Safety assessment and molecular genetic profiling by pulsed-field gel electrophoresis (PFGE) and PCR-based techniques of Enterococcus faecium strains of food origin

Enterococcus faecium is authorized as animal probiotic in the European Union, but this species has emerged as an important cause of nosocomial infections in humans. We investigated the safety of 14 potential probiotic E. faecium strains with antimicrobial activity, previously isolated from food, following the guidance proposed by EFSA. All the enterococci were susceptible to ampicillin, and none of them harbored the genes encoding the enterococcal surface protein (esp), putative glycosyl hydrolase (hylEfm), and insertion sequence IS16. The genetic relatedness of these enterococci was determined by pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus (ERIC-PCR), and restriction analysis of amplified 16S rDNA (ARDRA). PFGE analysis of SmaI patterns evidenced four subgroups, whereas RAPD and ERIC-PCR analysis gave nine and eight different subgroups, respectively. ERIC-PCR yielded the highest diversity, followed by RAPD and PFGE, while ARDRA achieved the lowest diversity. In conclusion, we demonstrated the absence of well-known enterococcal virulence markers in a collection of E. faecium strains from food, which renders them safe to be used in the food industry or as probiotics in animal production, and that ERIC-PCR is a reliable tool to be used for molecular genetic profiling of potential probiotic enterococci.Ministerio de Economía y CompetitividadComunidad de MadridEuropean Social FundDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaFALSEpu

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.Ministerio de Economía y CompetitividadComunidad de MadridSección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

Evaluation of Safety and Probiotic Traits from a Comprehensive Genome-Based In Silico Analysis of Ligilactobacillus salivarius P1CEA3, Isolated from Pigs and Producer of Nisin S

Author Contributions: Conceptualization, E.M.-A., P.E.H. and J.B.; methodology, E.S., N.P., I.L., P.E.H. and J.B.; investigation, E.S., N.P., I.L., E.M.-A. and J.B.; resources, L.M.C., E.M.-A., P.E.H. and J.B.; data curation, E.S., P.E.H. and J.B.; writing—original draft preparation, E.S.; writing—review and editing, P.E.H. and J.B.; supervision, E.M.-A., P.E.H. and J.B.; project administration, J.B.; funding acquisition, L.M.C., P.E.H. and J.B. All authors have read and agreed to the published version of the manuscript.Ligilactobacillus salivarius is an important member of the porcine gastrointestinal tract (GIT). Some L. salivarius strains are considered to have a beneficial effect on the host by exerting different probiotic properties, including the production of antimicrobial peptides which help maintain a healthy gut microbiota. L. salivarius P1CEA3, a porcine isolated strain, was first selected and identified by its antimicrobial activity against a broad range of pathogenic bacteria due to the production of the novel bacteriocin nisin S. The assembled L. salivarius P1CEA3 genome includes a circular chromosome, a megaplasmid (pMP1CEA3) encoding the nisin S gene cluster, and two small plasmids. A comprehensive genome-based in silico analysis of the L. salivarius P1CEA3 genome reveals the presence of genes related to probiotic features such as bacteriocin synthesis, regulation and production, adhesion and aggregation, the production of lactic acid, amino acids metabolism, vitamin biosynthesis, and tolerance to temperature, acid, bile salts and osmotic and oxidative stress. Furthermore, the strain is absent of risk-related genes for acquired antibiotic resistance traits, virulence factors, toxic metabolites and detrimental metabolic or enzymatic activities. Resistance to common antibiotics and gelatinase and hemolytic activities have been discarded by in vitro experiments. This study identifies several probiotic and safety traits of L. salivarius P1CEA3 and suggests its potential as a promising probiotic in swine production.Ministerio de Ciencia e Innovación (España)Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

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.Ministerio de Economía y CompetitividadComunidad de MadridSección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

Characterization of Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) 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.Ministerio de Economia y CompetitividadComunidad de MadridUniversidad Complutense de MadridFundação para a Ciência e a Tecnologia (FCT, Portugal)European Social FundDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

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.Comisión EuropeaAgência Nacional, PortugalSpanish-Portuguese Integrated ActionMinisterio de Ciencia e InnovaciónMinisterio de Educación, Cultura y DeporteMinisterio de Ciencia y TecnologíaComunidad de MadridDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

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

Background. The 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. Results. These 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. Conclusions. 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 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.Ministerio de Ciencia y Tecnología (AGL2009−08348-ALI)Universidad Complutense de Madrid/Banco Santander-Central Hispano (GR35/10-A)Dirección General de Universidades e Investigación, Consejería de Educación, Comunidad de Madrid (S−2009/AGR−1489)Ministerio de Ciencia e Innovación (HP2008-0070)Sección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer

Author Contributions: Conceptualization, E.M.-A., P.E.H. and J.B.; methodology, I.L., E.S., N.P. and A.C.; investigation, I.L., E.S. and N.P.; resources, A.C., L.M.C., P.E.H. and J.B.; writing—original draft preparation, I.L. writing—review and editing, P.E.H. and J.B.; supervision, E.M.-A. and J.B.; project administration, L.M.C., P.E.H., and J.B.; funding acquisition, L.M.C., P.E.H. and J.B. All authors have read and agreed to the published version of the manuscript.The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively.Ministerio de Ciencia e Innovación (España)Comunidad de MadridDepto. de Nutrición y Ciencia de los AlimentosFac. de VeterinariaTRUEpu

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.Ministerio de Ciencia y TecnologíaMinisterio de Ciencia e InnovaciónComunidad de MadridInstituto Nacional de Investigación y Tecnología Agraria y AlimentariaSección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu

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.Ministerio de Ciencia e InnovaciónMinisterio de Educación, Cultura y DeporteMinisterio de Ciencia y TecnologíaComunidad de MadridEuropean Commission and Agência Nacional from PortugalSección Dptal. de Nutrición y Ciencia de los Alimentos (Veterinaria)Fac. de VeterinariaTRUEpu