Enterocin DD14 can inhibit the infection of eukaryotic cells with enveloped viruses.

Bacteriocins are ribosomally synthesized bacterial peptides endowed with antibacterial, antiprotozoal, anticancer and antiviral activities. In the present study, we evaluated the antiviral activities of two bacteriocins, enterocin DD14 (EntDD14) and lacticaseicin 30, against herpes simplex virus type 1 (HSV-1), human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Vero, Huh7 and Vero E6 cells, respectively. In addition, the interactions of these bacteriocins with the envelope glycoprotein D of HSV-1 and the receptor binding domains of HCoV-229E and SARS-CoV-2 have been computationally evaluated using protein-protein docking and molecular dynamics simulations. HSV-1 replication in Vero cells was inhibited by EntDD14 and, to a lesser extent, by lacticaseicin 30 added to cells after virus inoculation. EntDD14 and lacticaseicin 30 had no apparent antiviral activity against HCoV-229E; however, EntDD14 was able to inhibit SARS-CoV-2 in Vero E6 cells. Further studies are needed to elucidate the antiviral mechanism of these bacteriocins.

Potential of Non-Saccharomyces Yeasts as Probiotics and Alternatives to Antibiotics in Animal Production.

Probiotics are live yeast or bacterial organisms that have beneficial effects on the host. Several microorganisms exhibit probiotic properties, the most common types being lactic acid bacteria, Bifidobacteria, spore-forming bacteria, and some yeast strains. Saccharomyces cerevisiae var. boulardii is the most important probiotic yeast species. However, another group of foodborne microorganisms, the so-called non-Saccharomyces yeasts (NSYs), has recently been re-evaluated and shown to have enormous potential in various fields of application, ranging from food fermentation to human and animal applications. NSYs are able to produce a range of bioactive compounds such as antimicrobials, mannoproteins, enzymes, polyunsaturated fatty acids, essential amino acids, vitamins, and ß-glucans, which increases their potential applications as a new class of probiotics and/or alternatives to antibiotics in animal husbandry. In this review, we aim to highlight the potential and benefits of NSYs as probiotics and natural antimicrobials to improve animal health. Furthermore, the use of NSYs as biological alternatives to antibiotics to control foodborne pathogens in animal production is discussed.

Advances in Characterizing the Transport Systems of and Resistance to EntDD14, A Leaderless Two-Peptide Bacteriocin with Potent Inhibitory Activity.

Enterocin DD14 (EntDD14) is a two-peptide leaderless bacteriocin produced by the Enterococcus faecalis 14 strain previously isolated from meconium. This bacteriocin is mainly active against Gram-positive bacteria. Leaderless bacteriocins do not undergo post-translational modifications and are therefore immediately active after their synthesis. As a result, the cells that produce such bacteriocins have developed means of protection against them which often involve transport systems. In this and our previous work, we constructed different mutants deleted in the genes involved in the transport functions, thus covering all the supposed components of this transport system, using Listeria innocua ATCC 33090 as the indicator strain to assess the activity of externalized EntDD14. We also assessed the self-resistance of the WT and all its engineered derivative mutants against EntDD14, provided extracellularly, in order to evaluate their self-resistance. The results obtained highlight that the ABC transporter constituted by the DdG, H, I, and J proteins contributes to EntDD14 export as well as resistance to an external supply of EntDD14. Our results also have established the essential role of the DdE and DdF proteins as primary transporters dedicated to the externalization of EntDD14. Moreover, the in silico data showed that DdE and DdF appear to assemble in a formation that forms an essential channel for the exit of EntDD14. This channel DdEF may interact with the ABC transporter DdGHIJ in order to control the flow of bacteriocin across the membrane, although the nature of this interaction remains to be elucidated.

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.

Vaginal Microbiota: Age Dynamic and Ethnic Particularities of Algerian Women.

The composition of the vaginal microbiota is a key element for maintaining gynecological and reproductive health. With the aim of obtaining an accurate overview of the vaginal microbiota of Algerian women, in terms of their age and ethnic group, we conducted a 16S rRNA gene targeted metagenomic analysis of 100 vaginal samples taken from healthy childbearing and menopausal women. These data were used to establish the pattern of the vaginal microbiota during reproductive and postreproductive phases. Hormone levels were correlated to changes in microbial composition for menopausal women. The ethnic comparison revealed a particular microbiota profile for Algerian women, with a dominance of CST III and CST I. A rapid qPCR method developed by the authors was successfully used to identify the vaginal bacterial pattern for a customized gynecological management.

Evidence for the Involvement of Pleckstrin Homology Domain-Containing Proteins in the Transport of Enterocin DD14 (EntDD14); a Leaderless Two-Peptide Bacteriocin.

Bacteriocins synthesis is initiated from an inactive precursor, which is composed of an N-terminal leader peptide attached to a C-terminal pro-peptide. However, leaderless bacteriocins (LLB) do not possess this N-terminal leader peptide nor undergo post-translational modifications. These atypical bacteriocins are observed to be immediately active after their translation in the cytoplasm. However, although considered to be simple, the biosynthetic pathway of LLB remains to be fully understood. Enterocin DD14 (EntDD14) is a two-peptide LLB produced by Enterococcus faecalis 14, which is a strain isolated from meconium. In silico analysis of DNA encoding EntDD14 located a cluster of 10 genes ddABCDEFGHIJ, where ddE and ddF encode the peculiar DdE and DdF proteins, carrying pleckstrin homology (PH) domains. These modules are quite common in Eucarya proteins and are known to be involved in intracellular signaling or cytoskeleton organization. To elucidate their role within the EntDD14 genetic determinants, we constructed deletion mutants of the ddE and ddF genes. As a result, the mutants were unable to export EntDD14 outside of the cytoplasm even though there was a clear expression of structural genes ddAB encoding EntDD14, and genes ddHIJ encoding an ABC transporter. Importantly, in these mutant strains (ΔddE and ΔddF), EntDD14 was detected by mass spectrometry in the intracellular soluble fraction exerting, upon its accumulation, a toxic effect on the producing strain as revealed by cell-counting and confocal microscopy analysis. Taken together, these results clearly indicate that PH domain-containing proteins, such as DdE and DdF, are involved in the transport of the leaderless two-peptide EntDD14.

Bioprospecting Antimicrobials from Lactiplantibacillus plantarum: Key Factors Underlying Its Probiotic Action.

Lactiplantibacillus plantarum (L. plantarum) is a well-studied and versatile species of lactobacilli. It is found in several niches, including human mucosal surfaces, and it is largely employed in the food industry and boasts a millenary tradition of safe use, sharing a long-lasting relationship with humans. L. plantarum is generally recognised as safe and exhibits a strong probiotic character, so that several strains are commercialised as health-promoting supplements and functional food products. For these reasons, L. plantarum represents a valuable model to gain insight into the nature and mechanisms of antimicrobials as key factors underlying the probiotic action of health-promoting microbes. Probiotic antimicrobials can inhibit the growth of pathogens in the gut ensuring the intestinal homeostasis and contributing to the host health. Furthermore, they may be attractive alternatives to conventional antibiotics, holding potential in several biomedical applications. The aim of this review is to investigate the most relevant papers published in the last ten years, bioprospecting the antimicrobial activity of characterised probiotic L. plantarum strains. Specifically, it focuses on the different chemical nature, the action spectra and the mechanisms underlying the bioactivity of their antibacterial and antiviral agents. Emerging trends in postbiotics, some in vivo applications of L. plantarum antimicrobials, including strengths and limitations of their therapeutic potential, are addressed and discussed.

Lactobacillus fermentum: a bacterial species with potential for food preservation and biomedical applications.

Lactic acid-producing bacteria are the most commonly used probiotics that play an important role in protecting the host against harmful microorganisms, strengthening the host immune system, improving feed digestibility, and reducing metabolic disorders. Lactobacillus fermentum (Lb. fermentum) is a Gram-positive bacterium belonging to Lactobacillus genus, and many reportedly to enhance the immunologic response as well as prevent community-acquired gastrointestinal and upper respiratory infections. Additionally, Lb. fermentum strains produce diverse and potent antimicrobial peptides, which can be applied as food preservative agents or as alternatives to antibiotics. Further functions attributed to probiotic Lb. fermentum strains are their abilities to decrease the level of blood stream cholesterol (as cholesterol-lowering agents) and to potentially help prevent alcoholic liver disease and colorectal cancer among humans. Finally, Lb. fermentum is a key microorganism in sourdough technology, contributing to flavor, texture, or health-promoting dough ingredients, and has recently been used to develop new foods stuffs such as fortified and functional foods with beneficial attributes for human health. Development of such new foodstuffs are currently taking important proportions of the food industry market. Furthermore, an increasing awareness of the consumers prompts the food-makers to implement alternative environmental friendly solutions in the production processes and/or suitable biological alternative to limit the use of antibiotics in feed and food. Here, we give an account on the application of Lb. fermentum strains in the biomedical and food preservation fields, with a focus on probiotic features such as bacteriocin production. We also summarize the use of Lb. fermentum as cell factories with the aim to improve the efficacy and health value of functional food.

Antibiotic resistance, genome analysis and further safe traits of Clostridium perfringens ICVB082; a strain capable of producing an inhibitory compound directed only against a closely related pathogenic strain.

Eleven strains of clostridia were isolated from chickens suffering from necrotic enteritis (NE) disease, and were identified by 16S rDNA sequencing as C. perfringens (Clin1, ICVB079, ICVB080, ICVB081, ICVB082, ICVB083, ICVB085, ICVB088, ICVB089, ICVB090), C. sporogenes (ICVB086) and C. cadaveris (ICVB087). These novel strains were then characterized for their pathoproperties including their sensitivity to different antibiotics, hemolytic activities and abilities to carry netB gene, which encodes the necrotic enteritis B-Like toxin (NetB); a key virulence factor involved in the NE. Whilst, no antibiotic resistance was detected for all these strains, C. perfringens ICVB081 and C. perfringens Clin1 have ß-hemolytic activities and carry DNA coding for the netB gene. Remarkably, cross-resistant assays performed between these Clostridium strains underpinned the capability of C. perfringens ICVB082 to inhibit the pathogenic C. perfringens DSM756, used as reference strain. This inhibition was exerted through production of an extracellular compound, which was sensitive to heat treatment, lipase and active at pH values ranging from 4 to 7. This report deals with the isolation of novel Clostridium strains from chicken origin and underlines the safety and inhibitory capability of C. perfringens ICVB082 through an extracellular metabolite.

Metabolic Shift of an Isogenic Strain of Enterococcus faecalis 14, Deficient in Its Own Bacteriocin Synthesis, as Revealed by a Transcriptomic Analysis.

The production of antimicrobial molecules often involves complex biological pathways. This study aimed at understanding the metabolic and physiological networks of enterocin EntDD14-associated function, in the bacteriocinogenic strain, Enterococcus faecalis 14. A global and comparative transcriptomic study was carried out on E. faecalis 14 and its isogenic mutant Δbac, inactivated in genes coding for EntDD14. The in vitro ability to form biofilm on polystyrene plates was assessed by the crystal violet method, while the cytotoxicity on human colorectal adenocarcinoma Caco-2 cells was determined by the Cell Counting Kit-8. Transcriptomic data revealed that 71 genes were differentially expressed in both strains. As expected, genes coding for EntDD14 were downregulated in the Δbac mutant, whereas the other 69 genes were upregulated. Upregulated genes were associated with phage-related chromosomal islands, biofilm formation capability, resistance to environmental stresses, and metabolic reprogramming. Interestingly, the Δbac mutant showed an improved bacterial growth, a high capacity to form biofilm on inanimate surfaces and a very weak cytotoxicity level. These multiple metabolic rearrangements delineate a new line of defense to counterbalance the loss of EntDD14.

New Bacteriocins from Lacticaseibacillus paracasei CNCM I-5369 Adsorbed on Alginate Nanoparticles Are Very Active against Escherichia coli.

Lacticaseibacillus paracasei CNCM I-5369, formerly Lactobacillus paracasei CNCM I-5369, produces bacteriocins that are remarkably active against Gram-negative bacteria, among which is the Escherichia coli-carrying mcr-1 gene that is involved in resistance to colistin. These bacteriocins present in the culture supernatant of the producing strain were extracted and semi-purified. The fraction containing these active bacteriocins was designated as E20. Further, E20 was loaded onto alginate nanoparticles (Alg NPs), leading to a highly active nano-antibiotics formulation named hereafter Alg NPs/E20. The amount of E20 adsorbed on the alginate nanoparticles was 12 wt.%, according to high-performance liquid chromatography (HPLC) analysis. The minimal inhibitory concentration (MIC) values obtained with E20 ranged from 250 to 2000 µg/mL, whilst those recorded for Alg NPs/E20 were comprised between 2 and 4 µg/mL, which allowed them to gain up to 500-fold in the anti-E. coli activity. The damages caused by E20 and/or Alg NPs/E20 on the cytology of the target bacteria were characterized by transmission electron microscopy (TEM) imaging and the quantification of intracellular proteins released following treatment of the target bacteria with these antimicrobials. Thus, loading these bacteriocins on Alg NPs appeared to improve their activity, and the resulting nano-antibiotics stand as a promising drug delivery system.

Impact of seasonality and environmental conditions on yeast diversity from camel's milk collected in Algeria.

During this study, we characterized the seasonality's impact and environmental conditions on the yeast diversity from raw camel's milk collected in Algeria. The yeast counts were estimated to 3.55 × 102 CFU mL-1, with a maximum of 6.3 × 102 CFU mL-1. The yeasts were categorized phenotypically by API 20C AUX, MALDI-TOF and genetically by sequencing 26S rDNA and ITS1-5.8S-ITS2. The rDNA sequencing approaches revealed 12 species including unusual ones such as Trichosporon asahii, Pichia fermentans, Millerozyma farinosa, Pichia galeiformis, Candida tartarivorans and Pichia manshurica. The most dominant species were T. asahii (23%), P. fermentans (19%) and Rhodotorula mucilaginosa (14%). The high occurrence and large diversity were registered in samples collected during the autumn season, in the semi-arid and arid highlands regions with 0.66 × 103 CFU mL-1 and 0.51 × 103 CFU mL-1, respectively. Interestingly, T. asahii, R. mucilaginosa, P. fermentans, C. parapsilosis and C. zeylanoides were detected during both spring and autumn.

Impact of growth temperature on the adhesion of colistin-resistant Escherichia coli strains isolated from pigs to food-contact-surfaces.

This study investigated the effect of the growth temperature (20 and 37 °C) of Escherichia coli strains isolated from pigs on their adhesion to stainless steel and polycarbonate. This study also evaluated the ability of the DLVO and XDLVO mathematical models to predict this adhesion. The rise of growth temperature from 20 to 37 °C significantly influenced the adhesion of studied E. coli strains. The data also underlined that the mathematical prediction did not fully match with the experimental bacterial adhesion to surfaces. Furthermore, results showed that the colistin-resistant and sensitive E. coli strains adhesion depends on the type of abiotic surface. Based on these results, the mathematical models are limited in the prediction of the bacterial adhesion to abiotic surfaces. The surface roughness is a major parameter of the bacterial adhesion and should be included in the future mathematical models predicting the bacterial adhesion.

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.

Microbial-derived products as potential new antimicrobials.

Due to the continuing global concerns involving antibiotic resistance, there is a need for scientific forums to assess advancements in the development of antimicrobials and their alternatives that might reduce development and spread of antibiotic resistance among bacterial pathogens. The objectives of the 2nd International Symposium on Alternatives to Antibiotics were to highlight promising research results and novel technologies that can provide alternatives to antibiotics for use in animal health and production, assess challenges associated with their authorization and commercialization for use, and provide actionable strategies to support their development. The session on microbial-derived products was directed at presenting novel technologies that included exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials, probiotics development via fecal microbiome transplants among monogastric production animals such as chickens and mining microbial sources such as bacteria or yeast to identify new antimicrobial compounds. Other research has included continuing development of antimicrobial peptides such as newly discovered bacteriocins as alternatives to antibiotics, use of bacteriophages accompanied by development of unique lytic proteins with specific cell-wall binding domains and novel approaches such as microbial-ecology guided discovery of anti-biofilm compounds discovered in marine environments. The symposium was held at the Headquarters of the World Organisation for Animal Health (OIE) in Paris, France during 12-15 December 2016.

Immunobiosis and probiosis: antimicrobial activity of lactic acid bacteria with a focus on their antiviral and antifungal properties.

Lactic acid bacteria (LAB), a heterogeneous group of bacteria that produce lactic acid as the main product of carbohydrate degradation, play an important role in the production and protection of fermented foods. Moreover, beside the technological use of these microorganisms added to control and steer food fermentations, their beneficial healthy properties are largely overt. Thus, numerous LAB strains have obtained the probiotic status, which entails the ability to maintain and promote a good health of consumers. In particular, increasing consideration is being focused on probiotic microorganisms that can improve the human immune response against dangerous viral and fungal enemies. For such beneficial microbes, the term "immunobiotics" has been coined. Together with an indirect host-mediated adverse effect against undesirable microorganisms, also a direct antagonistic activity of several LAB strains has been largely demonstrated. The purpose of this review is to provide a fullest possible overview of the antiviral and antifungal activities ascribed to probiotic LAB. The interest in this research field is substantiated by a large number of studies exploring the potential application of these beneficial microorganisms both as biopreservatives and immune-enhancers, aiming to reduce and/or eliminate the use of chemical agents to prevent the development of pathogenic, infectious, and/or degrading causes.

Correction to: Immunobiosis and probiosis: antimicrobial activity of lactic acid bacteria with a focus on their antiviral and antifungal properties.

There is an error in the original publication of this paper. The incorrect author name was captured as "Djamel Dridier" instead of "Djamel Drider". The original article has been corrected.

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.

Enterocin B3A-B3B produced by LAB collected from infant faeces: potential utilization in the food industry for Listeria monocytogenes biofilm management.

Enterococcus faecalis B3A-B3B produces the bacteriocin B3A-B3B with activity against Listeria monocytogenes, Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium perfringens, but apparently not against fungi or Gram-negative bacteria, except for Salmonella Newport. B3A-B3B enterocin has two different nucleotides but similar amino acid composition to the class IIb MR10A-MR10B enterocin. B3A-B3B consists of two peptides of predicted molecular mass of 5176.31 Da (B3A) and 5182.21 Da (B3B). Importantly, B3A-B3B impeded biofilm formation of the foodborne pathogen L. monocytogenes 162 grown on stainless steel. The antimicrobial treatment of stainless steel with nisin (1 or 16 mg ml-1) decreased the cell numbers by about 2 log CFU ml-1, thereby impeding the biofilm formation by L. monocytogenes 162 or its nisin-resistant derivative strain L. monocytogenes 162R. Furthermore, the combination of nisin and B3A-B3B enterocin reduced the MIC required to inhibit this pathogen grown in planktonic or biofilm cultures.

Fermentation of sarshir (kaymak) by lactic acid bacteria: antibacterial activity, antioxidant properties, lipid and protein oxidation and fatty acid profile.

BACKGROUND: The antioxidant and antibacterial activities of fermented sarshir (traditional dairy food), with three probiotic Lactobacillus plantarum strains (LP3, AF1, and LU5), were investigated. The oxidative stability and the lipid profile of non-fermented and fermented sarshir were compared, in addition to radical scavenging activity, as well as peroxide, anisidine and carbonyl values (PV, AnV and CV, respectively). RESULTS: The strong antibacterial activity of fermented sarshir against common pathogenic bacteria, including Gram-negative Escherichia coli O157: H7 ATCC 35150 and Pseudomonas aeruginosa ATCC 27853, as well as Gram-positive Bacillus cereus ATCC 10876 and Staphylococcus aureus ATCC 25923, was established. Among the strains examined, L. plantarum LP3 exhibited the highest radical scavenging activity (53.1 ± 1.8%) and lowest PV (3.0 meq kg-1 ), AnV (1.31 ± 0.06) and CV (1.4 ± 0.08). The pH of sarshir decreased from 6.2 ± 0 to 3.5 ± 0.1 during 14 h of fermentation. Incorporated bacterial cells exhibited notable viability during 10 days of cold storage (4 °C). CONCLUSION: The fermentation of sarshir by L. plantarum strains, especially LP3, resulted in beneficial changes in radical scavenging activity, as well as PV, AnV and carbonyl values, in addition to a broad spectrum of inhibitory activity against strains of P. aeruginosa, E. coli O157:H7, B. cereus and S. aureus. © 2017 Society of Chemical Industry.