Protective Effects of Novel Lactobacillaceae Strains Isolated from Chicken Caeca against Necrotic Enteritis Infection: In Vitro and In Vivo Evidences.

The present study aimed to show the benefits of novel lactic acid bacteria (LAB) strains isolated from the caeca of healthy chickens. These novel strains, identified as Limosilactobacillus reuteri and Ligilactobacillus salivarius, displayed high levels of lactic acid production, capability of biofilm formation, high aggregation and adhesion scores, and significant survival rates under conditions mimicking the chicken gastrointestinal tract (GIT). In addition, these novel Lactobacillaceae isolates were neither hemolytic nor cytotoxic. In vivo trials were able to establish their ability to reduce necrotic enteritis. Notably, a significant weight gain was registered, on day 10 of treatment, in the group of chickens fed with a mixture of L. reuteri ICVB416 and L. salivarius ICVB430 strains, as compared with the control group. This group has also shown a reduced number of lesions in the gut compared with other infected chicken groups. This study provides in vitro and in vivo evidence supporting the benefits of these novel Lactobacillaceae isolates for their use in poultry livestock as protective cultures to control the bacterial necrotic enteritis (NE) Clostridium perfringens.

Compatibility, Cytotoxicity, and Gastrointestinal Tenacity of Bacteriocin-Producing Bacteria Selected for a Consortium Probiotic Formulation to Be Used in Livestock Feed.

Bacteriocin-producing Escherichia coli ICVB442, E. coli ICVB443, Enterococcus faecalis ICVB497, E. faecalis ICVB501, and Pediococcus pentosaceus ICVB491 strains were examined for their pathogenic risks and compatibility and hence suitability as consortium probiotic bacteria. Except for E. coli ICVB442, all were inclined to form biofilm. All were gelatinase-negative, sensitive to most of the antibiotics tested and not cytotoxic to porcine intestinal epithelial cells (IPEC-1) when tested at a multiplicity of infection (MOI) of 1. P. pentosaceus ICVB491 stood apart by inhibiting the other four strains. Both E. coli strains and E. faecalis ICVB497 strain were ß-hemolytic. Survival in the TIM-1 dynamic model of the human digestive system was 139% for the tested E. coli ICVB443 strain, 46% for P. pentosaceus ICVB491, and 32% for the preferred E. faecalis ICVB501 strain. These three potential probiotics, which are bacteriocin-producing strains, will be considered for simultaneous use as consortium with synergistic interactions in vivo on animal model.

Biodiversity and Phylogenetic Relationships of Novel Bacteriocinogenic Strains Isolated from Animal's Droppings at the Zoological Garden of Lille, France.

This study aimed at exploring droppings of animals living in captivity in the zoological garden (Zoo) of Lille (France), as novel sources of bacteriocinogenic strains. A collection of 295 bacterial isolates was constituted from droppings of capybara, alpaca, muntjac, zebra, tapir, rhinoceros, binturong, armadillo, saki monkey and cockatoo. Of 295 isolates, 51 exhibited antagonism against a panel of pathogenic target bacteria like Escherichia coli MC4100, Clostridium perfringens DSM 756 and Salmonella enterica subsp. enterica Newport ATCC6962. Remarkably, within this collection, only 2 Gram-negative bacilli exhibited activity against E. coli MC4100 strain used as target organism. Then, the 16S rDNA sequencing revealed these thereafter cited species, Pediococcus pentosaceus, Weissella cibaria, E. coli, Lactobacillus reuteri, Enterococcus hirae and Enterococcus faecalis. Characterization of this antagonism has revealed 11 strains able producing extracellular protease-sensitive inhibitory compounds. These strains included E. coli ICVB442 and ICVB443, Ent. faecalis ICVB472, ICVB474, ICVB477 ICVB479, ICVB481, ICVB497 and ICVB501 and Ped. pentosaceus ICVB491 and ICVB492. The genomes of the 5 most promising bacteriocinogenic strains were sequenced and analysed with Bagel4 software. Afterwards, this bioinformatics analysis permitted to locate genes encoding bacteriocins like colicin Y (E. coli), enterocin 1071A, enterocin 107 B (Ent. faecalis) and penocin A (Ped. pentosaceus), associating the above-mentioned antibacterial activity of proteinaceous nature to possible production of bacteriocins. All these results enabled us to select different bacteriocinogenic strains for a further characterization in terms of beneficial traits.

In silico analyses of the genomes of three new bacteriocin-producing bacteria isolated from animal's faeces.

Here, we have analysed and explored the genome sequences of three newly isolated bacteria that were recently characterised for their probiotic activities and ability to produce bacteriocins. These strains, isolated from faeces of animals living in captivity at the zoological garden of Lille (France), are Escherichia coli ICVB443, Enterococcus faecalis ICVB501 and Pediococcus pentosaceus ICVB491. Their genomes have been analysed and compared to those of their pathogenic or probiotic counterparts. The genome analyses of E. coli ICVB443 and Ent. faecalis ICVB501 displayed similarities to those of probiotics E. coli 1917 Nissle, and Ent. faecalis Symbioflor 1, respectively. Furthermore, E. coli ICVB443 shares at least 89 genes with the enteroaggregative E. coli 55989 (EAEC), and Ent. faecalis ICVB501 shares at least 315 genes with the pathogenic Ent. faecalis V583 strain. Unlike Ped. pentosaceus ICVB491, which is devoid of virulence genes, E. coli ICVB443 and Ent. faecalis ICVB501 both carry genes encoding virulence factors on their genomes. Of note, the bioinformatics analysis of these two genomes located the bsh gene, which codes for bile salt hydrolase (BSH). The presence of BSH is of major importance, as it can help to increase the viability of these two strains in the gastrointestinal tract (GIT). The genome analysis of Ped. pentosaceus ICVB491 confirmed its GRAS status (Generally Recognised As Safe), as no genomic virulence factor determinant was found.

Benefits and Inputs From Lactic Acid Bacteria and Their Bacteriocins as Alternatives to Antibiotic Growth Promoters During Food-Animal Production.

Resistance to antibiotics is escalating and threatening humans and animals worldwide. Different countries have legislated or promoted the ban of antibiotics as growth promoters in livestock and aquaculture to reduce this phenomenon. Therefore, to improve animal growth and reproduction performance and to control multiple bacterial infections, there is a potential to use probiotics as non-antibiotic growth promoters. Lactic acid bacteria (LAB) offer various advantages as potential probiotics and can be considered as alternatives to antibiotics during food-animal production. LAB are safe microorganisms with abilities to produce different inhibitory compounds such as bacteriocins, organic acids as lactic acid, hydrogen peroxide, diacetyl, and carbon dioxide. LAB can inhibit harmful microorganisms with their arsenal, or through competitive exclusion mechanism based on competition for binding sites and nutrients. LAB endowed with specific enzymatic functions (amylase, protease…) can improve nutrients acquisition as well as animal immune system stimulation. This review aimed at underlining the benefits and inputs from LAB as potential alternatives to antibiotics in poultry, pigs, ruminants, and aquaculture production.

High-throughput optimization of medium components and culture conditions for the efficient production of a lipopeptide pseudofactin by Pseudomonas fluorescens BD5.

BACKGROUND: Lipopeptides are a promising group of surface-active compounds of microbial origin (biosurfactants). These diverse molecules are produced mainly by Bacillus and Pseudomonas strains. Because of their attractive physiochemical and biological properties, biosurfactants are considered to be "green and versatile molecules of the future". The main obstacles in widespread use of biosurfactants are mainly their low yields and high production costs. Pseudofactin (PF) is a lipopeptide produced by Pseudomonas fluorescens BD5. Recently, we identified two analogues, PF1 (C16-Val) and PF2 (C16-Leu), and reported that PF2 has good emulsification and foaming activities, as well as antibacterial, antifungal, anticancer, and antiadhesive properties. Reported production of PF in a mineral salt medium was approximately 10 mg/L. RESULTS: Here, we report successful high-throughput optimization of culture medium and conditions for efficient PF production using P. fluorescens BD5. Compared with production in minimal medium, PF yield increased almost 120-fold, up to 1187 ± 13.0 mg/L. Using Plackett-Burman and central composite design methodologies we identified critical factors that are important for efficient PF production, mainly high glycerol concentration, supplementation with amino acids (leucine or valine) and complex additives (e.g. tryptone), as well as high culture aeration. We also detected the shift in a ratio of produced PF analogues in response to supplementation with different amino acids. Leucine strongly induces PF2 production, while valine addition supports PF1 production. We also reported the identification of two new PF analogues: PF3 (C18-Val) and PF4 (C18-Leu). CONCLUSIONS: Identification of critical culture parameters that are important for lipopeptide production and their high yields can result in reduction of the production costs of these molecules. This may lead to the industrial-scale production of biosurfactants and their widespread use. Moreover, we produced new lipopeptide pure analogues that can be used to investigate the relationship between the structure and biological activity of lipopeptides.

Polynucleotide phosphorylase is involved in the control of lipopeptide fengycin production in Bacillus subtilis.

Bacillus subtilis is a wealth source of lipopeptide molecules such as iturins, surfactins and fengycins or plipastatins endowed with a range of biological activities. These molecules, designated secondary metabolites, are synthesized via non-ribosomal peptides synthesis (NRPS) machinery and are most often subjected to a complex regulation with involvement of several regulatory factors. To gain novel insights on mechanism regulating fengycin production, we investigated the effect of the fascinating polynucleotide phosphorylase (PNPase), as well as the effect of lipopeptide surfactin. Compared to the wild type, the production of fengycin in the mutant strains B. subtilis BBG235 and BBG236 altered for PNPase has not only decreased to about 70 and 40%, respectively, but also hampered its antifungal activity towards the plant pathogen Botrytis cinerea. On the other hand, mutant strains BBG231 (srfAA-) and BBG232 (srfAC-) displayed different levels of fengycin production. BBG231 had registered an important decrease in fengycin production, comparable to that observed for BBG235 or BBG236. This study permitted to establish that the products of pnpA gene (PNPase), and srfAA- (surfactin synthetase) are involved in fengycin production.

Study of the correlation between fengycin promoter expression and its production by Bacillus subtilis under different culture conditions and the impact on surfactin production.

This work aimed to rely expression of the fengycin promoter to fengycin production under different culture conditions. To this end, Bacillus subtilis BBG208, derived from BBG21, which is a fengycin overproducing strain carrying the green fluorescent protein (GFP) under the control of fengycin promoter, was used to assess the effects of different carbon and nitrogen sources on surfactin and fengycin production and the fengycin promoter expression. The data showed that some carbon sources oriented synthesis of one family of lipopeptides, while most of the nitrogen sources allowed high co-production of fengycin and surfactin. High expressions of promoter Pfen and fengycin synthesis were obtained with urea or urea + ammonium mixture as nitrogen source and mannitol as carbon source. Moreover, temperature, pH and oxygenation influenced their biosynthesis based on the nutrition conditions. Optimization of the production medium increased the fengycin production to 768 mg L-1, which is the highest level reported for this strain. This study defines the suitable nutrient conditions allowing as well the highest expression of the fengycin promoter and portrays the conditions relying on the fengycin and surfactin production.

Lactobacillus plantarum and Its Probiotic and Food Potentialities.

The number of studies claiming probiotic health effects of Lactobacillus plantarum is escalating. Lb. plantarum is a lactic acid bacterium found in diverse ecological niches, highlighting its particular capabilities of adaptation and genome plasticity. Another function that needs to be underlined is the capabilities of Lb. plantarum to produce diverse and potent bacteriocins, which are antimicrobial peptides with possible applications as food preservative or antibiotic complementary agents. Taken together, all these characteristics design Lb. plantarum as a genuine model for academic research and viable biological agent with promising applications. The present review aims at shedding light on the safety of Lb. plantarum and run through the main studies underpinning its beneficial claims. The mechanisms explaining probiotic-related features are discussed.

Influence of promoters on the production of fengycin in Bacillus spp.

Fengycin is a promising antifungal lipopeptide from Bacillus spp. synthesized by non-ribosomal peptide synthetases (NRPS). In this work, fengycin production of a spontaneous fengycin overproducing strain, Bacillus subtilis BBG21, was first compared to those of B. subtilis BBG111 (a 168 derivative), B. subtilis ATCC 21332 and Bacillus amyloliquefaciens FZB42 under two different experimental conditions. In both conditions, very high fengycin yields were obtained from strain BBG21 (480 mg/L) in comparison to its counterparts. The high efficiency of the fengycin promoter (Pfen) of BBG21 compared to the promoter of BBG111 and FZB42 was confirmed using a GFP reporter gene. Under all tested conditions, this promoter showed highest expression in comparison to the other strains. The highest fluorescence rate was obtained with mannitol as carbon source. In addition, when the Ppps promoter from B. subtilis BBG111 was replaced by promoter Pfen from BBG21, fengycin production increased about 10-fold, while no fengycin overproduction was observed when replacement was performed with Pfen from ATCC 21332. Comparative sequence analysis of these different promoters revealed one nucleotide modification in the UP element known for its importance in the regulation process. This point mutation is thus responsible for overproduction of fengycin in BBG21.

Selective fengycin production in a modified rotating discs bioreactor.

Production of lipopeptides fengycin and surfactin in rotating discs bioreactor was studied. The effects of rotation velocity and the addition of agitators between the discs on volumetric oxygen transfer coefficient k L a were firstly studied in model media. Then the production of lipopeptides was also studied at different agitation conditions in the modified bioreactor (with agitators). The effect of agitation on dissolved oxygen, on submerged and immobilized biomass, on lipopeptide concentrations and yields and on the selectivity of the bioreaction was elucidated and discussed. The proposed modified rotating discs bioreactor allowed to obtain high fengycin concentrations (up to 787 mg L(-1)), but also better selectivity of the bioreaction towards fengycin (up to 88%) and better yields of fengycin per glucose (up to 62.9 mg g(-1)), lipopeptides per glucose (up to 71.5 mg g(-1)), fengycin per biomass (up to 309 mg g(-1)) and lipopeptides per biomass (up to 396 mg g(-1)) than those reported in the literature. Highest fengycin production and selectivity were obtained at agitation velocity of 30 min(-1). The proposed non-foaming fermentation process could contribute to the scale-up of lipopeptide fermentors and promote the industrial production of fengycin. The proposed bioreactor and bioprocess could be very useful also for the production of other molecules using bioprocesses requiring bubbleless oxygen supply.

Impact of energy supply and oxygen transfer on selective lipopeptide production by Bacillus subtilis BBG21.

The influence of power dissipation and volumetric oxygen transfer coefficient k(L)a on Bacillus subtilis productivity of lipopeptides surfactin and fengycin was studied in shake flasks in view of scaling-up of this fermentation process. The experiments performed with different flask sizes, relative filling volumes, and shaking frequencies confirmed clearly that lipopeptide production changed in function of power dissipation, via interfacial gas-liquid contact surface and oxygen supply. It was demonstrated that k(L)a is the key parameter controlling the productivity and the selectivity of the bioreaction. Varying the oxygen transfer conditions, the synthesis could be oriented to mixed production or to surfactin mono-production. The fraction of surfactin towards total lipopeptides produced and the maximal surfactin production both increased with k(L)a increase (surfactin concentration about 2 g L(-1) at k(L)a=0.04-0.08 s(-1)), while the maximal fengycin production (fengycin concentration about 0.3 g L(-1)) was obtained at moderate oxygen supply (k(L)a=0.01 s(-1)).

Effect of different Bacillus subtilis lipopeptides on surface hydrophobicity and adhesion of Bacillus cereus 98/4 spores to stainless steel and Teflon.

Various lipopeptides produced by Bacillus subtilis were examined for their ability to modify the surface hydrophobicity of two substrata, stainless steel (SS) and Teflon. These modifications were evaluated by water contact angle measurements. The effects depended on the lipopeptide, its concentration, and the tested substratum. Treatment of SS with different concentrations of surfactin S1 showed an increase of the hydrophobicity between 1 and 100 mg l(-1). On the same substratum, fengycin increased hydrophobicity up to its critical micelle concentration (6.25 mg l(-1)). With higher concentrations of fengycin, hydrophobicity decreased. Surfactin, mycosubtilin, and iturin A decreased hydrophobicity on Teflon. The different effects of these three families of lipopeptides were related to their structural differences. A good correlation was shown between hydrophobicity modifications of surfaces and the attachment of B. cereus 98/4 spores. Enhancement in the hydrophobicity of the surfaces increased the number of adhering spores.

Differentiated pellicle organization and lipopeptide production in standing culture of Bacillus subtilis strains.

Pellicle formation and lipopeptide production was analysed in standing cultures of different Bacillus subtilis strains producing two or three families of lipopeptides. Despite its ability to produce surfactin, B. Subtilis ATCC 6633 was unable to form stable pellicle at air-water interface. For the ATTC 21332 and ATCC 9943 strains, it was shown for the first time that the lipopeptides were also produced in standing cultures at productivities similar or lower than those obtained when the culture medium is agitated. A differentiated behaviour was observed between these strains in repetitive batch cultures. B. subtilis 9943 formed a wrinkled, thinner and more resistant pellicle than B. subtilis 21332. The structure of the pellicle determined by electron microscopy observations showed that cells of B. subtilis 9943 formed microcolonies whereas those of B. subtilis 21332 rapidly died. Under these conditions, surfactin production by strain 21332 decreased after 2 days whereas it remained stable for B. subtilis 9943 during the 6 days of the cultures. These data indicate that cells of B. subtilis strains growing in pellicle can produce lipopeptides differently depending on their cellular organisation.

Mycosubtilin overproduction by Bacillus subtilis BBG100 enhances the organism's antagonistic and biocontrol activities.

A Bacillus subtilis derivative was obtained from strain ATCC 6633 by replacement of the native promoter of the mycosubtilin operon by a constitutive promoter originating from the replication gene repU of the Staphylococcus aureus plasmid pUB110. The recombinant strain, designated BBG100, produced up to 15-fold more mycosubtilin than the wild type produced. The overproducing phenotype was related to enhancement of the antagonistic activities against several yeasts and pathogenic fungi. Hemolytic activities were also clearly increased in the modified strain. Mass spectrometry analyses of enriched mycosubtilin extracts showed similar patterns of lipopeptides for BBG100 and the wild type. Interestingly, these analyses also revealed a new form of mycosubtilin which was more easily detected in the BBG100 sample. When tested for its biocontrol potential, wild-type strain ATCC 6633 was almost ineffective for reducing a Pythium infection of tomato seedlings. However, treatment of seeds with the BBG100 overproducing strain resulted in a marked increase in the germination rate of seeds. This protective effect afforded by mycosubtilin overproduction was also visualized by the significantly greater fresh weight of emerging seedlings treated with BBG100 compared to controls or seedlings inoculated with the wild-type strain.

Effect of different temperature downshifts on protein synthesis by Aeromonas hydrophila.

The psychrotrophic bacterium Aeromonas hydrophila 7966 was subjected to cold shocks from 30 degrees C to 20 degrees C, 15 degrees C, 10 degrees C, or 5 degrees C, or were incubated at low temperature to determine its adaptative response. The cell protein patterns analyzed by two-dimensional electrophoresis revealed that only a few proteins were underexpressed, whereas numerous new proteins appeared with the decrease of temperature, and some others were overexpressed. Among them, a few constituted cold shock proteins because they were transiently induced, whereas others belong to the acclimatation family proteins. Two cold shock proteins of 11 kDa were synthesized at low level because they were visualized only after radiolabeling or silver staining. Moreover, under our experimental conditions, no major cold shock protein of a molecular mass similar to that of E. coli (7.4 kDa) could be identified.