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 bacteria 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 Lactic acid bacteria (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%Vagococcus). 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