523–527 Biosci Biotechnol Biochem 73:1479–1483 Ringø E, Birkbeck TH (1999) Intestinal microflora of fish larvae and fry. Holland RD, Duffy CR, Rafii F, Sutherland JB, Heinze TM, Holder CL, Aquac Res 30:73–93 Voorhees KJ, Lay JO (1999) Identification of bacterial proteins ob- Robertson PAW, Dowd CO, Burells C, Williams P, Austin B (2000) Use served in MALDI TOF mass spectra from whole cells. Anal Chem – of Carnobacterium sp. as a probiotic for Atlantic salmon (Salmon 71:3226 3230 solar L.) and rainbow trout (Oncorhynchus mykiss,Walbaum). Hosseini SV,Arlindo S, Böhme K, Fernandez-No C, Calo-Mata P, Barros Aquaculture 185:235–243 Velázquez J (2009) Molecular and probiotic characterization of bac- Saito N, Nei M (1987) The neighbor-joining method: a new method for teriocin producing Enterococcus faecium strains isolated from reconstructing phylogenetic trees. Mol Biol Evol 4:406–425 nonfermented animal foods. J Appl Microbiol ISSN 1364-5072 Shnit-Orland M, and Kushmaro A (2008) Coral mucus bacteria as a Hugenholtz J, Kleerebezem M (1999) Metabolic engineering of lactic source for antibacterial activity. Proceedings of the 11th acid bacteria: overview of the approaches and results of pathway International Coral Reef Symposium, Ft. Lauderdale, Florida, 7– rerouting involved in food fermentations. Curr Opin Biotechnol 11 July 2008 – 10:492 497 Sica MG, Olivera NL, Brugnoni LI, Marruci PL, López Cazorla AC, Kumar S, Nei M, Dudley N, Tamura K (2008) MEGA: a biologist-centric Cubitto MA (2010) Isolation, identification and antimicrobial activ- software for evolutionary analysis of DNA and protein sequences. ity of lactic acid bacteria from the Bahia Blanca estuary. Rev Biol Brief Bioinform 9:299–306 Mar Oceanogr 45:389–397 Jini R, Swapna HC, Amit KR, Vrinda R, Halami PM, Sachindra NM, Soccol CR, De Souza Vandenberghe LP, Spier MR, Pedroni Medeiros Bhaskar N (2011) Isolation and characterization of potential lactic AB, Yamaguishi CT, De Dea Lindner J, Pandey A, Thomaz-Soccol acid bacteria from freshwater fish processing wastes for application V (2010) The potential of probiotics: a review. Food Technol in fermentatative utilization of fish processing waste. Braz J Biotechnol 48:413–434 Microbiol 42:1516–1525 Sorroza L, Padilla D, Acosta F, Román L, Grasso V,Vega J, Real F (2012) Majamaa H, Isolauri E (1997) Probiotics: a novel approach in the man- Characterization of the probiotic strain Vagococcus fluvialis in the agement of food allergy. J Allergy Clin Immunol 99:179–185 protection of European sea bass (Dicentrarchus labrax) against vib- Manero A, Blanch AR (1999) Identification of Enterococcus ssp. with a riosis by Vibrio anguillarum. Vet Microbiol 155:369–373 biochemical key. Appl Environ Microbiol 65:4425–4430 Stevens KA, Sheldon BW, Klapes NA, Klaenhammer TR (1991) Nisin Mathara JM, Schillinger U, Kutima PM, Mbugua SK, Holzapfel WH treatment for inactivation of Salmonella species and other gram- (2004) Isolation, identification and characterisation of the dominant negative bacteria. Appl Environ Microbiol 57:3613–3615 Ann Microbiol (2019) 69:1–15 15

Stiles ME, Holzapfel W (1997) Lactic acid bacteria of foods and their Torodov SD, Dicks LMT (2004) Effect of medium components on bac- current . Int J Food Microbiol 36:1–29 teriocin production by Lactobacillus pentosus ST151BR, a strain Spanggaard, B., Huber, I., Nielsen, J., B. Sick, E., B. Pipper, C. isolated from beer produced by the fermentation of maize, barley Martinussen, T., J. Slierendrecht, W. & Gram, L (2001) The probi- and soy flour. World J Micobiol Biotechnol 20:643–650 otic potential againt vibriosis of the indigenous microflora of rain- Vàzquez JA, Gonzàlez M, Murado MA (2005) Effects of lactic acid bow trout. Environ Microbiol 3: 755–765 bacteria cultures on pathogenic microbiota from fish. Aquaculture Tanigawa K, Kawabata H, Watanabe K (2010) Identification and typing 245:149–161 of Lactococcus lactis by matrix-assisted laser desorption ionization– Vandamme P, Pot B, Gillis M, de Vos P, Kersters K, Swings J (1996) time of flight mass spectrometry. Appl Environ Microbiol 76:4055– Polyphasic taxonomy, a consensus approach to bacterial systemat- 4062 ics. Microbiol Rev 60:407–438 Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW: improving Vendrell D, Luis Balcazar J, de Blas I, Ruiz-Zarzuela I, Girones O, the sensitivity of progressive multiple sequence alignment through Muzquiz JL (2008) Protection of rainbow trout (Oncorhynchus sequence weighting, position-specific gap penalties and weight ma- mykiss) from lactococcosis by probiotic bacteria. Comp Immunol trix choice. Nucleic Acids Res 22:4673–4680 Microbiol Infect Dis 31:337–345