Cultivable epiphytic bacteria of the Chlorophyta Ulva sp.: diversity, antibacterial, and biofilm-modulating activities.

AIMS: Macroalgae harbor a rich epiphytic microbiota that plays a crucial role in algal morphogenesis and defense mechanisms. This study aims to isolate epiphytic cultivable microbiota from Ulva sp. surfaces. Various culture media were employed to evaluate a wide range of cultivable microbiota. Our objective was to assess the antibacterial and biofilm-modulating activities of supernatants from isolated bacteria. METHODS AND RESULTS: Sixty-nine bacterial isolates from Ulva sp. were identified based on 16S rRNA gene sequencing. Their antibacterial activity and biofilm modulation potential were screened against three target marine bacteria: 45%, mostly affiliated with Gammaproteobacteria and mainly grown on diluted R2A medium (R2Ad), showed strong antibacterial activity, while 18% had a significant impact on biofilm modulation. Molecular network analysis was carried out on four bioactive bacterial supernatants, revealing new molecules potentially responsible for their activities. CONCLUSION: R2Ad offered the greatest diversity and proportion of active isolates. The molecular network approach holds promise for both identifying bacterial isolates based on their molecular production and characterizing antibacterial and biofilm-modulating activities.

Anti-Biofilm Extracts and Molecules from the Marine Environment.

Pathogenic bacteria and their biofilms are involved in many diseases and represent a major public health problem, including the development of antibiotic resistance. These biofilms are known to cause chronic infections for which conventional antibiotic treatments are often ineffective. The search for new molecules and innovative solutions to combat these pathogens and their biofilms has therefore become an urgent need. The use of molecules with anti-biofilm activity would be a potential solution to these problems. The marine world is rich in micro- and macro-organisms capable of producing secondary metabolites with original skeletons. An interest in the chemical strategies used by some of these organisms to regulate and/or protect themselves against pathogenic bacteria and their biofilms could lead to the development of bioinspired, eco-responsible solutions. Through this original review, we listed and sorted the various molecules and extracts from marine organisms that have been described in the literature as having strictly anti-biofilm activity, without bactericidal activity.

Microbiota of the Digestive Glands and Extrapallial Fluids of Clams Evolve Differently Over Time Depending on the Intertidal Position.

The Manila clam (Ruditapes philippinarum) is the second most exploited bivalve in the world but remains threatened by diseases and global changes. Their associated microbiota play a key role in their fitness and acclimation capacities. This study aimed at better understanding the behavior of clam digestive glands and extrapallial fluids microbiota at small, but contrasting spatial and temporal scales. Results showed that environmental variations impacted clam microbiota differently according to the considered tissue. Each clam tissue presented its own microbiota and showed different dynamics according to the intertidal position and sampling period. Extrapallial fluids microbiota was modified more rapidly than digestive glands microbiota, for clams placed on the upper and lower intertidal position, respectively. Clam tissues could be considered as different microhabitats for bacteria as they presented different responses to small-scale temporal and spatial variabilities in natural conditions. These differences underlined a more stringent environmental filter capacity of the digestive glands.

Screening for antimicrobial and proteolytic activities of lactic acid bacteria isolated from cow, buffalo and goat milk and cheeses marketed in the southeast region of Brazil.

Lactic acid bacteria (LAB) can be isolated from different sources such as milk and cheese, and the lipolytic, proteolytic and glycolytic enzymes of LAB are important in cheese preservation and in flavour production. Moreover, LAB produce several antimicrobial compounds which make these bacteria interesting for food biopreservation. These characteristics stimulate the search of new strains with technological potential. From 156 milk and cheese samples from cow, buffalo and goat, 815 isolates were obtained on selective agars for LAB. Pure cultures were evaluated for antimicrobial activities by agar antagonism tests and for proteolytic activity on milk proteins by cultivation on agar plates. The most proteolytic isolates were also tested by cultivation in skim milk followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the fermented milk. Among the 815 tested isolates, three of them identified as Streptococcus uberis (strains FT86, FT126 and FT190) were bacteriocin producers, whereas four other ones identified as Weissella confusa FT424, W. hellenica FT476, Leuconostoc citreum FT671 and Lactobacillus plantarum FT723 showed high antifungal activity in preliminary assays. Complementary analyses showed that the most antifungal strain was L. plantarum FT723 that inhibited Penicillium expansum in modified MRS agar (De Man, Rogosa, Sharpe, without acetate) and fermented milk model, however no inhibition was observed against Yarrowia lipolytica. The proteolytic capacities of three highly proteolytic isolates identified as Enterococcus faecalis (strains FT132 and FT522) and Lactobacillus paracasei FT700 were confirmed by SDS-PAGE, as visualized by the digestion of caseins and whey proteins (ß-lactoglobulin and α-lactalbumin). These results suggest potential applications of these isolates or their activities (proteolytic activity or production of antimicrobials) in dairy foods production.

Characterization of the antifungal activity of Lactobacillus harbinensis K.V9.3.1Np and Lactobacillus rhamnosus K.C8.3.1I in yogurt.

Few antifungal protective cultures adapted to fermented dairy products are commercially available because of the numerous constraints linked to their market implementation. Consumer's demand for naturally preserved food products is growing and the utilization of lactic acid bacteria is a promising way to achieve this goal. In this study, using a 2(5-1) factorial fractional design, we first evaluated the effects of fermentation time, of initial sucrose concentration and of the initial contamination amount of a spoilage yeast, on antifungal activities of single and mixed cultures of Lactobacillus rhamnosus K.C8.3.1I and Lactobacillus harbinensis K.V9.3.1Np in yogurt. L. harbinensis K.V9.3.1Np, the most relevant strain with regard to antifungal activity was then studied to determine its minimal inhibitory inoculation rate, its antifungal stability during storage and its impact on yogurt organoleptic properties. We showed that L. harbinensis K.V9.3.1Np maintained a stable antifungal activity over time, which was not affected by initial sucrose, nor by a reduction of the fermentation time. This inhibitory activity was an all-or-nothing phenomenon. Once L. harbinensis K.V9.3.1Np reached a population of ∼ 2.5 × 10(6) cfu/g of yogurt at the time of contamination, total inhibition of the yeast was achieved. We also showed that an inoculation rate of 5 × 10(6) cfu/ml in milk had no detrimental effect on yogurt organoleptic properties. In conclusion, L. harbinensis K.V9.3.1Np is a promising antifungal bioprotective strain for yogurt preservation.

Screening of Lactobacillus spp. for the prevention of Pseudomonas aeruginosa pulmonary infections.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that significantly increases morbidity and mortality in nosocomial infections and cystic fibrosis patients. Its pathogenicity especially relies on the production of virulence factors or resistances to many antibiotics. Since multiplication of antibiotic resistance can lead to therapeutic impasses, it becomes necessary to develop new tools for fighting P. aeruginosa infections. The use of probiotics is one of the ways currently being explored. Probiotics are microorganisms that exert a positive effect on the host's health and some of them are known to possess antibacterial activities. Since most of their effects have been shown in the digestive tract, experimental data compatible with the respiratory environment are strongly needed. The main goal of this study was then to test the capacity of lactobacilli to inhibit major virulence factors (elastolytic activity and biofilm formation) associated with P. aeruginosa pathogenicity. RESULTS: Sixty-seven lactobacilli were isolated from the oral cavities of healthy volunteers. These isolates together with 20 lactobacilli isolated from raw milks, were tested for their capacity to decrease biofilm formation and activity of the elastase produced by P. aeruginosa PAO1. Ten isolates, particularly efficient, were accurately identified using a polyphasic approach (API 50 CHL, mass-spectrometry and 16S/rpoA/pheS genes sequencing) and typed by pulsed-field gel electrophoresis (PFGE). The 8 remaining strains belonging to the L. fermentum (6), L. zeae (1) and L. paracasei (1) species were sensitive to all antibiotics tested with the exception of the intrinsic resistance to vancomycin. The strains were all able to grow in artificial saliva. CONCLUSION: Eight strains belonging to L. fermentum, L. zeae and L. paracasei species harbouring anti-elastase and anti-biofilm properties are potential probiotics for fighting P. aeruginosa pulmonary infections. However, further studies are needed in order to test their innocuity and their capacity to behave such as an oropharyngeal barrier against Pseudomonas aeruginosa colonisation in vivo.

Extracts from Wallis Sponges Inhibit Vibrio harveyi Biofilm Formation.

Pathogenic bacteria and their biofilms are involved in many human and animal diseases and are a major public health problem with, among other things, the development of antibiotic resistance. These biofilms are known to induce chronic infections for which classical treatments using antibiotic therapy are often ineffective. Sponges are sessile filter-feeding marine organisms known for their dynamic symbiotic partnerships with diverse microorganisms and their production of numerous metabolites of interest. In this study, we investigated the antibiofilm efficacy of different extracts from sponges, isolated in Wallis, without biocidal activity. Out of the 47 tested extracts, from 28 different genera, 11 showed a strong activity against Vibrio harveyi biofilm formation. Moreover, one of these extracts also inhibited two quorum-sensing pathways of V. harveyi.

Mechanically Enhanced Salmo salar Gelatin by Enzymatic Cross-linking: Premise of a Bioinspired Material for Food Packaging, Cosmetics, and Biomedical Applications.

Marine animal by-products of the food industry are a great source of valuable biomolecules. Skins and bones are rich in collagen, a protein with various applications in food, cosmetic, healthcare, and medical industries in its native form or partially hydrolyzed (gelatin). Salmon gelatin is a candidate of interest due to its high biomass production available through salmon consumption, its biodegradability, and its high biocompatibility. However, its low mechanical and thermal properties can be an obstacle for various applications requiring cohesive material. Thus, gelatin modification by cross-linking is necessary. Enzymatic cross-linking by microbial transglutaminase (MTG) is preferred to chemical cross-linking to avoid the formation of potentially cytotoxic residues. In this work, the potential of salmon skin gelatin was investigated, in a comparative study with porcine gelatin, and an enzymatic versus chemical cross-linking analysis. For this purpose, the two cross-linking methods were applied to produce three-dimensional, porous, and mechanically reinforced hydrogels and sponges with different MTG ratios (2%, 5%, and 10% w/w gelatin). Their biochemical, rheological, and structural properties were characterized, as well as the stability of the material, including the degree of syneresis and the water-binding capacity. The results showed that gelatin enzymatically cross-linked produced material with high cross-linking densities over 70% of free amines. The MTG addition seemed to play a crucial role, as shown by the increase in mechanical and thermal resistances with the production of a cohesive material stable above 40 °C for at least 7 days and comparable to porcine and chemically cross-linked gelatins. Two prototypes were obtained with similar thermal resistances but different microstructures and viscoelastic properties, due to different formation dynamics of the covalent network. Considering these results, the enzymatically cross-linked salmon gelatin is a relevant candidate as a biopolymer for the production of matrix for a wide range of biotechnological applications such as food packaging, cosmetic patch, wound healing dressing, or tissue substitute.

Set up of a new in vitro model to study dietary fructans fermentation in formula-fed babies.

A new in vitro fermentation model with immobilised infant faecal microbiota simulating the proximal colon of a formula-fed baby was developed and used to test the effects of known prebiotic fructans. Intestinal fermentation, based on a previously developed colonic fermentation model, using a new feeding medium simulating a formula-fed infant ileal chyme, was carried out for seventy-one consecutive days divided into four stabilisation periods intercalated with four prebiotic treatment periods. At the end of the first stabilisation period, total bacterial concentration in colonised beads and in faecal sample was similar, metabolite concentrations returned to stabilisation values after each treatment period. As expected, the four prebiotic treatments significantly increased the bifidobacterial populations, whereas they decreased bacteroides and clostridia. No difference was observed in the prebiotic effect of these substrates selected. The treatments significantly increased total production of SCFA and decreased ammonia compared to stabilisation periods. Long-term stability of the system together with the reproducibility of the known prebiotic effects highlights the potential of the present model to quantify and compare the effects of different substrates in a formula-fed infant microbiota within the same fermentation experiment.

Glycerol induces reuterin production and decreases Escherichia coli population in an in vitro model of colonic fermentation with immobilized human feces.

Lactobacillus reuteri ATCC 55730 is a probiotic strain that produces, in the presence of glycerol, reuterin, a broad-spectrum antimicrobial substance. This strain has been shown to prevent intestinal infections in vivo; however, its mechanisms of action, and more specifically whether reuterin production occurs within the intestinal tract, are not known. In this study, the effects of L. reuteri ATCC 55730 on intestinal microbiota and its capacity to secrete reuterin from glycerol in a novel in vitro colonic fermentation model were tested. Two reactors were inoculated with adult immobilized fecal microbiota and the effects of daily addition of L. reuteri into one of the reactors (c.10(8) CFU mL(-1)) without or with glycerol were tested on major bacterial populations and compared with addition of glycerol or reuterin alone. The addition of glycerol alone or with L. reuteri increased numbers of the Lactobacillus-Enterococcus group and decreased Escherichia coli. The addition of reuterin significantly and selectively decreased E. coli without affecting other bacterial populations. The observed decrease in E. coli concentration during the addition of glycerol (in presence or absence of L. reuteri) could be due to in situ reuterin production because 1,3-propanediol, a typical product of glycerol fermentation, was detected during the addition of glycerol.

Novel Antifungal Compounds, Spermine-Like and Short Cyclic Polylactates, Produced by Lactobacillus harbinensis K.V9.3.1Np in Yogurt.

Lactobacillus harbinensis K.V9.3.1Np was described as endowed with high antifungal activity. Most of the studies associated this activity to the produced organic acids, i.e., lactic acid, acetic acid, and hexanoic acid. The aim of this study was to purify and identify, other not yet described, antifungal molecules produced by L. harbinensis K.V9.3.1Np when used in yogurt fermentation. Active compounds were extracted through several extraction processes using organic solvents and protein precipitation. The fractions of interest were purified using flash chromatography and preparative HPLC for specific characterization. The bioactive compounds identification was performed using Nuclear Magnetic Resonance and Mass Spectrometry. Activity tests against Penicillium expansum and Yarrowia lipolytica showed that the active compounds from L. harbinensis K.V9.3.1Np are benzoic acid and a polyamine identified as a spermine analog, which has not been reported earlier. However, the highest activity was shown by a mixture of short (n = 2-5) polycyclic lactates. Our overall results demonstrate the efficiency of the proposed extraction/purification approach. The new compounds described here have promising antifungal activities but further studies are still needed to decipher their mode of action and production pathways. Even though, they present an interesting potential application in food, feed, as well as, in pharmaceutical industries and could serve as alternative to chemical additives.

Inhibitory activity spectrum of reuterin produced by Lactobacillus reuteri against intestinal bacteria.

BACKGROUND: Reuterin produced from glycerol by Lactobacillus reuteri, a normal inhabitant of the human intestine, is a broad-spectrum antimicrobial agent. It has been postulated that reuterin could play a role in the probiotic effects of Lb. reuteri. Reuterin is active toward enteropathogens, yeasts, fungi, protozoa and viruses, but its effect on commensal intestinal bacteria is unknown. Moreover reuterin's mode of action has not yet been elucidated. Glutathione, a powerful antioxidant, which also plays a key role in detoxifying reactive aldehydes, protects certain bacteria from oxidative stress, and could also be implicated in resistance to reuterin. The aim of this work was to test the activity of reuterin against a representative panel of intestinal bacteria and to study a possible correlation between intracellular low molecular weight thiols (LMW-SH) such as glutathione, hydrogen peroxide and/or reuterin sensitivity. Reuterin was produced by Lb. reuteri SD2112 in pure glycerol solution, purified and used to test the minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC). Hydrogen peroxide sensitivity and intracellular LMW-SH concentration were also analysed. RESULTS: Our data showed that most tested intestinal bacteria showed MIC below that for a sensitive indicator Escherichia coli (7.5-15 mM). Lactobacilli and Clostridium clostridioforme were more resistant with MIC ranging from 15 to 50 mM. No correlation between bacterial intracellular concentrations of LMW-SH, including glutathione, and reuterin or hydrogen peroxide sensitivities were found. CONCLUSION: Our data showed that intestinal bacteria were very sensitive to reuterin and that their intracellular concentration of LMW-SH was not directly linked to their capacity to resist reuterin or hydrogen peroxide. This suggests that detoxification by LMW-SH such as glutathione is not a general mechanism and that other mechanisms are probably involved in bacterial tolerance to reuterin and hydrogene peroxide.

Action mechanisms involved in the bioprotective effect of Lactobacillus harbinensis K.V9.3.1.Np against Yarrowia lipolytica in fermented milk.

The use of lactic acid bacteria (LAB) as bioprotective cultures can be an alternative to chemical preservatives or antibiotic to prevent fungal spoilage in dairy products. Among antifungal LAB, Lactobacillus harbinensis K.V9.3.1Np showed a remarkable antifungal activity for the bioprotection of fermented milk without modifying their organoleptic properties (Delavenne et al., 2015). The aim of the present study was to elucidate the action mechanism of this bioprotective strain against the spoilage yeast Yarrowia lipolytica. To do so, yeast viability, membrane potential, intracellular pH (pHi) and reactive oxygen species (ROS) production were assessed using flow cytometry analyses after 3, 6 and 10days incubation in cell-free supernatants. The tested supernatants were obtained after milk fermentation with yogurt starter cultures either in co-culture with L. harbinensis K.V9.3.1Np (active supernatant) or not (control supernatant). Scanning-electron microscopy (SEM) was used to monitor yeast cell morphology and 9 known antifungal organic acids were quantified in both yogurt supernatants using high-performance liquid chromatograph (HPLC). Yeast growth occurred within 3days incubation in control supernatant, while it was prevented for up to 10days by the active supernatant. Interestingly, between 66 and 99% of yeast cells were under a viable but non-cultivable (VNC) state despite an absence of membrane integrity loss. While ROS production was not increased in active supernatant, cell physiological changes including membrane depolarization and pHi decrease were highlighted. Moreover, morphological changes including membrane collapsing and cell lysis were observed. These effects could be attributed to the synergistic action of organic acids. Indeed, among the 8 organic acids quantified in active supernatant, five of them (acetic, lactic, 2-pyrrolidone-5-carboxylic, hexanoic and 2-hydroxybenzoic acids) were at significantly higher concentrations in the active supernatant than in the control one. In conclusion, this study has provided new information on the physiological mechanisms induced by an antifungal LAB that could be used as part of the hurdle technology to prevent fungal spoilage in dairy products.

Comparative effects of exopolysaccharides from lactic acid bacteria and fructo-oligosaccharides on infant gut microbiota tested in an in vitro colonic model with immobilized cells.

The aim of this study was to compare the effects of purified exopolysaccharides from Lactobacillus rhamnosus RW-9595M with those of a well-known prebiotic (short-chain fructo-oligosaccharides) on infant colonic microbiota using a new three-stage chemostat model with immobilized infant faecal microbiota. Two continuous cultures with different faecal inocula were tested with different compositions of carbohydrate media. During the first fermentation (F1), fructo-oligosaccharides tested at a concentration of 9.8 g L(-1) increased the number of lactobacilli and decreased coliforms both in gel beads and in effluent from all three reactors, in agreement with data from the literature. During the second fermentation (F2), the effect of fructo-oligosaccharides tested at a lower concentration (7.5 g L(-1)) was reduced compared with F1. Fructo-oligosaccharides also increased total organic acid concentration and decreased ammonia production. Results obtained for exopolysaccharide tested at 1.5 g L(-1) indicate that exopolysaccharides from L. rhamnosus RW-9595M was not metabolized by infant microbiota and lacked any prebiotic effect.

New three-stage in vitro model for infant colonic fermentation with immobilized fecal microbiota.

The development and validation of a new three-stage culture system with immobilized fecal microbiota to simulate infant colonic ecosystem is described. Two continuous cultures with different fecal inocula were used to assess the validity and stability of the intestinal model. The total anaerobe populations measured in beads and effluent fermentations reached high concentrations similar to infant feces. Fluorescence in situ hybridization analyses and denaturing gradient gel electrophoresis profiles of effluent samples from the three reactors revealed complex patterns similar to that observed in the inoculum, indicating that fecal bacterial diversity was well-preserved and that dominant bacterial populations showed good stability among reactors. For both experiments, the bacterial populations and fermentation product concentrations were in the range of published data for infant feces. These results demonstrate that this new three-stage continuous culture with immobilized cells provides a useful tool for studying the infant colon ecosystem.

Identification and quantification of antifungal compounds produced by lactic acid bacteria and propionibacteria.

Fungal growth in bakery products represents the most frequent cause of spoilage and leads to economic losses for industrials and consumers. Bacteria, such as lactic acid bacteria and propionibacteria, are commonly known to play an active role in preservation of fermented food, producing a large range of antifungal metabolites. In a previous study (Le Lay et al., 2016), an extensive screening performed both in vitro and in situ allowed for the selection of bacteria exhibiting an antifungal activity. In the present study, active supernatants against Penicillium corylophilum and Aspergillus niger were analyzed to identify and quantify the antifungal compounds associated with the observed activity. Supernatant treatments (pH neutralization, heating and addition of proteinase K) suggested that organic acids played the most important role in the antifungal activity of each tested supernatant. Different methods (HPLC, mass spectrometry, colorimetric and enzymatic assays) were then applied to analyze the supernatants and it was shown that the main antifungal compounds corresponded to lactic, acetic and propionic acids, ethanol and hydrogen peroxide, as well as other compounds present at low levels such as phenyllactic, hydroxyphenyllactic, azelaic and caproic acids. Based on these results, various combinations of the identified compounds were used to evaluate their effect on conidial germination and fungal growth of P. corylophilum and Eurotium repens. Some combinations presented the same activity than the bacterial culture supernatant thus confirming the involvement of the identified molecules in the antifungal activity. The obtained results suggested that acetic acid was mainly responsible for the antifungal activity against P. corylophilum and played an important role in E. repens inhibition.

Comparative detection of bacterial adhesion to Caco-2 cells with ELISA, radioactivity and plate count methods.

Different methods are used to study bacterial adhesion to intestinal epithelial cells, which is an important step in pathogenic infection as well as in probiotic colonization of the intestinal tract. The aim of this study was to compare the ELISA-based method with more conventional plate count and radiolabeling methods for bacterial adhesion detection. An ELISA-based assay was optimized for the detection of Bifidobacterium longum and Escherichia coli O157:H7, which are low and highly adherent bacteria, respectively. In agreement with previous investigations, a percentage of adhesion below 1% was obtained for B. longum with ELISA. However, high nonspecific background and low positive signals were measured due to the use of polyclonal antibodies and the low adhesion capacity with this strain. In contrast, the ELISA-based method developed for E. coli adhesion detected a high adhesion percentage (15%). For this bacterium the three methods tested gave similar results for the highest bacterial concentrations (6.8 Log CFU added bacteria/well). However, differences among methods increased with the addition of decreased bacterial concentration due to different detection thresholds (5.9, 5.6 and 2.9 Log CFU adherent bacteria/well for radioactivity, ELISA and plate count methods, respectively). The ELISA-based method was shown to be a good predictor for bacterial adhesion compared to the radiolabeling method when good quality specific antibodies were used. This technique is convenient and allows handling of numerous samples.

Ruminococcus luti sp. nov., isolated from a human faecal sample.

A strain of an unidentified strictly anoxic, gram-postive, non-motile Ruminococcus-like bacterium was isolated from a human faecal sample. The organism used carbohydrates as fermentable substrates, produced acetate, succinate, and hydrogen as the major products of glucose metabolism, and possessed a G + C content of 43.3 mol%. The morphological and biochemical characteristics of the organism were consistent with its assignment to the genus Ruminococcus but it did not correspond to any recognized species of this genus. Comparative 16S rRNA gene sequencing showed the unidentified bacterium represents a previously unrecognised sub-line within the Clostridium coccoides rRNA group of organisms. The nearest relative of the unknown bacterium corresponded to Ruminococcus obeum but a 16S rRNA sequence divergence value of >3% demonstrated it represents a different species. Based on the presented findings a new species, Ruminococcus luti, is described. The type strain of Ruminococcus luti is BInIX(T) (DSM 14534T, CCUG 45635T).

In vitro inhibition of Escherichia coli O157:H7 by bifidobacterial strains of human origin.

The ability of bifidobacteria isolated from infant feces to inhibit enterohemorrhagic Escherichia coli serotype O157:H7 in vitro and reduce its adhesion to human enterocyte-like Caco-2 cells was evaluated in comparison to American Type Culture Collection bifidobacterial reference strains. Five Bifidobacterium isolates from infant feces were identified and characterized by morphology, fructose-6-phosphate phosphoketolase (F6PPK) assay, polymerase chain reaction using bifidobacterial 16S rDNA specific primers, carbohydrate fermentation patterns, resistance to lysozyme, acid, bile and hydrogen peroxide as well as their ability to inhibit E. coli O157:H7 using the agar spot technique. Infant isolates showed greater resistance to bile, acid, lysozyme and more antimicrobial activity against E. coli O157:H7 than ATCC strains. Two infant isolates identified as B. bifidum RBL 71 and B. bifidum RBL 460 showed good adhesion and significant potential for reducing adhesion of E. coli O157:H7 to Caco-2 cells. This effect was dependent on bifidobacterial cell concentration. These results show that bifidobacteria isolated from infants may be useful for improving probiotic formulae with respect to protection against E. coli O157:H7 infection.

Stability and Inhibitory Activity of Pediocin PA-1 Against Listeria sp. in Simulated Physiological Conditions of the Human Terminal Ileum.

Listeria monocytogenes is responsible for severe foodborne infections, which can be life-threatening especially for infants and elderly populations. The emergence of antibiotic-resistant pathogens has stimulated the search for new strategies, such as the use of bacteriocins, to prevent or cure foodborne infectious diseases in the intestine. In this study, we evaluated the efficacy of the bacteriocin pediocin PA-1 from Pediococcus acidilactici UL5 to inhibit Listeria ivanovii, used as a surrogate for L. monocytogenes, under physiological conditions of the terminal ileum, simulated in a continuous in vitro fermentation model. A fecal sample from a healthy adult was immobilized and propagated for 30 days in a continuous stirred tank reactor, fed with a nutritive medium simulating the ileal chime (pH 7.5). After reaching a pseudo-steady state, the reactor was inoculated five times with L. ivanovii to reach a final concentration of 10(7) CFU/ml within the reactor. Two spikes of L. ivanovii without adjunction of pediocin PA-1 served as control assays, and three other spikes were done to test the effects of three concentrations of pediocin PA-1 corresponding to 2, 3, and 5× the minimum inhibitory concentration (MIC) active against L. ivanovii. The concentration of L. ivanovii in the reactor was followed for 8 h using the PALCAM selective medium. The different groups of commensal bacteria were enumerated on selective medium or using fluorescence in situ hybridization. Our data showed that pediocin PA-1 is stable in the ileum conditions and that it is able to exert its inhibition activity against L. ivanovii in a dose-dependent manner. The addition of pediocin PA-1 at 5 × MIC induced a complete disappearance of L. ivanovii (5 log reduction) within 5 h, compared to a reduction of 2 logs, corresponding to the washout phenomenon, when no pediocin PA-1 was added. Reduction of 0.8 and 1.3 logs within 8 h was also obtained with the addition of 2 and 3 × MIC, respectively. The same experiment has shown that addition of pediocin-PA1 in the reactor had a negligible effect on the balance of commensal bacteria.