Impairment of Listeria monocytogenes biofilm developed on industrial surfaces by Latilactobacillus curvatus CRL1579 bacteriocin.

The effect of lactocin AL705, bacteriocin produced by Latilactobacillus (Lat.) curvatus CRL1579 against Listeria biofilms on stainless steel (SS) and polytetrafluoroethylene (PTFE) coupons at 10 °C was investigated. L. monocytogenes FBUNT showed the greatest adhesion on both surfaces associated to the hydrophobicity of cell surface. Partially purified bacteriocin (800 UA/mL) effectively inhibited L. monocytogenes preformed biofilm through displacement strategy, reducing the pathogen by 5.54 ± 0.26 and 4.74 ± 0.05 log cycles at 3 and 6 days, respectively. The bacteriocin-producer decreased the pathogen biofilm by ∼2.84 log cycles. Control and Bac- treated samples reached cell counts of 7.05 ± 0.18 and 6.79 ± 0.06 log CFU/cm2 after 6 days of incubation. Confocal scanning laser microscopy (CLSM) allowed visualizing the inhibitory effect of lactocin AL705 on L. monocytogenes preformed biofilms under static and hydrodynamic flow conditions. A greater effect of the bacteriocin was found at 3 days independently of the surface matrix and pathogen growth conditions at 10 °C. As a more realistic approach, biofilm displacement strategy under continuous flow conditions showed a significant loss of biomass, mean thickness and substratum coverage of pathogen biofilm. These findings highlight the anti-biofilm capacity of lactocin AL705 and their potential application in food industries.

Bioprotective cultures and bacteriocins as food preservatives.

Food preservation technologies face the challenge of extending product shelf life applying different factors to prevent the microbiological spoilage of food and inhibit/inactivate food borne pathogens maintaining or even enhancing its quality. One such preservation strategy is the application of bacteriocins or bacteriocin-producer cultures as a kind of food biopreservation. Bacteriocins are ribosomally synthesized small polypeptide molecules that exert antagonistic activity against closely related and unrelated bacteria without harming the producing strain by specific immunity proteins. This chapter aims to contribute to current knowledge about innovative natural preservative agents and their application in the food industry. Specifically, its purpose is to analyze the classification of bacteriocins from lactic acid bacteria (LAB), desirable characteristics of bacteriocins that position them in a privileged place in food biopreservation technology, their success story as well as the bacteriocinogenic LAB in various food systems. Finally, challenges and barrier strategies used to enhance the efficiency of the bacteriocins antimicrobial effect are presented in this chapter.

A multi-strain probiotic promoted recovery of puppies from gastroenteritis in a randomized, double-blind, placebo-controlled study.

Objective and animals: Acute diarrhea is among the most common causes of veterinary consultations for dogs. A double-blind, placebocontrolled intervention trial was done with 120 puppies with gastroenteritis. These dogs were 1 to 4 mo old, male and female, of various breeds and sizes. Procedure: Dogs were randomly allocated into 2 groups: Those in the treated group (TG) received a multi-strain probiotic with Lactobacillus johnsonii CRL1693, Ligilactobacillus murinus CRL1695, Limosilactobacillus mucosae CRL1696, and Ligilactobacillus salivarius CRL1702 (1 × 109 CFU/mL) daily for 7 d, whereas those in the control group (CG) received a placebo. All puppies received intravenous fluids, an antiparasitic, amoxicillin PO, and enrofloxacin SC. Results: At the start of the trial, the 2 groups were similar. Probiotic administration for 7 d normalized fecal consistency, with 69, 50, and 80% of small, medium, and large puppies in the TG achieving a fecal score of 1 (separate hard lumps) at 7 d, significantly better than puppies in the CG. After 7 d of treatment, most puppies (70%) in the TG had an excellent recovery, whereas in the CG, recoveries were 35.7% "bad" and 30.4% "fair." Therefore, treatment with probiotics hastened recovery (P < 0.0001). At the end of the trial, there was a significant increase of cultivable lactobacilli in the feces of TG puppies, but no significant differences between the 2 groups in numbers of total mesophylls, enterobacteria, or Gram-positive cocci. Total mortality was 5.8%, including 4 puppies from the CG and 3 from the TG. Conclusion: In a randomized, double-blind, placebo-controlled study, puppies with gastroenteritis symptoms receiving a multi-strain probiotic had rapid improvement, implying beneficial effects on the microbiota and its functionality.

Un probiotique multi-souches a favorisé la guérison des chiots de la gastro-entérite dans une étude randomisée, en double aveugle et vérifiée par placebo. Objectif et animaux: La diarrhée aiguë fait partie des causes les plus fréquentes de consultations vétérinaires pour les chiens. Un essai d'intervention en double aveugle et vérifié par placebo a été réalisé avec 120 chiots atteints de gastro-entérite. Ces chiens étaient âgés de 1 à 4 mois, mâles et femelles, de différentes races et tailles. Procédure: Les chiens ont été répartis au hasard en 2 groupes : ceux du groupe traité (TG) ont reçu un probiotique multisouches contenant Lactobacillus johnsonii CRL1693, Ligilactobacillus murinus CRL1695, Limosilactobacillus mucosae CRL1696 et Ligilactobacillus salivarius CRL1702 (1 × 109 UFC/mL) quotidiennement pendant 7 j, tandis que ceux du groupe témoin (CG) ont reçu un placebo. Tous les chiots ont reçu des liquides intraveineux, un antiparasitaire, de l'amoxicilline PO et de l'enrofloxacine SC. Résultats: Au début de l'essai, les 2 groupes étaient similaires. L'administration de probiotiques pour une durée de 7 j a normalisé la consistance fécale, avec 69, 50 et 80 % des chiots petits, moyens et grands dans le TG obtenant un score fécal de 1 (morceaux durs séparés) à 7 jours, ce qui était significativement meilleur que les chiots dans le CG. Après 7 jours de traitement, la plupart des chiots (70 %) dans le TG ont eu une excellente récupération, alors que dans le CG, les récupérations étaient de 35,7 % « mauvaises ¼ et 30,4 % « passables ¼. Par conséquent, le traitement avec des probiotiques a accéléré la récupération (P < 0,0001). À la fin de l'essai, il y avait une augmentation significative des lactobacilles cultivables dans les fèces des chiots TG, mais aucune différence significative entre les 2 groupes en nombre de mésophylles totaux, d'entérobactéries ou de coques à Gram positif. La mortalité totale était de 5,8 %, dont 4 chiots du CG et 3 du TG. Conclusion: Dans une étude randomisée, en double aveugle et vérifiée par placebo, des chiots présentant des symptômes de gastro-entérite recevant un probiotique multi-souches ont présenté une amélioration rapide, impliquant des effets bénéfiques sur le microbiote et sa fonctionnalité.(Traduit par Dr Serge Messier).

Identification and characterization of potential probiotic lactic acid bacteria isolated from pig feces at various production stages.

Lactic acid bacteria (LAB) were isolated, identified, and characterized from pig feces at various growth stages and feed rations in order to be used as probiotic feed additives. Lactic acid bacteria numbers ranged from 7.10 ± 1.50 to 9.40 log CFUs/g for growing and lactating pigs, respectively. Isolates (n = 230) were identified by (GTG)5-polymerase chain reaction and partial sequence analysis of 16S rRNA. Major LAB populations were Limosilactobacillus reuteri (49.2%), Pediococcus pentosaceus (20%), Lactobacillus amylovorus (11.4%), and L. johnsonii (8.7%). In-vitro assays were performed, including surface characterization and tolerance to acid and bile salts. Several lactobacilli exhibited hydrophobic and aggregative characteristics and were able to withstand gastrointestinal tract conditions. In addition, lactobacilli showed starch- and phytate-degrading ability, as well as antagonistic activity against Gram-negative pathogens and the production of bacteriocin-like inhibitory substances. When resistance or susceptibility to antibiotics was evaluated, high phenotypic resistance to ampicillin, gentamicin, kanamycin, streptomycin, and tetracycline and susceptibility towards clindamycin and chloramphenicol was observed in the assayed LAB. Genotypic characterization showed that 5 out of 15 resistance genes were identified in lactobacilli; their presence did not correlate with phenotypic traits. Genes erm(B), strA, strB, and aadE conferring resistance to erythromycin and streptomycin were reported among all lactobacilli, whereas tet(M) gene was harbored by L. reuteri and L. amylovorus strains. Based on these results, 6 probiotic LAB strains (L. reuteri F207R/G9R/B66R, L. amylovorus G636T/S244T, and L. johnsonii S92R) can be selected to explore their potential as direct feed additives to promote swine health and replace antibiotics.

Des bactéries lactiques (LAB) ont été isolées, identifiées et caractérisées à partir de matières fécales de porc à différents stades de croissance et de rations alimentaires afin d'être utilisées comme additifs alimentaires probiotiques. Le nombre de bactéries lactiques variait de 7,10 ± 1,50 à 9,40 log UFC/g pour les porcs en croissance et en lactation, respectivement. Les isolats (n = 230) ont été identifiés par réaction d'amplification en chaîne par la (GTG)5-polymérase et analyse partielle de la séquence de l'ARNr 16S. Les principales populations de LAB étaient Limosilactobacillus reuteri (49,2 %), Pediococcus pentosaceus (20 %), Lactobacillus amylovorus (11,4 %) et L. johnsonii (8,7 %). Des tests in vitro ont été effectués, y compris la caractérisation de surface et la tolérance aux acides et aux sels biliaires. Plusieurs lactobacilles présentaient des caractéristiques hydrophobes et agrégatives et étaient capables de résister aux conditions du tractus gastro-intestinal. De plus, les lactobacilles ont montré une capacité de dégradation de l'amidon et des phytates, ainsi qu'une activité antagoniste contre les agents pathogènes à Gram négatif et la production de substances inhibitrices de type bactériocine. Lorsque la résistance ou la sensibilité aux antibiotiques a été évaluée, une résistance phénotypique élevée à l'ampicilline, à la gentamicine, à la kanamycine, à la streptomycine et à la tétracycline et une sensibilité à la clindamycine et au chloramphénicol ont été observées dans les LAB testés. La caractérisation génotypique a montré que cinq gènes de résistance sur 15 ont été identifiés dans les lactobacilles; leur présence n'était pas corrélée aux traits phénotypiques. Les gènes erm(B), strA, strB et aadE conférant une résistance à l'érythromycine et à la streptomycine ont été signalés parmi tous les lactobacilles, tandis que le gène tet(M) était hébergé par les souches L. reuteri et L. amylovorus. Sur la base de ces résultats, six souches probiotiques LAB (L. reuteri F207R/G9R/B66R, L. amylovorus G636T/S244T et L. johnsonii S92R) peuvent être sélectionnées pour explorer leur potentiel en tant qu'additifs alimentaires directs pour promouvoir la santé des porcs et remplacer les antibiotiques.(Traduit par Docteur Serge Messier).

Effect of probiotic lactobacilli supplementation on growth parameters, blood profile, productive performance, and fecal microbiology in feedlot cattle.

Lactic acid bacteria (LAB) selected on the basis of probiotic characteristics were administered to beef feedlot catlle and the effect on body condition/growth and nutritional-metabolic status as well as on E. coli O157:H7 fecal shedding, were investigated. A feeding trials involving 126 steers were used to evaluate the effects of Lactobacillus acidophilus CRL2074, Limosilactobacillus fermentum CRL2085 and Limosilactobacillus mucosae CRL2069 and their combinations (5 different probiotic groups and control) when 107-108 CFU/animal of each probiotic group were in-feed supplemented. Cattle were fed a high energy corn-based diet (16 to 88%) and samples from each animal were taken at 0, 40, 104 and 163 days. In general, animals body condition and sensorium state showed optimal muscle-skeletal development and behavioral adaption to confinement; no nasal/eye discharges and diarrheic feces were observed. The nutritional performance of the steers revealed a steady increase of biometric parameters and weight. Animals supplied with L. mucosae CRL2069 for 104 days reached the maximum mean live weight (343.2 kg), whereas the greatest weight daily gain (1.27 ± 0.16 Kg/day) was obtained when CRL2069 and its combination with L. fermentum CRL2085 (1.26 ± 0.11 kg/day) were administered during the complete fattening cycle. With several exceptions, bovine cattle blood and serum parameters showed values within referential ranges. As a preharvest strategy to reduce Escherichia coli O157:H7 in cattle feces, CRL2085 administered during 40 days decreased pathogen shedding with a reduction of 43% during the feeding period. L. fermentum CRL2085 and L. mucosae CRL2069 show promise for feedlot cattle feeding supplementation to improve metabolic-nutritional status, overall productive performance and to reduce E. coli O157:H7 shedding, thus decreasing contamination chances of meat food products.

Bioprotective Lactic Acid Bacteria and Lactic Acid as a Sustainable Strategy to Combat Escherichia coli O157:H7 in Meat.

Human infection by Enterohemorrhagic Escherichia coli (EHEC) constitutes a serious threat to public health and a major concern for the meat industry. Presently, consumers require safer/healthier foods with minimal chemical additives, highlighting the need for sustainable solutions to limit and prevent risks. This work evaluated the ability of two antagonistic lactic acid bacteria (LAB) strains, Lactiplantibacillus plantarum CRL681 and Enterococcus mundtii CRL35, and their combination in order to inhibit EHEC in beef (ground and vacuum sealed meat discs) at 8 °C during 72 h. The effect of lower lactic acid (LA) concentrations was evaluated. Meat color was studied along with how LAB strains interfere with the adhesion of Escherichia coli to meat. The results indicated a bacteriostatic effect on EHEC cells when mixed LAB strains were inoculated. However, a bactericidal action due to a synergism between 0.6% LA and LAB occurred, producing undetectable pathogenic cells at 72 h. Color parameters (a*, b* and L*) did not vary in bioprotected meat discs, but they were significantly modified in ground meat after 24 h. In addition, LAB strains hindered EHEC adhesion to meat. The use of both LAB strains plus 0.6% LA, represents a novel, effective and ecofriendly strategy to inactivate EHEC in meat.

Flavonoids and Phenylethylamides Are Pivotal Factors Affecting the Antimicrobial Properties of Stingless Bee Honey.

Despite the recent approval of stingless bee honey to the Argentine Food Code, there are still many gaps in information. Likely, the main reason for this is that multiple ecological and chemical factors influence their production and antimicrobial properties. This work combined metabolomic, microbiological, and physicochemical analyses to characterize the honey ofTetragonisca fiebrigifrom Northeastern Argentina. The antimicrobial activity tests showed that honey samples (n = 24) inhibited some Gram-positive and Gram-negative bacteria at different sensitivity levels. Furthermore, samples selected for their high bioactivity revealed crystallizations, a positive correlation with fungal growth, and the presence of flavonoids. The major polyphenols annotated by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis and supported by metabolomic tools were quercetin 3,4'-dimethyl ether, pachypodol, jaceoside, irigenin trimethyl ether, corymboside, chrysoeriol 7-neohesperidoside, and corymboside. In contrast, samples missing antimicrobial activity did not crystallize, lacked flavonoids, and were enriched in phenylethylamides. Based on these findings, we discuss the significance of flavonoids and phenylethylamides on honey's antimicrobial activity and food quality and how they may indeed reflect essential parameters of the hive, such as microbial balance and eubiosis.

Administration of probiotic lactic acid bacteria to modulate fecal microbiome in feedlot cattle.

Modulation of animal gut microbiota is a prominent function of probiotics to improve the health and performance of livestock. In this study, a large-scale survey to evaluate the effect of lactic acid bacteria probiotics on shaping the fecal bacterial community structure of feedlot cattle during three experimental periods of the fattening cycle (163 days) was performed. A commercial feedlot located in northwestern Argentina was enrolled with cattle fed mixed rations (forage and increasing grain diet) and a convenience-experimental design was conducted. A pen (n = 21 animals) was assigned to each experimental group that received probiotics during three different periods. Groups of n = 7 animals were sampled at 40, 104 and 163 days and these samples were then pooled to one, thus giving a total of 34 samples that were subjected to high-throughput sequencing. The microbial diversity of fecal samples was significantly affected (p < 0.05) by the administration period compared with probiotic group supplementation. Even though, the three experimental periods of probiotic administration induced changes in the relative abundance of the most representative bacterial communities, the fecal microbiome of samples was dominated by the Firmicutes (72-98%) and Actinobacteria (0.8-27%) phyla, while a lower abundance of Bacteroidetes (0.08-4.2%) was present. Probiotics were able to modulate the fecal microbiota with a convergence of Clostridiaceae, Lachnospiraceae, Ruminococcaceae and Bifidobacteriaceae associated with health and growth benefits as core microbiome members. Metabolic functional prediction comparing three experimental administration periods (40, 104 and 163 days) showed an enrichment of metabolic pathways related to complex plant-derived polysaccharide digestion as well as amino acids and derivatives during the first 40 days of probiotic supplementation. Genomic-based knowledge on the benefits of autochthonous probiotics on cattle gastrointestinal tract (GIT) microbiota composition and functions will contribute to their selection as antibiotic alternatives for commercial feedlot.

Inhibition of a spoilage exopolysaccharide producer by bioprotective extracts from Lactobacillus acidophilus CRL641 and Latilactobacillus curvatus CRL705 in vacuum-packaged refrigerated meat discs.

The effect of bioprotective extracts (BEs) from Lactobacillus acidophilus CRL641 (BE-1) and Latilactobacillus curvatus CRL705 (BE-2) against the exopolysaccharide producer Latilactobacillus sakei CRL1407 in vacuum-packaged meat discs at 4 °C was evaluated. Lat. sakei CRL1407 was able to grow in control samples from 2.80 to 7.77 log CFU/g after 38 days. BE-1 and BE-2 reduced bacterial growth by 2.11 and 1.35 log CFU/g, respectively, but their combination led to a greater growth reduction (3.31 log CFU/g). The antimicrobial activity was detected in treated samples with BE-1 and BE-1 + BE-2 until day 16, while with BE-2 only at the initial time. The pH values remained constant in the discs treated with the BEs combination, whereas the greatest drop in pH was observed in control samples. The minor lipid oxidation without perceptible color changes was detected in the presence of BE-1 and BE-1 + BE-2. The combination of BEs as biocontrol agent plus conventional preservation barriers could extend the fresh meat shelf-life without quality loss.

Bioprotective extracts from Lactobacillus acidophilus CRL641 and Latilactobacillus curvatus CRL705 inhibit a spoilage exopolysaccharide producer in a refrigerated meat system.

The effect of bioprotective extracts (BEs) from Latilactobacillus curvatus CRL705 and Lactobacillus acidophilus CRL641 against Latilactobacillus sakei CRL1407 was evaluated in a refrigerated meat model system under vacuum and aerobic conditions at 4 and 10 °C. As shown by culturing, the BE-1 from L. acidophilus completely inhibited the spoilage strain, while that from Lat. Curvatus CRL705 (BE-2) and its combination with BE-1 exerted a bacteriostatic effect. The antimicrobial activity and exopolysaccharide production correlated with the efficacy of inhibitory treatment while final pH decrease was higher in control samples. When flow cytometry was applied, a lack of correlation with plate counting was found; counts under the detection limit for BE-1 at 21 and 28 days at 4 and 10 °C represented between 64.15 and 73.70% of dead cells. Thus, the concurrence of lactic acid bacteria as biocontrol agents and the use of more accurate tools to prevent the growth of deteriorating species will contribute to the extension of fresh meat shelf-life without quality loss.

Proteomic Analysis of Listeria monocytogenes FBUNT During Biofilm Formation at 10°C in Response to Lactocin AL705.

Listeria monocytogenes is one of the major food-related pathogens and is able to survive and multiply under different stress conditions. Its persistence in industrial premises and foods is partially due to its ability to form biofilm. Thus, as a natural strategy to overcome L. monocytogenes biofilm formation, the treatment with lactocin AL705 using a sublethal dose (20AU/ml) was explored. The effect of the presence of the bacteriocin on the biofilm formation at 10°C of L. monocytogenes FBUNT was evaluated for its proteome and compared to the proteomes of planktonic and sessile cells grown at 10°C in the absence of lactocin. Compared to planktonic cells, adaptation of sessile cells during cold stress involved protein abundance shifts associated with ribosomes function and biogenesis, cell membrane functionality, carbohydrate and amino acid metabolism, and transport. When sessile cells were treated with lactocin AL705, proteins' up-regulation were mostly related to carbohydrate metabolism and nutrient transport in an attempt to compensate for impaired energy generation caused by bacteriocin interacting with the cytoplasmic membrane. Notably, transport systems such as ß-glucosidase IIABC (lmo0027), cellobiose (lmo2763), and trehalose (lmo1255) specific PTS proteins were highly overexpressed. In addition, mannose (lmo0098), a specific PTS protein indicating the adaptive response of sessile cells to the bacteriocin, was downregulated as this PTS system acts as a class IIa bacteriocin receptor. A sublethal dose of lactocin AL705 was able to reduce the biofilm formation in L. monocytogenes FBUNT and this bacteriocin induced adaptation mechanisms in treated sessile cells. These results constitute valuable data related to specific proteins targeting the control of L. monocytogenes biofilm upon bacteriocin treatment.

Hydrolysis of raw fish proteins extracts by Carnobacterium maltaromaticum strains isolated from Argentinean freshwater fish.

Lactic acid bacteria (LAB) isolated from freshwater fish (hatcheries and captures) from Paraná river (Argentina) were analyzed by using culture-dependent approaches. The species belonging to Carnobacterium (C.) divergens, C. inhibens, C. maltaromaticum, C. viridans and Vagococcus (V.) salmoninarum were identify as predominant by RAPD-PCR and 16 s rRNA gene sequencing. C. maltaromaticum (H-17, S-30, B-42 and S-44) grew in raw fish extract and slightly reduced the medium pH (5.81-5.91). These strains exhibited moderate fish sarcoplasmic protein degradation (≤ 73 %) releasing small peptides and free amino acids, being alanine, glycine, asparagine and arginine concentrations increased in a higher extent (17.84, 1.47, 1.26 and 0.47 mg/100 mL, respectively) by S-44 strain at 96 h incubation. Interestingly C. maltaromaticum H-17 was able to inhibit Listeria monocytogenes. Results suggest that these strains would contribute to the development of new safe and healthy fishery products with improved nutritional and sensory characteristics.

The Ability of Riboflavin-Overproducing Lactiplantibacillus plantarum Strains to Survive Under Gastrointestinal Conditions.

Riboflavin, vitamin B2, is essential for humans and has to be obtained from the diet. Some lactic acid bacteria (LAB) produce this vitamin, and they can be used for in-situ fortification of foods. This could be an alternative to supplementation with chemically synthesized vitamin, to palliate riboflavin deficiencies in specific groups of people. Moreover, if the producing LAB could survive in the gastrointestinal stress (GIT) they could be added as probiotics in this environment. In the present study we tested two riboflavin-overproducing Lactiplantibacillus plantarum strains (M5MA1-B2 and M9MG6-B2), spontaneous mutants of LAB isolated from chicha, a traditional Andean beverage. These two LAB, and also their isogenic strains M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12], expressing the mCherry protein from the pRCR12 plasmid, were evaluated in vitro under simulated GIT conditions. Among other, specifically developed protein fluorescence assays were used. The four LAB showed similar levels of adhesion (>6.0%) to Caco-2 cells, higher than that of the probiotic Lacticaseibacillus rhamnosus GG strain (4.51%). Thus, LAB biofilm formation was assessed in the labeled cells by intracellular mCherry fluorescence and in the unlabeled parental strains by crystal violet staining. Both methods detected the formation of consistent biofilms by the L. plantarum strains. The quantification of mCherry fluorescence was also used to analyze LAB auto-aggregation properties. High levels of auto-aggregation were detected for both M5MA1-B2[pRCR12] and M9MG6-B2[pRCR12]. Survival of LAB included in a commercial cereal-based food matrix (Incaparina) under GIT conditions was also evaluated. The four LAB were resistant in vitro to the stomach and intestinal stresses, and proliferated in this environment, indicating a protective and nutritional effect of the Incaparina on the bacteria. Also, M9MG6-B2 survival in the presence or absence of Incaparina was evaluated in vivo in a BALB/c mouse model. The administration of the M9MG6-B2 strain alone or together with Incaparina had no adverse effect on the health, growth and/or well-being of the rodents. In addition, an increment in the villus length/crypt depth ratio was observed. The overall results obtained indicate that the LAB studied have probiotic characteristics of interest for the development of functional foods.

Erratum: Fukuyama, K., et al. Evaluation of the Immunomodulatory Ability of Lactic Acid Bacteria Isolated from Feedlot Cattle Against Mastitis Using a Bovine Mammary Epithelial Cells In Vitro Assay. Pathogens 2020, 9, 410.

The authors would like to make the following corrections about the published paper [...].

Evaluation of the Immunomodulatory Ability of Lactic Acid Bacteria Isolated from Feedlot Cattle Against Mastitis Using a Bovine Mammary Epithelial Cells In Vitro Assay.

Bovine mastitis, the inflammation of the mammary gland, affects the quality and quantity of milk yield. Mastitis control relies on single or multiple combinations of antibiotic therapy. Due to increasing antibiotic resistance in pathogens, the intramammary infusion of lactic acid bacteria (LAB) has been considered as a potential alternative to antibiotics for treating and preventing bovine mastitis through the improvement of the host immunity. Probiotic effects are a strain-dependent characteristic; therefore, candidate LAB strains have to be evaluated efficiently to find out the ones with the best potential. Here, we investigated LAB strains originally isolated from feedlot cattle's environment regarding their ability in inducing the Toll-like receptor (TLR)-triggered inflammatory responses in bovine mammary epithelial (BME) cells in vitro. The BME cells were pre-stimulated with the LAB strains individually for 12, 24, and 48 h and then challenged with Escherichia coli-derived lipopolysaccharide (LPS) for 12 h. The mRNA expression of selected immune genes-interleukin 1 alpha (IL-1α), IL-1ß, monocyte chemotactic protein 1 (MCP-1), IL-8, chemokine (C-X-C motif) ligand 2 (CXCL2), and CXCL3 were quantified by real-time quantitative PCR (RT-qPCR). Results indicated that pretreatment with some Lactobacillus strains were able to differentially regulate the LPS inflammatory response in BME cells; however, strain-dependent differences were found. The most remarkable effects were found for Lactobacillus acidophilus CRL2074, which reduced the expression of IL-1α, IL-1ß, MCP-1, IL-8, and CXCL3, whereas Lactobacillus rhamnosus CRL2084 diminished IL-1ß, MCP-1, and IL-8 expression. The pre-stimulation of BME cells with the CRL2074 strain resulted in the upregulated expression of three negative regulators of the TLRs, including the ubiquitin-editing enzyme A20 (also called tumor necrosis factor alpha-induced protein 3, TNFAIP3), single immunoglobin IL-1 single receptor (SIGIRR), and Toll interacting protein (Tollip) after the LPS challenge. The CRL2084 pre-stimulation upregulated only Tollip expression. Our results demonstrated that the L. acidophilus CRL2074 strain possess remarkable immunomodulatory abilities against LPS-induced inflammation in BME cells. This Lactobacillus strain could be used as candidate for in vivo testing due to its beneficial effects in bovine mastitis through intramammary infusion. Our findings also suggest that the BME cells immunoassay system could be of value for the in vitro evaluation of the immunomodulatory abilities of LAB against the inflammation resulting from the intramammary infection with mastitis-related pathogens.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented chia sourdough.

Chia, is a gluten-free, rich in proteins, oilseed that is "on trend" as an alternative ingredient in food production, adding nutritional value. As a reservoir of natural biodiversity, lactic acid bacteria development, during spontaneous chia flour fermentation (sourdough) for 10 days, were investigated by culturing and high throughput sequencing (HTS). Culture-dependent analysis showed a rapid increase in total LAB numbers from the second day of sourdough refreshment. Taxonomical identification of LAB isolates by rep-PCR and further 16S rRNA sequencing was performed. Besides Among identified LAB by culture-dependent approach, species from genus Enterococcus were the most abundant; Lactococcus (Lc. lactis), Lactobacillus (L. rhamnosus) and Weissella (W. cibaria) species were also isolated. By HTS, twelve OTUs belonging to LAB genera were identified during chia sourdough fermentation with an increased Lactobacillus diversity. Enterococcus (E.) faecium, E. mundtii, W. cibaria and L. rhamnosus were detected as dominant species in the final propagation stages while Bacillus and Clostridium were mostly present during first fermentation stages. The investigation of biotechnological and safety traits (acidification ability, protein hydrolysis, exopolysaccharides production, antimicrobial activity and antibiotic resistance) of 15 representative LAB strains was performed. Strains characterization led to the selection of Lc. lactis CH179, L. rhamnosus CH34 and W. cibaria CH28 as candidates to be used as novel functional starter culture for gluten-free chia fermented products. As far as we know, this is the first study providing information on the molecular inventory of LAB population during spontaneous fermentation of chia sourdough.

Physiological and proteomic response of Escherichia coli O157:H7 to a bioprotective lactic acid bacterium in a meat environment.

The enterohemorrhagic Escherichia (E.) coli (EHEC) is a pathogen of great concern for public health and the meat industry all over the world. The high economic losses in meat industry and the high costs of the illness highlight the necessity of additional efforts to control this pathogen. Previous studies have demonstrated the inhibitory activity of Enterococcus mundtii CRL35 towards EHEC, showing a specific proteomic response during the co-culture. In the present work, additional studies of the EHEC-Ent. mundtii interaction were carried out: i) differential protein expression of E. coli O157:H7 NCTC12900 growing in co-culture with Ent. mundtii in a meat environment, ii) the reciprocal influence between these two microorganisms in the adhesion to extracellular matrix (ECM) proteins and iii) the possible induction of the phage W933, coding for Shiga toxin (Stx1), by Ent. mundtii CRL35. Proteomic analysis showed a significant repression of a number of E. coli NCTC12900 proteins in co-culture respect to its single culture, these mostly related to the metabolism and transport of amino acids and nucleotides. On the other hand, statistically significant overexpression of EHEC proteins involved in stress, energy production, amino acid metabolism and transcription was observed at 30 h respect to 6 h when EHEC grew in co-culture. Data are available via ProteomeXchange with identifier PXD014588. Besides, EHEC showed a decreased adhesion capacity to ECM proteins in the presence of the bioprotective strain. Finally, Ent. mundtii CRL35 did not induce the lytic cycle of W933 bacteriophage, thus indicating its potential safe use for eliminating this pathogen. Overall, this study expands the knowledge of EHEC- Ent. mundtii CRL35 interaction in a meat environment, which will certainly contribute to find out effective biological strategies to eliminate this pathogen.

Mechanistically Inspired Kinetic Approach to Describe Interactions During Co-Culture Growth of Carnobacterium maltaromaticum and Listeria monocytogenes.

Lactic acid bacteria and Listeria monocytogenes are psychotropic organisms that can grow and compete in food such as lightly preserved fishery products. Predictive microbiology is nowadays one of the leading tools to assess the behavior of bacteria in food and to predict food spoilage. Mathematical models can be used to predict the growth, inactivation or growth probability of bacteria. Currently, the efforts in microbial modeling are oriented towards extrapolation of results beyond experiments in order to predict the growth of interacting microorganisms and develop new food preservation processes. In the present work, a model combining both heterogeneous population and quasi-chemical approaches to describe the different phases of the bacterial growth curve is presented. The model was applied to both monoculture and co-culture cases of lactic acid bacteria, Carnobacterium maltaromaticum H-17, and two Listeria monocytogenes strains in a raw fish extract. It is a highlight that our model includes novel inhibition reactions due to the accumulation of metabolites, and a general equation to take into account the effect of chemical compounds during the lag or physiological adaptation phase of the cells. Our results show that the proposed model can accurately describe the experimental data when the curve shape is a sigmoid, and when it presents a maximum. Besides, the parameters have biological interpretability since the model is mechanistically inspired.