Estefanía Muñoz-Atienza

Antimicrobial activity, antibiotic susceptibility and virulence factors of Lactic Acid Bacteria of aquatic origin intended for use as probiotics in aquaculture

BackgroundThe microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for the aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate the antimicrobial/bacteriocin activity against fish pathogens, the antibiotic susceptibility, and the prevalence of virulence factors and detrimental enzymatic activities in 99 Lactic Acid Bacteria (LAB) (59 enterococci and 40 non-enterococci) isolated from aquatic animals regarded as human food.ResultsThese LAB displayed a broad antimicrobial/bacteriocin activity against the main Gram-positive and Gram-negative fish pathogens. However, particular safety concerns based on antibiotic resistance and virulence factors were identified in the genus Enterococcus (86%) (Enterococcus faecalis, 100%; E. faecium, 79%). Antibiotic resistance was also found in the genera Weissella (60%), Pediococcus (44%), Lactobacillus (33%), but not in leuconostocs and lactococci. Antibiotic resistance genes were found in 7.5% of the non-enterococci, including the genera Pediococcus (12.5%) and Weissella (6.7%). One strain of both Pediococcus pentosaceus and Weissella cibaria carried the erythromycin resistance gene mef(A/E), and another two P. pentosaceus strains harboured lnu(A) conferring resistance to lincosamides. Gelatinase activity was found in E. faecalis and E. faecium (71 and 11%, respectively), while a low number of E. faecalis (5%) and none E. faecium exerted hemolytic activity. None enterococci and non-enterococci showed bile deconjugation and mucin degradation abilities, or other detrimental enzymatic activities.ConclusionsTo 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 in vitro subtractive screening presented in this work constitutes a valuable strategy for the large-scale preliminary selection of putatively safe LAB intended for use as probiotics in aquaculture.

In vitro and in vivo evaluation of lactic acid bacteria of aquatic origin as probiotics for turbot (Scophthalmus maximus L.) farming

Turbot (Scophthalmus maximus L.) is an important commercial marine flatfish. Its production may be affected by bacterial diseases that cause severe economical losses, mainly tenacibaculosis and vibriosis, provoked by Tenacibaculum maritimum and Vibrio splendidus, respectively. An alternative or complementary strategy to chemotherapy and vaccination for the control of these diseases is the use of probiotics. In this work, we report the in vitro and in vivo potential of eight lactic acid bacteria (LAB), previously isolated from fish, seafood and fish products intended for human consumption, as turbot probiotics. Seven out of the eight LAB exerted direct antimicrobial activity against, at least, four strains of T. maritimum and V. splendidus. All LAB survived in seawater at 18 °C for 7 days, and withstood exposure to pH 3.0 and 10% (v/v) turbot bile; however, they differed in cell surface hydrophobicity (8.2-21.7%) and in their ability to adhere to turbot skin (1.2-21.7%) and intestinal (0.7-2.1%) mucus. Most of the tested strains inhibited the binding of turbot pathogens to the mucus. Leuconostoc mesenteroides subsp. cremoris SMM69 and Weissella cibaria P71 were selected based on their strong antimicrobial activity against T. maritimum and V. splendidus, good probiotic properties, and different adhesion ability to skin mucus and capacity to inhibit the adhesion of turbot pathogens to mucus. These two LAB strains were harmless when administered by bath to turbot larvae and juveniles; moreover, real-time PCR on the transcription levels of the immunity-related genes encoding IL-1β, TNF-α, lysozyme, C3, MHC-Iα and MHC-IIα in five organs (head-kidney, spleen, liver, intestine and skin) revealed the ability of these LAB to stimulate their expression in turbot juveniles, especially the non-specific immunity associated genes in mucosal tissues. Based on our results, Lc. cremoris SMM69 and W. cibaria P71 may be considered as suitable probiotic candidates for turbot farming.

Phenotypic and genetic evaluations of biogenic amine production by lactic acid bacteria isolated from fish and fish products

In this work, biogenic amine production (histamine, tyramine and putrescine) by a collection of 74 lactic acid bacteria of aquatic origin has been investigated by means of amino acid decarboxylation by growth on decarboxylase differential medium, biogenic amine detection by thin-layer chromatography (TLC) and decarboxylase gene detection by PCR. None of the evaluated strains showed neither production of histamine and putrescine, nor presence of the genetic determinants encoding the corresponding decarboxylase activities. However, the tyrosine decarboxylase gene (tdc) was present in all the enterococcal strains, and tyramine production was detected by TLC in all of them but Enterococcus faecium BCS59 and MV5. Analysis of the tyrosine decarboxylase operon of these strains revealed the presence of an insertion sequence upstream tdc that could be responsible for their lack of tyrosine decarboxylase activity.

Antimicrobial activity and occurrence of bacteriocin structural genes in Enterococcus spp. of human and animal origin isolated in Portugal

The main objective of this study was to detect the antimicrobial activity and the presence of bacteriocin structural genes in 224 enterococcal isolates from fecal origin obtained from humans, pets, wild animals and birds. Direct antimicrobial activity against Listeria monocytogenes CECT4032 was detected in 102 (45.6%) of the tested isolates. From these, only 22 displayed bacteriocin activity against this indicator. The bacteriocinogenic strains contained one or more of the bacteriocin structural genes tested in this study, with those of enterocins P, A and L50 (L50A and L50B) being the most abundant. Our results show a high occurrence of the combination of different bacteriocin structural genes in the enterococcal isolates analyzed, indicating an elevated genetic potential of these strains to produce various bacteriocins.

Different impact of heat-inactivated and viable lactic acid bacteria of aquatic origin on turbot (Scophthalmus maximus L.) head-kidney leucocytes

In aquaculture, several criteria should be considered to select an appropriate probiotic, including the aquatic origin and safety of the strain and its ability to modulate the host immune response. The properties and effects of probiotics are strain-specific and some factors such as viability, dose and duration of diet supplementation may regulate their immunomodulatory activities. In this study, we assessed the in vitro effect of eight heat-inactivated and viable lactic acid bacteria (LAB) of aquatic origin belonging to the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and Weissella on the viability and innate immune response of turbot (Scophthalmus maximus L.) leucocytes. Head-kidney leucocytes were incubated with viable and heat-inactivated LAB at different concentrations. After incubation, the viability of leucocytes was evaluated using colorimetric assays (MTT and LDH) and flow cytometry (annexin V/propidium iodide). Heat-inactivated LAB showed no cytotoxic effect while viable LAB exerted variable influence on apoptosis of turbot phagocytes and lymphocytes. Leucocyte respiratory burst activity and phagocytosis were also differentially activated, as viable LAB stimulated leucocytes more efficiently than the heat-inactivated LAB. Our results suggest diverse strain-specific mechanisms of interaction between the evaluated LAB and turbot leucocytes. Furthermore, our work sets up in vitro systems to evaluate the effect of LAB as potential probiotics, which will be useful to develop efficient screening.

Identification of bacteriocin genes in enterococci isolated from game animals and saltwater fish.

Bacteriocins produced by enterococci, referred to as enterocins, possess great interest for their potential use as biopreservatives in food and feed, as well as alternative antimicrobials in humans and animals. In this context, the aim of the present study was to determine the antimicrobial activity and the presence of bacteriocin structural genes in fecal enterococcal isolates from animal origins. Evaluation of the direct antimicrobial activity of 253 isolates from wild boars (Sus scrofa, n = 69), mullets (Liza ramada, n = 117), and partridges (Perdix perdix, n = 67) against eight indicator bacterial strains (including Listeria monocytogenes, Pediococcus pentosaceus, and Enterococcus spp.) showed that 177 (70%) exerted antimicrobial activity against at least one indicator microorganism. From these isolates, 123 were further selected on the basis of their inhibition group, and 81 were found to be producers of bacteriocins active against Listeria monocytogenes. Analysis of the presence of enterocin structural genes in a subset of 36 isolates showed that 70% harbored one or more of the evaluated genes, those of enterocin P and hiracin JM79 being the most prevalent. These results show that wild animals constitute an appropriate source for the isolation of bacteriocinogenic enterococci.

Bacteriocin production by lactic acid bacteria isolated from fish, seafood and fish products

Analysis of 1245 lactic acid bacteria (LAB) isolates obtained from fish, seafood and fish products showed that 197 exerted direct antimicrobial activity against 20 spoilage and food-borne pathogenic microorganisms. Further evaluation of 64 LAB isolates selected on the basis of their direct antimicrobial activity revealed 25 secreted bacteriocins. Biochemical characterization, PCR analysis and/or DNA sequencing of the superoxide dismutase gene (sodA) and/or 16S rRNA gene (16S rDNA), and/or SDS-PAGE analyses of the 64 selected isolates allowed the identification of 24 of them as Enterococcus faecium, 22 as Enterococcus faecalis, seven as Pediococcus pentosaceus, five as Weissella cibaria, three as Lactobacillus sakei subsp. carnosus, one as L. sakei subsp. sakei, one as Lactobacillus curvatus subsp. curvatus and one as Leuconostoc mesenteroides subsp. cremoris. PCR analyses for the detection of genes encoding previously described bacteriocins performed on the 25 bacteriocinogenic strains showed that 19 strains (18 enterococci and 1 lactobacilli) amplified, at least, one of the tested genes, and up to four and two target genes were simultaneously detected in a single enterococcal and lactobacilli strain, respectively. Moreover, in vitro safety evaluation of E. faecium strains was carried out by detection of potential virulence factors, analysis of hemolysin, gelatinase and protease production, and susceptibility testing to antibiotics of relevance for human and veterinary industry. The results reported herein demonstrate the suitability of fish, seafood and fish products for the isolation of LAB (mainly enterococci), including (multi)bacteriocinogenic strains, encoding bacteriocins active against Listeria monocytogenes and other food-borne pathogens of interest for the food industry.

Characterization of Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss) feed and larvae: safety, DNA fingerprinting, and bacteriocinogenicity

The use of lactic acid bacteria (LAB) as probiotics constitutes an alternative or complementary strategy to chemotherapy and vaccination for disease control in aquaculture. The objectives of this work were (1) the in vitro safety assessment of 8 Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) feed and larvae; (2) the evaluation of their genetic relatedness; (3) the study of their antimicrobial/bacteriocin activity against fish pathogens; and (4) the biochemical and genetic characterization of the bacteriocin produced by the strain displaying the greatest antimicrobial activity. Concerning the safety assessment, none of the pediococci showed antibiotic resistance nor produced hemolysin or gelatinase, degraded gastric mucin, or deconjugated bile salts. Four strains (50%) produced tyramine or putrescine, but the corresponding genes were not amplified by PCR. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting allowed clustering of the pediococci into 2 well-defined groups (68% similarity). From the 8 pediococci displaying direct antimicrobial activity against at least 3 out of 9 fish pathogens, 6 strains (75%) were identified as bacteriocin producers. The bacteriocin produced by P. acidilactici L-14 was purified, and mass spectrometry and DNA sequencing revealed its identity to pediocin PA-1 (PedPA-1). Altogether, our results allowed the identification of 4 (50%) putatively safe pediococci, including 2 bacteriocinogenic strains. ERIC-PCR fingerprinting was a valuable tool for genetic profiling of P. acidilactici strains. This work reports for the first time the characterization of a PedPA-1-producing P. acidilactici strain isolated from an aquatic environment (rainbow trout larvae), which shows interesting properties related to its potential use as a probiotic in aquaculture.