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Characterization of two novel lactic acid bacteria isolated from the intestine of rainbow trout (Oncorhynchus mykiss, Walbaum) in Slovakia

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An effective probiotic must comply with criteria, which determines its effect. The aim of this work was to isolate lactic acid bacteria (LAB) from the intestinal content of rainbow trout (Oncorhynchus mykiss), subsequently potentially used as
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  Contents lists available at ScienceDirect Aquaculture  journal homepage: www.elsevier.com/locate/aquaculture Characterization of two novel lactic acid bacteria isolated from the intestineof rainbow trout ( Oncorhynchus mykiss,  Walbaum) in Slovakia Adriána Fe č kaninová a , Jana Ko šč ová b , Dagmar Mudro ň ová b , Petra Schusterová b ,Ivana Cinge ľ ová Maru šč áková b , Peter Popelka a, ⁎ a  Department of Food Hygiene and Technology, University of Veterinary Medicine and Pharmacy in Ko  š  ice, Komenského 73, Ko  š  ice 041 81, Slovakia b  Department of Microbiology and Immunology, University of Veterinary Medicine and Pharmacy in Ko  š  ice, Komenského 73, Ko  š  ice 041 81, Slovakia A R T I C L E I N F O  Keywords: Aquaculture Oncorhynchus mykiss ProbioticsLactic acid bacteriaIsolationSelection criteria A B S T R A C T An e ff  ective probiotic must comply with criteria, which determines its e ff  ect. The aim of this work was to isolatelactic acid bacteria (LAB) from the intestinal content of rainbow trout ( Oncorhynchus mykiss ), subsequentlypotentially used as probiotics in order to improve health status of  󿬁 sh during 󿬁 sh farming. Selection criteria wereused to obtain suitable probiotic candidates for aquaculture of salmonid. A total of 6 lactic acid bacteria isolatedfrom intestines of rainbow trout belonging to the genus  Lactobacillus  were included in this study, and en-compassed the following species:  L. plantarum  (n=3),  L. fermentum  (n=2), and  L. brevis  (n=1). Antimicrobialsusceptibility test has been performed on the basis of Guidance on the assessment of bacterial susceptibility toantimicrobials of human and veterinary importance by the EFSA. Sensitivity or intrinsic resistance of the isolatedLAB to a recommended set of antibiotics make them safe for use as probiotics in aquaculture. All six auto-chthonous isolates showed the antagonistic activity against both salmonid pathogens  Aeromonas salmonicida subsp.  salmonicida  CCM 1307 and  Yersinia ruckeri  CCM 6093. Determined  in vitro  survival conditions in thegastrointestinal tract of rainbow trout, and taking into account the previous results, two isolates -  Lactobacillus plantarum  (R2) and  Lactobacillus fermentum  (R3), which showed the highest level of tolerance to di ff  erent pHvalues, bile, temperature, and the best growth properties, were selected as candidates for probiotics. These twostrains have been sent to the Czech Collection of Microorganisms (CCM) of Masaryk University in Brno forpurposes of Patent Procedure under the Budapest Treaty. 1. Introduction Aquaculture is facing a new era of expansion in Europe (theEuropean Commission, 2015a). EU aquaculture is renowned for its highquality, sustainability and consumer protection standards (theEuropean Commission, 2017). The main aquaculture-producing coun-tries in terms of volume are Spain, the United Kingdom, France, Greeceand Italy. Slovak aquaculture produces 0.09%  󿬁 sh of the total EUaquaculture production, in terms of volume and 0.08% in terms of value (the European Commission, 2016).Fish are produced in  󿬁 sh farming facilities (tanks, cages, nurseries,hatcheries and recirculation systems) with a capacity of 140,503m 3 and in 485  󿬁 sh ponds, covering an area of about 2000ha (the EuropeanCommission, 2015b).Despite the aquaculture sector presenting a relatively small con-tribution to the national economy, it has the important non-productionfunctions that are instrumental to environmental protection andenhancement. Examples are water management,  󿬂 ood control, land-scaping, biodiversity preservation, and recreational  󿬁 shing.Aquaculture in Slovakia can be split into two groups: farming of sal-monids ( e.g.  trout and grayling) and of lowland  󿬁 sh species ( e.g.  carp,pike, tench and cat 󿬁 sh). Trout accounts for 70% of both volume andvalue of total production, carp for 23% of volume and 20% of value,and other freshwater species for 7% of volume and 10% of value (theEuropean Commission, 2015b).As intensive  󿬁 sh farming is facing the problem of massive losscaused by diseases, there are a range of approaches available to protectfarmed aquatic animals against the e ff  ect of pathogens. Of these ap-proaches, probiotics have become widely used for the control of disease(Hai, 2015). Lactic acid bacteria (LAB) such as  Lactobacillus  spp.,  Ped-iococcus  spp.,  Lactococcus  spp., Carnobacterium spp., and genus  Leuco-nostoc  are often used as probiotics in aquaculture because of theirbene 󿬁 cial e ff  ects (Merri 󿬁 eld et al., 2010). Potential probiotics may becommonly obtained from various sources, such as the gastrointestinal https://doi.org/10.1016/j.aquaculture.2019.03.026Received 2 October 2018; Received in revised form 31 January 2019; Accepted 14 March 2019 ⁎ Corresponding author.  E-mail address:  peter.popelka@uvlf.sk (P. Popelka). Aquaculture 506 (2019) 294–301Available online 15 March 20190044-8486/ © 2019 Elsevier B.V. All rights reserved.    tract (Ramesh et al., 2015), and  󿬁 sh mucus (Tapia Paniagua et al.,2012) of aquatic animals. The sources can also be the aquatic en-vironment such as water or sediment (Del'duca et al., 2013), or isolatedfrom microbial bio  󿬂 akes (Ferreira et al., 2015).Desirable characteristics for the selection of potential probioticsinclude (i) no harm to the host; (ii) acceptance by the host throughingestion, and colonization and proliferation within the host; (iii)ability to reach targeted organs where they can work; and (iv) novirulent resistance or antibacterial resistance genes (Verschuere et al.,2000; Kesarcodi-Watson et al., 2008). The reasons for selecting poten- tial probiotics are based on their inhibitory activity against targetedpathogens  in vitro  (Jöborn et al., 1997, 1999; Bourouni et al., 2007; Cao et al., 2012). They have to be evaluated for safety (Verschuere et al.,2000), or for pathogenicity (Chythanya et al., 2002) to the hosts. Pro- biotics should be tested for their inhibitory activity against targetedpathogens (Vijayan et al., 2006; Hai et al., 2007), or for their protection of hosts when challenged with pathogens (Irianto and Austin, 2002b;Vaseeharan et al., 2004). Theoretically, the probiotic candidate thatful 󿬁 ls more of these characteristics than others shall be considered anappropriate probiotic. Some  in vivo  tests should be carried out(Verschuere et al., 2000) before application on a large scale.So far only one probiotic was authorized for the use in aquaculturein the European Union, namely  Pediococcus acidilactici  CNCM MA 18/5M, a member of the lactic acid group bacteria (Regulation (EC) 911/,2009; Ramos et al., 2013). Bactocell ®  Aquaculture (Lallemand Inc.,Canada) therefore becomes the  󿬁 rst probiotic authorized for such use inaquaculture in the European Union, it is already authorized for severalspecies including salmonids (EFSA, 2012a). In which it is able to im-prove the quality of the  󿬁 nal  󿬁 sh products by increasing the number of well-conformed  󿬁 sh  ( prevention of Vertebral Compression Syndrome,VCS). This syndrome, which is thought to a ff  ect over 20% of rainbowtrout harvested constitutes an important economic loss for  󿬁 sh farmers.The use of Bactocell ®  in the prevention of VCS in salmonids is thesubject of an international patent  󿬁 led by IFREMER and INRA in 2006.In shrimps, Bactocell ®  is able to increase survival and growth perfor-mance. The exploratory trials on the possible use of Bactocell ®  inaquaculture started in 2002, with  󿬁 rst, feasibility trials on live preys(Gatesoupe, 2002). This was followed by numerous  󿬁 eld trials and in-depth studies, on shrimps, salmonids and other marine  󿬁 shes, some of which will be subject to the EU authorization following steeply fromthis  󿬁 rst. The application dossiers for the use of Bactocell ®  in shrimpsand salmonids are therefore the fruit of several years of intellectualinvestment as well as, research and development conducted in closepartnership with renowned researchers, institutions and leading privatecompanies in aquaculture (Bactocell, http://www.aquafeed.com/ buyers-guide/suppliers-news-article/2934/Bactocell-the- 󿬁 rst-probiotic-authorized-for-use-in-aquaculture-in-the-European-Union/).The aim of this study was to isolate lactic acid bacteria from in-testinal content of rainbow trout and to investigate the properties of isolated strains according to selection criteria for potential use of thestrains as probiotics in aquaculture. 2. Materials and methods  2.1. Sample collection Sixty samples of healthy juvenile rainbow trout (150 – 250g) werecollected from the commercial  󿬁 sh farm Rybárstvo Po ž ehy s.r.o. in theSlovak Republic, and transferred alive to the laboratory of theUniversity of Veterinary Medicine and Pharmacy in Ko š ice within 4h.The  󿬁 sh were sacri 󿬁 ced using a mechanical stunning followed by spinalcord section according to the University of Veterinary Medicine andPharmacy Ethics Committee recommendations. The whole intestineswere taken aseptically from each  󿬁 sh and the intestinal content werecollected into the sterile  󿬂 asks.  2.2. Isolation of lactic acid bacteria For isolation, 1g of intestinal content from each  󿬁 sh specimen wasremoved under aseptic conditions, and suspended in 9ml sterile salineand homogenized for 1min in stomacher (Steward Stomacher, UnitedKingdom). Afterwards a volume of 0.1ml of serial dilutions(10 − 1 – 10 − 8 ) were spread on plates of de Man Rogosa and Sharp (MRS)agar (HiMedia, India) and incubated anaerobically (Oxoid Gas PackAnaerobic system) at 37°C for 48h. After incubation, the total numberof lactic acid bacteria was determined. Well isolated colonies with ty-pical characteristics with entire margins were picked from each plateand transferred to an MRS broth.  2.3. Identi  󿬁 cation of isolates The cultures were identi 󿬁 ed according to their morphological, cul-tural, physiological and biochemical characteristics. The used testswere: Gram staining, catalase test, API 50 CH (bioMérieux SA, France),matrix-assisted laser desorption ionization-time-of- 󿬂 ight mass spek-trometry (MALDI-TOF MS) (Ultra 󿬂 ex III, Bruker Daltonics, Germany)and polymerase chain reaction (PCR) /16S ribosomal RNA (rRNA).  2.3.1. MALDI-TOF MS For MALDI-TOF analysis, a solitary bacterial colony of each isolatewas resuspended in 300 μ l distilled water. Then, 900 μ l of absoluteethanol was added, and the mixture was centrifuged at 13,000  g   for2min, after which the supernatant was discarded. Fifty microlitres of formic acid (70% v/v) was added to the pellet, and thoroughly mixedby pipetting before the addition of 50 μ l of acetonitrile to the mixture.The mixture was centrifuged again at 13,000  g   for 2min. One microlitreof the supernatant was placed onto a spot of steel target plate and airdried at room temperature. Both the microbial  󿬁 lm and the supernatantof the extracted proteins were overlaid with 1 μ l of matrix solution(saturated solution of   α -cyano-4-hydroxycinnamic acid in 50% acet-onitrile, and 2.5% tri- 󿬂 uoroacetic acid). The matrix sample was co-crystallized by air drying at room temperature (Ferreira et al., 2011).MALDI-TOF was performed on Micro 󿬂 ex LT instrument (BrukerDaltonics GmbH, Leipzig, Germany) using FlexControl software (ver-sion 3.0). Spectra were recorded in the positive linear mode. The rawspectra obtained from each isolate were imported into Biotyper soft-ware version 3.0 (Bruker Daltonics GmbH, Leipzig, Germany, databaseversion 3.3.1.0) and analyzed by standard pattern matching with de-fault settings without any user intervention. Scores of  ≥ 2.0 were con-sidered high-con 󿬁 dence (secure species) identi 󿬁 cation, scores between1.7 and 2.0 were considered intermediate con 󿬁 dence (genus only)identi 󿬁 cation, and scores<1.7 were considered unacceptable identi- 󿬁 cation.  2.3.2. API 50 CH  Fermentation of carbohydrates was determined using API 50 CH, astandardized system, consisting of 50 biochemical tests for the study of carbohydrate metabolism by microorganisms. API 50 CH is used inconjunction with API 50 CHL medium for the identi 󿬁 cation of the genus  Lactobacillus  and related genera. The test was performed according tothe manufacturer's instructions. The biochemical pro 󿬁 le obtained foreach isolate was identi 󿬁 ed using api web ™  identi 󿬁 cation software withdatabase (V5.1). Accurate identi 󿬁 cation using the API system wascon 󿬁 rmed when the percentage of identi 󿬁 cation was>90%.  2.3.3. PCR 2.3.3.1. Genomic DNA preparation . Preparation of genomic DNA wasperformed as follows. The single colonies of lactobacilli cultured onMRS agar were washed in PBS and resuspended in 50 μ l of steriledistilled water. After overnight incubation at  − 80°C, bacterialsuspension was boiled for 10min. Subsequently, cell debris washarvested by centrifugation for 5min (14,000rpm). Supernatant with  A. Fe č  kaninová, et al.  Aquaculture 506 (2019) 294–301 295  bacterial DNA was used as template for PCR reactions.  2.3.3.2. PCR conditions . The 20 μ l reaction mixture contained 1 xDreamTaq Green PCR Master Mix (Thermo Fisher Scienti 󿬁 c), 1 μ M of each primer and 2 μ l of DNA template. The PCR reaction was performedin a thermo-cycler GenePro (BIOER, China). The ampli 󿬁 cation programconsisted of (i) denaturation at 95°C for 10min, (ii) 35cycles of denaturation at 95°C for 30s, annealing at 53°C (  L. plantarum ) or55°C (  L. fermentum ) for 30s or 60°C for 45s (  L. brevis ), elongation at72°C for 1min, (iii) followed by a  󿬁 nal extension at 72°C for 10min.Five microliters of PCR products were electrophoresed at constantvoltage (100V) on 2% agarose gel stained using GoodView for 36min.Subsequently, PCR products were visualized by UV light in Kodak GelLogic System (Table 1).  2.4. Antibiotic susceptibility test  Antimicrobial susceptibility has been tested on nine antibioticslisted by EFSA (2012b) (ampicillin, vancomycin, gentamicin, kana-mycin, streptomycin, erythromycin, clindamycin, tetracycline andchloramphenicol) and was evaluated using commercial  E  -test  –  MICTest Strip (Lio 󿬁 lchem ® , Italy). The concentration on the strips was from0.016 to 256 μ g/ml with the exception of streptomycin(0.064 – 1024 μ g/ml). Bacterial cultures in the exponential growth phasewere diluted in a sterile saline to a turbidity of McFarland standard 1and used to inoculate a melted and cooled Iso-Sensitest agar (90% w/v,Oxoid, UK) supplemented with MRS agar (10% w/v). MIC Test stripswere placed on the surface of the inoculated agar and incubated underanaerobic conditions at 37°C for 48h. The MICs was interpreted as thepoint at which the ellipse intersected the strip as described in the MICTest Strip technical guide.  2.5. Antagonistic activity  The inhibitory e ff  ect of lactic acid isolates on selected two salmonidpathogens (  Aeromonas salmonicida  subsp.  salmonicida  CCM 1307 and Yersinia ruckeri  CCM 6093) was noticed through the combination of discdi ff  usion method, and double layer di ff  usion assay. These pathogenswere obtained from the Czech Collection of Microorganisms (CCM),Brno, the Czech Republic and from the State of Veterinary and FoodInstitute in Dolný Kubín, Slovakia. On the plates with 20ml of PYG agarwith pH6.5 were transferred sterile discs of 6mm (BBL, BectonDickinson, USA), which were inoculated of 10 μ l 24h broth cultures of each isolates. After incubation at 37°C for 48h under anaerobic con-ditions, the discs were removed aseptically, and the plates were ex-posed to chloroform vapour for 30min and then left semi-open for 2hto allow evaporation of residual chloroform. Afterwards the plates wereoverlayed with 3ml of 0.7% tryptone soya agar containing 0.3ml of 18h pathogen cultures and further incubated for 24h at the optimumtemperature for each pathogen. The sensitivity of pathogens in thepresence of each probiotic isolates was determined by measuring theclear zone of inhibition (ZOI). The experiment was carried out intriplicates and activity was reported as diameter of ZOI ± SD.  2.6. Growth properties and viability testing  The numbers of lactobacilli were enumerated by Bacteria countingkit for  󿬂 ow cytometry (Molecular Probes, Life Technologies, USA) afterthe growth in MRS broth (Himedia, India) at 37°C and 12°C for 24h.Subsequently, viability of lactobacilli was stated after the staining withcarboxy 󿬂 uorescein diacetate (cFDA)  –  a non- 󿬂 uorescent substratewhich is cell permeant and is hydrolysed intracellularly by non-speci 󿬁 cesterases in live cells to positively charged  󿬂 uorescent carboxy- 󿬂 uorescein that is retained in the cell when the membrane is intact(Bunthof et al., 2001; Hoefel et al., 2003). A stock solution of cFDA (Sigma-Aldrich, Germany) was prepared and stored as described byAmor et al. (2002). Bacterial suspensions in the amount of 50 μ l weremixed with 5 μ l of 1mM solution of cFDA and with 445 μ l of PBScontaining 1mM dithiothreitol (Sigma-Aldrich). The samples were in-cubated for 30min at 37°C. Flow cytometric assessment of viability of lactobacilli was performed on a BD FACSCanto ™  󿬂 ow cytometer(Becton Dickinson Biosciences, the USA) and analyzed with BD FACSDiva ™  software. FSC  vs.  SSC dot plot was used to state a position of bacteria. Fluorescence measurements were carried out using the488nm blue laser with FL-1  󿬁 lter (530/30nm). The numbers of viableand dead bacteria (in percentage) were evaluated on the basis of FL-1vs .  count histograms. The results are expressed as an arithmetic averagefrom 3 replicates±SD.  2.7. Acid-, bile- and water-tolerance tests Isolates of lactobacilli were tested in the arti 󿬁 cial gastric juice andin the presence of 10% trout bile. Gastric juice was prepared by re-suspending the pepsin (3g/l; Sigma) in 0.5% NaCl and adjusting the pHto 2.0, 2.5 and 3.0 with HCl. The viability of bacteria in gastric juicewas measured before incubation and after 30, 60, 90 and 120min of incubation at 12°C.Bile tolerance was tested in fresh trout bile received aseptically fromgall bladders of trout immediately after killing. The viability of lacto-bacilli was measured before incubation and after 1, 2, 3 and 4h of incubation at 12°C in 10% bile in phosphate bu ff  ered saline (PBS; MPBiomedicals, France) where pH was adjusted to 7.2.Water for water-tolerance test was taken directly from breedingtanks at the  󿬁 sh farm. The viability of lactobacilli was measured beforeincubation and after 4h of incubation at 12°C.Water as well as gastric juice and bile solutions were  󿬁 lteredthrough 0.22 μ m syringe  󿬁 lter (Merck, Germany), and then they wereinoculated in a ratio 50:1 with overnight cultures of lactobacilli con-taining approximately 1×10 9 bacteria/ml. The viability was testedafter cFDA staining as was described above.  2.8. Statistical analysis Data were analyzed in statistical program GraphPadPrism version Table 1 Sequence of the oligo-nucleotide primers used for PCR ampli 󿬁 cations in this study.  Lactobacillus  spp. Primer sequence (5 ′ -3 ′ ) PCR product size ReferenceForward primerReverse primer  Lactobacillus plantarum  5 ′ -ATGAGGTATTCAACTTATG-3 ′  220bp Berthier and Ehrlich (1998)5 ′ -GCTGGATCACCTCCTTTC-3 ′  Lactobacillus fermentum  5 ′ -GTTGTTCGCATGAACAACGCTTAA-3 ′  889bp Chagnaud et al. (2001)5 ′ -CGACGACCATGAACCACCTGT-3 ′  Lactobacillus brevis  5 ′ -AATTGATTTTCATACCGCAGAA-3 ′  145bp Fusco et al. (2016)5 ′ -TTGGCACCGCATGATGTG-3 ′  A. Fe č  kaninová, et al.  Aquaculture 506 (2019) 294–301 296  3.00 (GraphPad Software, San Diego California USA).Viability tests were analyzed in the  󿬁 rst stage by two-one analysisvariance (ANOVA) and subsequently by one-way ANOVA followed byTukey's multiple comparison test. 3. Results 3.1. Isolation and identi  󿬁 cation of lactic acid bacteria In this study, various LAB isolates were obtained from 60 samples of the gut contents of healthy rainbow trout,  Oncorhynchus mykiss . Theisolated strains had the typical characteristics of lactic acid bacteria(LAB), which are Gram-positive, rod shaped; non-spore forming, non-motile, and catalase negative (Salvetti et al., 2012). The LAB isolateswere classi 󿬁 ed into the genus  Lactobacillus  based on their morphologyand biochemical characters. The six  Lactobacillus  strains isolated fromthe gut contents of   Oncorhynchus  in the study were identi 󿬁 ed as  L. plantarum  (R1, R2, R4),  L. fermentum  (R3, R5) and L.  brevis  (R6) by API50 CH and MALDI-TOF MS (Table 2). Finally, the identi 󿬁 cation of LABwas con 󿬁 rmed by PCR. 3.1.1. Antibiotic susceptibility test  The minimum inhibitory concentrations values of the antimicrobialsof the tested isolates were lower than the established cut-o ff   values byEFSA (2012b). In the case of vancomycin, it presents intrinsic resistanceof all tested LAB (R1 – R6), as well as isolates of   Lactobacillus plantarum (R1, R2, R4) which are intrinsic resistant to streptomycin (Table 3). 3.1.2. Antagonistic activity  All six autochthonous isolates showed antagonistic activity againstselected pathogens, which was determined by measuring the size of inhibition zones. No signi 󿬁 cant di ff  erences were found between theisolates (Fig. 1). 3.1.3. Growth properties and viability testing  All tested  Lactobacillus  isolates showed very good growth properties.They reached counts 5×10 9 – 1×10 10 bacteria/ml at 37°C and6×10 7 – 3×10 8 bacteria/ml at 12°C with viability around 90%. Thehighest tolerance to pH2 has been noted in L.  fermentum  R3 and R5,and L.  plantarum  R1 was the most susceptible (Fig. 2). Isolates R2 – R5showed similar high resistance to pH2.5 reaching viability higher than90% after 120min of incubation and isolates R1 and R6 were sig-ni 󿬁 cantly more sensitive (Fig. 3). In the gastric juice with pH3 allisolates survived in high percentages (>85%; Fig. 4). Viability of lac-tobacilli R1, R2, R3 and R5 in 10% trout bile was higher than 90% evenafter 4h of incubation. Isolates R4 and R6 survived in signi 󿬁 cantlylower numbers (Fig. 5). In water, the highest viability was recorded inthe case of isolates R2 and R4, and the R6 isolate survived in the lowestnumbers (Fig. 6). 4. Discussion When developing microorganisms for use as probiotics in aqua-culture, it is important they should be regarded as safe, not only for theaquatic hosts, but also for their surrounding environments and humans(Muñoz – Atienza et al., 2013). A list of characteristics for potentialprobiotic bacteria to be used in aquaculture species was reported byVine et al. (2006) and extended by Merri 󿬁 eld et al. (2010). First, thecandidate microorganism should be naturally occurring and non-pa-thogenic in the host organisms' native habitat. The microorganismshould also be easily cultured with advantageous growth character-istics, such as a short lag period and generation time, as well as stronggrowth at the host organisms' rearing temperature. The candidateprobiotic should have positive traits to culture the host gut, such asresistance to bile salts and low pH values, as well as the adherencewithin intestinal mucus, and the ability to colonize the intestinal epi-thelial surface. It is also essential that there are no health risks; there-fore it is important the microorganisms do not possess any plasmid-encoded antibiotic resistance genes. Finally, the candidate probioticshould have positive e ff  ects on 󿬁 sh health and/or nutrition. An examplewould be the exhibition of antagonistic properties towards one or morekey pathogens (e.g.  Aeromonas salmonicida  and  Yersinia ruckeri ). Theprobiotic may also have relevant nutritional bene 󿬁 ts, such as produc-tion of extracellular digestive enzymes. For example, production of chitinase or cellulase in cases where  󿬁 sh are being fed chitin-rich orplant-based diets, or production of vitamins.The aim of this work was to obtain a suitable candidate for pro-biotics in salmonid aquaculture on the basis of selection criteria. Theisolates were tested by microbiological, biochemical, and genetic ex-amination to identify and select the most suitable candidates for furtherassessment as probiotics in sustainable aquaculture. Although membersof the genus  Lactococcus  have been identi 󿬁 ed, we have decided tocontinue testing with lactobacilli R1  –  R6, as these are more commonlyused in aquaculture preparations according to their bene 󿬁 cial proper-ties which were described by Farzanfar (2006). A total of 6 lactic acidbacteria isolates from intestinal content of rainbow trout belonging tothe genus  Lactobacillus  were included in this study and encompassed thefollowing species  L. plantarum  (R1, R2, R4),  L. fermentum  (R3, R5), and  L. brevis  (R6).According to the EFSA (2017), the lactobacilli species tested in thiswork (  L. plantarum ,  L. fermentum , and  L. brevis ) are included in the QPSlist and, therefore, demonstration of their safety only requires con- 󿬁 rmation of the absence of determinants of resistance to antibiotics of human and veterinary clinical signi 󿬁 cance.All tested isolates of lactobacilli were susceptible to the antibioticsrecommended by the EFSA (2012b); therefore, no further testing isrequired. However, all isolates (R1 – R6) were resistant to vancomycin,although this is an intrinsic resistance and the EFSA did not impose cut-o ff   values for this antibiotic. Heterofermentative lactobacilli are natu-rally resistant to glycopeptide antibiotics, including vancomycin,whereas strictly homo-fermentative lactobacilli are susceptible to van-comycin (Egervärn, 2009). In the present study  L. plantarum  isolates Table 2 Identi 󿬁 cation of lactic acid bacteria isolates. Isolate Top species best match Score value of MALDI-TOF MS API 50 CH percentage of similarityR1  Lactobacillus plantarum  1.833 99.8R2  Lactobacillus plantarum  2.403 99.8R3  Lactobacillus fermentum  2.140 98.9R4  Lactobacillus plantarum  2.354 99.8R5  Lactobacillus fermentum  2.038 98.9R6  Lactobacillus brevis  2.350 99.6 MALDI-TOF MS Scores of  ≥ 2.0 were considered high-con 󿬁 dence (secure species) identi 󿬁 cation, scores between 1.7 and 2.0 were considered intermediate con 󿬁 dence(genus only) identi 󿬁 cation.The percentage of API identi 󿬁 cation of>90% was considered accurate identi 󿬁 cation.  A. Fe č  kaninová, et al.  Aquaculture 506 (2019) 294–301 297  (R1, R2, R4) were also resistant to streptomycin. Intrinsic resistance tovancomycin and amino-glycosides, such as streptomycin, has been re-ported as a general feature for lactobacilli and stems from the absenceof the peptidoglycan D-alanine target precursor and the lack of a cy-tochrome-mediated transport system required for aminoglycoside up-take, respectively (Egervärn, 2009).Survival of the bacteria in the conditions of gastrointestinal tract isanother selection criterion for probiotic suitability in aquaculture.Probiotics resistant to acid and bile are more likely to survive passagethrough the gastrointestinal tract and may colonize the intestine(Allameh et al., 2012; Sica et al., 2012). For carnivorous  󿬁 sh, such assalmonids, the digestive process starts in the stomach where a strongacidic environment (pH2 – 4) is induced by the production of HCl, and issuitable for activating the pepsin enzyme. The digestion continues inthe basal intestinal environment (pH8 – 9) by the action of the pan-creatic enzyme trypsin and intestinal proteases (Ko šť  anová, 2001; Bone Table 3 Determination of lactic acid bacteria isolates antibiotic susceptibility by  E  -test (n=3). Isolate Ampicillin VancomycinMIC (mg/L) MCV (mg/L) DS MIC (mg/L) MCV (mg/L) DSR1 0.09 ± 0.05 2 S  –  n.r. RR2 0.10 ± 0.05 2 S  –  n.r. RR3 0.05 ± 0.02 2 S  –  n.r. RR4 0.06 ± 0.01 2 S  –  n.r. RR5 0.04 ± 0.01 2 S  –  n.r. RR6 0.10 ± 0.00 2 S  –  n.r. RIsolate Gentamicin KanamycinMIC (mg/L) MCV (mg/L) DS MIC (mg/L) MCV (mg/L) DSR1 1.00 ± 0.00 a 16 S 40.00 ± 21.17 64 SR2 2.33 ± 0.58 e 16 S 48.00 ± 16.00 64 SR3 1.17 ± 0.29 16 S 13.33 ± 4.62 32 SR4 1.67 ± 0.76 16 S 24.00 ± 8.00 64 SR5 1.67 ± 0.29 16 S 30.67 ± 18.04 32 SR6 0.63 ± 0.21 16 S 26.67 ± 2.18 32 SIsolate Streptomycin ErythromycinMIC (mg/L) MCV (mg/L) DS MIC (mg/L) MCV (mg/L) DSR1 21.33 ± 4.62 n.r. R 0.36 ± 0.16 1 SR2 24.00 ± 8.00 n.r. R 0.24 ± 0.14 1 SR3 10.67 ± 2.31 64 S 0.29 ± 0.17 1 SR4 16.00 ± 8.00 n.r. R 0.29 ± 0.19 1 SR5 53.33 ± 18.48 64 S 0.53 ± 0.38 1 SR6 6.00 ± 0.00 64 S 0.30 ± 0.25 1 SIsolate Clindamycin TetracyclineMIC (mg/L) MCV (mg/L) DS MIC (mg/L) MCV (mg/L) DSR1 0.02 ± 0.00 2 S 8.00 ± 0.00 c, d, e 32 SR2 0.02 ± 0.00 2 S 10.67 ± 2.31 b, c, d, e 32 SR3 0.03 ± 0.02 1 S 4.67 ± 2.31 8 SR4 0.04 ± 0.02 2 S 2.67 ± 1.16 32 SR5 0.03 ± 0.00 1 S 1.58 ± 0.72 8 SR6 0.03 ± 0.00 1 S 1.50 ± 0.00 8 SChloramphenicolIsolate MIC (mg/L) MCV (mg/L) DSR1 2.67 ± 0.58 8 SR2 3.67 ± 0.58 e 8 SR3 3.33 ± 0.58 e 4 SR4 2.50 ± 0.87 8 SR5 3.00 ± 0.00 e 4 SR6 1.00 ± 0.43 4 S MIC - minimum inhibitory concentration, MCV - microbiological cut-o ff  value, n.r.- not required, DS - determination of susceptibility, S - susceptible,R  –  resistant. a Signi 󿬁 cant di ff  erence to isolate R2. b Signi 󿬁 cant di ff  erence to isolate R3. c Signi 󿬁 cant di ff  erence to isolate R4. d Signi 󿬁 cant di ff  erence to isolate R5. e Signi 󿬁 cant di ff  erence to isolate R6.  A. Fe č  kaninová, et al.  Aquaculture 506 (2019) 294–301 298
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