In this work, the antimicrobial activity against fish pathogens and the in vitro safety of 99 LAB previously isolated from fish, seafood and fish products
 have been assayed by using microbiological, biochemical and genetic assays in order to identify and select the most suitable candidates to be further evaluated as probiotics for a sustainable aquaculture. LAB are widely known for their ability to inhibit bacterial pathogens by the production of antimicrobial compounds such as organic acids, oxygen peroxide and ribosomally-synthesized peptides referred to as bacteriocins, which constitutes a desirable property for probiotics and a sustainable alternative to antibiotics
[9, 18]. In this respect, most of the LAB of aquatic origin tested in this work displayed a broad antimicrobial spectrum against the main Gram-positive and Gram-negative fish pathogens, being remarkable that a high number of strains (24 out of 49 strains, 49%) were identified as potential bacteriocin producers. Recently, bacteriocin production ability has been proposed as a key property for selection of probiotic LAB to be used in aquaculture as an alternative to antibiotics to fight against fish pathogen infections
, similarly as proposed for human and farm animal probiotics
[20–22]. In aquaculture farming, lactococcosis produced by the zoonotic agent L. garvieae, causing hemorrhagic septicaemia and meningoencephalitis, is one of the most serious diseases affecting several marine and fresh water fish species
. With regard to this, our work shows that putative bacteriocinogenic LAB active against this relevant fish pathogen are common amongst the microbiota isolated from aquatic animals (10 strains, 20%).
The application of probiotics in aquaculture may modify the microbial ecology of the aquatic hosts and their surrounding environment, and thus the assessment of their safety to the target aquatic species, the environment and humans constitutes an essential issue
. To date, several studies describing the screening and evaluation of LAB as probiotic candidates for aquaculture have been reported
[25–28]; however, the safety assessment of the strains is generally limited to in vivo challenge tests and rearing trials in order to confirm their lack of toxicity to the aquatic hosts
[24, 25, 28–31]. Strikingly, in vitro safety assessment studies have not been generally addressed, despite they have lower economic and ethic costs and result very effective to evaluate the safety of a high number of candidate probiotic strains not only for the host species, but also for humans and the environment. According to EFSA
, most of the LAB species tested in this work (P. pentosaceus, Lb. curvatus, L. lactis, Lc. mesenteroides) are included in the QPS list and, therefore, demonstration of their safety only requires confirmation of the absence of determinants of resistance to antibiotics of human and veterinary clinical significance. However, in the case of enterococci, a more thorough, strain-specific evaluation is required to assess the risk associated to their intentional use in the food chain, while no guidelines are given for the safety assessment of the species W. cibaria.
Our results show that enterococcal virulence factors were more frequently found in E. faecalis than in E. faecium, which is in concordance with previous reports
[32–34]. In this respect, most of the E. faecalis (95%) and a large percentage of the E. faecium (53%) strains evaluated in this work showed, at least, one virulence factor, being efaAfs, gelE and agg the most frequently detected genes. With regard to gelE, which encodes for an extracellular zinc endopeptidase that hydrolyzes gelatin, collagen, hemoglobin, and other bioactive compounds, this gene was detected at high frequency in E. faecalis, with all the gelE
strains showing gelatinase activity. However, five out of nine E. faecium strains harbouring gelE were unable to degrade gelatin, suggesting the carriage of a non-functional gene, as previously reported
[32, 33]. Likewise, in the case of E. faecium P68 and E. faecium GM29 harbouring cylL
, the lack of hemolytic activity may be explained by the absence of cylM, whose product is involved in the post-translational modification of cytolysin. On the other hand, esp and hyl, which encode a cell wall-associated protein involved in immune evasion and an hyaluronidase enzyme, respectively, were not found in any of the tested LAB. Previous studies have reported that esp and hyl are more common in ampicillin-resistant/vancomycin-resistant E. faecium (VREF) than in ampicillin-susceptible/VREF strains
. In this context, the increase in the incidence of VREF at hospital settings has been attributed mainly to the spread of ampicillin-resistant VREF exhibiting esp and/or hyl[36, 37]. Therefore, the fact that the E. faecium strains evaluated in this work lack these genes might be related with their non-clinical origin and absence of ampicillin resistance.
The use and frequent overuse of antibiotics, including those used in human medicine, in fish farming has resulted in the emergence and spread of antibiotic-resistant bacteria in the aquaculture environment. This possesses a threat to human and animal health due to the increase of acquired antibiotic resistance in fish pathogens, the transfer of their genetic determinants to bacteria of terrestrial animals and to human pathogens, and the alterations of the bacterial microbiota of the aquatic environment
[11, 29]. In our study, the percentage of enterococcal strains showing acquired antibiotic resistance was 68%. Interestingly, the results found in E. faecium (71%) and E. faecalis (62%) were similar, however, higher percentages of resistance to ciprofloxacin and/or norfloxacin, rifampicin, and glycopeptides were observed in E. faecalis. Nevertheless, the occurrence of erythromycin and tetracycline resistance was frequently detected amongst E. faecium (45%) but only in one E. faecalis strain (5%). In spite of the high prevalence of acquired antibiotic resistance found in enterococci of aquatic origin, they showed low incidence or absence of resistance to the clinically relevant antibiotics vancomycin (8.5%) and ampicillin, penicillin and gentamicin, respectively, which is in agreement with previous studies
[33, 38]. Moreover, the percentages of strains showing antibiotic resistance in the genera Weissella, Pediococcus and Lactobacillus were 60, 44 and 33%, respectively, while none of the leuconostocs and lactococci showed this phenotype. In this regard, our results indicate that the LAB susceptibility patterns of MIC values to clinically relevant antibiotics are species-dependent, similarly as previously described by other authors
[39, 40]. Moreover, multiple antibiotic resistance was commonly found in strains within the genus Enterococcus (37%), mainly in E. faecalis, while being very infrequent in the non-enterococcal strains (5%).
According to EFSA
, the determination of MICs above the established breakpoint levels, for one or more antibiotic, requires further investigation to make the distinction between added genes (genes acquired by the bacteria via gain of exogenous DNA) or to the mutation of indigenous genes. According to our results, acquired antibiotic resistance likely due to added genes is not a common feature amongst the non-enterococcal LAB of aquatic origin (7.5%). In this respect, this genotype was only found in the genera Pediococcus (12.5%) and Weissella (6.7%). Although P. pentosaceus LPV57 and LPM78 showed resistance to kanamycin (MIC of 128 mg/L), the respective resistance gene aac(6´ )-Ie-aph(2´ ´ )-Ia was not found in these strains. Similarly, P. pentosaceus TPP3 and SMF120 were phenotypically resistant to tetracycline (MIC of 16 mg/L), but did not contain tet(K), tet(L) or tet(M). In this respect, Ammor et al. reported that pediococci are intrinsically resistant to the latter two antibiotics, as well as to glycopeptides (vancomycin and teicoplanin), streptomycin, ciprofloxacin and trimethoprim-sulphamethoxazole. Other authors proposed a MIC for tetracycline in pediococci ranging between 8 and 16 mg/L
, or of 32 mg/L for oxytetracycline in P. pentosaceus. The tetracycline breakpoints suggested for pediococci by EFSA are lower than the MICs observed in our work and others
[17, 42]. On the other hand, the only antibiotic resistance detected in Leuconostoc strains was for vancomycin, which is an intrinsic property of this genus. It has been previously reported that Leuconostoc strains display poor, if any, resistance to antibiotics of clinical interest
. With regard to lactococci, the three L. cremoris strains evaluated were susceptible to all the antibiotics; however, relatively high MICs for rifampicin (16–32 mg/L) and trimethoprim (≥ 64 mg/L) were detected. In fact, most lactococcal species are resistant to trimethoprim
. As expected, all strains of heterofermentative Lactobacillus spp. were resistant to vancomycin but susceptible to the rest of the assayed antibiotics, except Lb. carnosus B43, which showed the highest MIC for ampicillin and penicillin (MICs of 8 and 4 mg/L, respectively). In this context, the presence of modifications in the low affinity penicillin-binding protein (PBP) that confers resistance to penicillin and β-lactams in E. faecium and Streptococcus pneumoniae, has been reported
[43, 44]. Moreover, nine PBPs have been described in Lb. casei ATCC 393
, which leads us to suggest that a similar mechanism may be also responsible for the ampicillin and penicillin resistance found in Lb. carnosus B43. The resistance to vancomycin detected in Pediococcus, Leuconostoc and Lactobacillus species in this study might be due to the presence of D-Ala-D-Lactate in their peptidoglycan rather than D-Ala-D-Ala dipeptide
. In this context, all tested W. cibaria strains showed MICs ≥ 128 mg/L for vancomycin, suggesting that vancomycin resistance is an intrinsic property of this species. In relation to Weissella spp., studies on antibiotic resistance profiles are very limited
 and breakpoints have not been defined by EFSA
. In our study, most W. cibaria strains showed low MIC values; however W. cibaria BCS50 showed relatively high MICs for penicillin (8 mg/L) and kanamycin (64 mg/L), and W. cibaria SMA25 showed MICs of 128 mg/L for kanamycin, 8 mg/L for gentamicin, erythromycin and neomycin, and 2 mg/L for clindamycin. Therefore, these two strains were discarded of this study, while W. cibaria P50, P61, P64, P73, SMA14, SDM381 and SDM389 were not included in the final selection due to their MICs for kanamycin (32–64 mg/L). According to these results, as a rule of thumb, we propose for W. cibaria the breakpoints assigned to Leuconostoc spp. by EFSA
, until further studies establish the wild-type MIC ranges within this species. In spite of that, different MICs for rifampicin and trimethoprim for W. cibaria and Lc. cremoris were found in this study. The reduced susceptibility of W. cibaria towards trimethoprim could indicate an intrinsic resistance to this antibiotic
. In our work, the only antibiotic resistance genes found were mef(A/E), which encodes a drug efflux pump conferring a low to moderate level of resistance to 14 (erythromycin and clarithromycin)- and 15 (azithromycin)-membered macrolides but not to lincosamide or streptogramin B antibiotics
, and lnu(A), encoding the lincosamide O-nucleotidyltransferase that inactivates lincomycin and clindamycin
. In this respect, P. pentosaceus LPM78 and W. cibaria SMA25, displaying erythromycin resistance (MIC = 8 and ≥ 8 mg/L, respectively), carried the gene mef(A/E), which can be found in a variety of Gram-positive bacteria, including corynebacteria, enterococci, micrococci, and several streptococcal species
[51, 52]. On the other hand, two pediococci (P. pentosaceus LPM78 and LPM83) that showed resistance to clindamycin (MIC = 4 and 2 mg/L, respectively) carried the gene lnu(A), which had been only previously found in staphylococci, streptococci, enterococci and lactobacilli of animal origin and in staphylococci isolated from humans
[50, 53]. Strikingly, the clindamycin resistant strains P. pentosaceus LPP32 and B5 and W. cibaria SMA25 (MIC = 4 and 2 mg/L, respectively) did not harbour this gene nor lnu(B). To our knowledge, this is the first description of mef(A/E) in the genera Pediococcus and Weissella, and lnu(A) in the genus Pediococcus. The detection of resistance genes for macrolide and lincosamide in non-enterococcal strains suggests a wider distribution of this group of genes than previously anticipated.
The in vitro subtractive screening proposed in this work also include the assessment of bile salts deconjugation, mucin degradation, biogenic amine production and other potentially detrimental enzymatic activities such as the β-glucuronidase activity, which should be absent in probiotic candidates
[54–56]. Excessive deconjugation of bile salts may be unfavourable in animal production since unconjugated bile acids are less efficient than their conjugated counterparts in the emulsification of dietary lipids. In addition, the formation of micelles, lipid digestion and absorption of fatty acids and monoglycerides could be impaired by deconjugated bile salts
. Similarly, excessive degradation of mucin may be harmful as it may facilitate the translocation of bacteria to extraintestinal tissues
. In this respect, it is worthy to note that none of the 49 tested LAB deconjugated bile salts nor exhibited mucinolytic activity, the latter indicating their low invasive and toxigenic potential at the mucosal barrier. These results are in accordance with previous findings showing that LAB do not degrade mucin in vitro[58, 59]. Moreover, β-glucuronidase activity has been associated with the generation of potential carcinogenic metabolites
; however, none of the LAB tested in our study displayed this harmful enzymatic activity. In a previous work
, we demonstrated that none of the 40 non-enterococcal strains evaluated herein produced histamine, tyramine or putrescine. With regard to enterococci, the nine E. faecium strains only produced tyramine, being E. faecium CV1 a low producer of this biogenic amine. Although the lack of biogenic amine production by probiotic strains is a desirable trait, it should be borne in mind that tyramine production by enterococci is a very frequent trait
[60, 61]. Finally, several studies have suggested that probiotic microorganisms might exert a beneficial effect in the digestion process of fish due to the production of extracellular enzymes
[62–65]. In our work, the LAB strains of aquatic origin within the genera Pediococcus, Enterococcus and Lactobacillus showed a higher number of enzymatic activities than Lactococcus, Leuconostoc and Weissella, being the enzymatic profiles similar amongst strains within the same genus. In this respect, nearly all the strains produced phosphatases, which might be involved in nutrient absorption
, and peptidases and glucosidases that breakdown peptides and carbohydrates, respectively. However, the tested LAB showed weak lipolytic activity and no proteolytic activity.