Tetracycline resistance of Escherichia coli isolated from water, human stool, and fish gills from the Lake Sevan basin.

The ecological state of Lake Sevan, the largest drinking water reservoir for the South Caucasus, formed under the influence of climatic and social changes. This study assesses the bacteriological quality of water in the rivers of the Lake Sevan basin and tetracycline-resistant bacteria isolated from fish and people living near the rivers of the Lake Sevan basin in Armenia in autumn 2019 and spring 2020. No differences have been shown for the tetracycline resistance of the investigated E. coli isolated from the human gut and the Masrik, Argichi, and Gavaraget Rivers. Horizontal gel electrophoresis revealed the same plasmid bands in most of the investigated E. coli with the same tetracycline resistance from the different sources of the Argichi River (obtained from people/fish/water sources where the fish were caught). The results also showed that most of the waters carried Edwardsiella spp., Erwinia spp., Morganella spp., and Proteus spp. in addition to E. coli; the coliform index did not exceed the standard level of 5 × 104 CFU mL-1 there. These findings highlight the importance of multidisciplinary studies of bacteria from "interacting" ecosystems, which might serve as a basis for the suggestion of microbial antibiotic resistance as another indicator of water pollution.

Probiotics for immunomodulation in prevention against respiratory viral infections with special emphasis on COVID-19.

COVID-19 pandemic is currently causing high mortality and economic crisis, and several drugs-based therapeutic strategies and vaccines are unfortunately used with little efficiency. Therefore, here is an urgent need to provide additives therapies that prevent or improve symptoms in populations infected by SARS-CoV-2 variants. This review aimed to examine relevant scientific information related to SARS-CoV-2 and host antiviral immunity, as well the possible role of probiotics in gut-lung cross talk pathways to promote lung immune response to COVID-19 infection. We searched online databases such as PubMed, Embase, Chinese databases, and selected articles and studies with relevant data reported on COVID-19 and other respiratory viral infections. Recent research highlighted potential immunomodulatory activities of probiotics assessed in animal models and clinical trials. However, the role of probiotics and gut microbiome in COVID-19 management, and approaches with significant understanding in molecular mechanism of probiotic action remain poorly investigated. Clinical investigations as well as animal model studies published have demonstrated that probiotics such as Lactobacillus rhamnosus and Bifidumbacterium lactis HN019, may influence positively not only microbiota balance but also antiviral immunity by improving both innate and adaptive responses and controlling inflammatory reaction in respiratory viral infection. Given the immunological interactions in gut-lung axis and the crucial role of probiotics in modulating immune responses by promoting dendritic cells (DCs) to regulate T cell responses, we hypothesized that application of probiotics may be successful in prevention or treatment of both intestinal disorders and airway diseases in patients with COVID-19.

Antioxidant and antimutagenic properties of probiotic Lactobacilli determined using LUX-biosensors.

The initial screening of probiotic strains in vitro, carried out by different methods, may omit strains that are promising from the point of view of biotechnology or, conversely, mark as promising strains those that will lose activity when transferred in vivo. It is known that the release of metabolites by probiotic bacteria, in particular, lactobacilli, is highly dependent on the biochemical context. In this work, we modified the method that was previously successfully used for the selection of probiotics for poultry, based on their antioxidant and DNA-protective properties. A comparison was made of this activity on standard media and on an artificial intestinal medium that mimics the intestines of a bird. As a result, three Lactobacillus strains were selected, which not only exhibit antioxidant and DNA-protective properties but also do not lose these activities in an artificial intestinal medium.

Coronavirus Infections of Animals: Future Risks to Humans.

Coronaviruses have tremendous evolutionary potential, and three major outbreaks of new human coronavirus infections have occurred in the recent history of humankind. In this paper, the patterns of occurrence of new zoonotic coronavirus infections and the role of bioveterinary control in preventing their potential outbreaks in the future are determined. The possibility of SARS-CoV-2 infection in companion animals is considered. Diverse human activities may trigger various interactions between animal species and their viruses, sometimes causing the emergence of new viral pathogens. In addition, the possibility of using probiotics for the control of viral infections in animals is discussed.

Blood protein polymorphisms and the gut bacteria: impact of probiotic Lactobacillus acidophilus Narine on Salmonella carriage in sheep.

Related to previous reports on correlations between an animal's genotype, its commensal microbiota, and the ability to resist infections, the aim of the current study was to investigate the associations between sheep genotype and 5-methylcytosine (5-mC) DNA methylation patterns, sheep genotype and cell surface hydrophobicity of sheep gut commensal bacteria. In addition, the effect of the probiotic Lactobacillus acidophilus strain INMIA 9602 Er 317/402 (probiotic formulation Narine) on Salmonella carriage in sheep at Armenian farms was also investigated. Allelotypes and genotypes of different pathogen-sensitive sheep breeds from Armenian farms were studied based on genetic markers of blood transferrin, albumin, and ceruloplasmin. Additionally, the differences between the breeds of Mazekh, Balbas, and Mazekh/Balbas hybrids were reported. The relationship between host sheep blood transferrin and albumin polymorphisms and cell surface hydrophobicity/5-mC DNA methylation patterns from the predominant gut commensal bacteria was shown. The Narine probiotic eliminates Salmonella from the sheep gut microbiota. At the same time, no significant changes in the percentage of 5-mC DNA methylation of predominant gut bacteria after probiotic administration were observed. The evaluation of bacterial cell surface hydrophobicity, the most significant factor affecting bacterial adhesion, as well as 5-mC DNA methylation, might be used for specific sheep husbandry/breeding programs. This study suggests that the commercial probiotic Narine could potentially be used to reduce Salmonella carriage in sheep.

Method of preparation, visualization and ultrastructural analysis of a formulation of probiotic Bacillus subtilis KATMIRA1933 produced by solid-phase fermentation.

Probiotic preparations are used in medical treatment and in agricultural practice. They modulate numerous activities in eukaryotic hosts, such as: inhibition of pathogenic microbiota; stimulation of immunological responses; and production of antioxidants, anti-mutagens, and DNA protectors. Also, probiotic bacteria are used as a preventive measure to prevent bacterial diseases of the gastrointestinal tract. Solid-phase fermentation is reported as being used in the production of probiotic formulations where a solid substratum, such as soy and oil meal, is utilized for the growth of beneficial microorganisms. However, there are insufficient reports in the literature related to methodological approaches enabling evaluation of the final products of solid-phase fermentation. We suggest a novel method enabling evaluation of probiotic solid-state fermentation dry powders and observation of their morphology, ultrastructure, and elucidation of the quantitative distribution of probiotic microorganisms in solid substrates using electron microscopy. •The method is intended for ultrastructure microphotography of dry substances - for example, ultrastructure of solid-phase fermentation products.•The method allows preserving the ultrastructure of substrates that are damaged when soaking.•The method does not require additional equipment and reagents and can be used in all laboratories using electron microscopy.

Effect of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 on the productivity, reproductive aging, and physiological characteristics of hens and roosters.

The study aims at investigating the effect of preparations of two bacilli strains on laying hens and roosters. Preparations were based on the strains Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895. Several groups of roosters and hens received a preparation based on either strain, or a mixture of both, from the first day to the last day of poultry in production. These preparations improved egg production, quality of sperm production, quality/hatchery of eggs, and slowed down the reproductive aging of hens. These observations were confirmed by the mathematical model proposed herein. At the molecular level, the slowing down of aging was confirmed by a decrease in the amount of mitochondrial DNA damage. Monitoring the physiological parameters of the experimental and control groups of birds showed that live weight gain in all experimental groups was higher than in the control group, and the reproductive organs of hens were more developed. There was also an improvement in the biochemical parameters of blood, the quality of the sperm of roosters, the laying of laying hens, and the morphological and biochemical parameters of the eggs. One of the most significant results is an increase in egg fertilization and a decrease in embryo death during the first 7 days of incubation.

Lactobacillus acidophilus INMIA 9602 Er-2 strain 317/402 probiotic regulates growth of commensal Escherichia coli in gut microbiota of familial Mediterranean fever disease subjects.

Previously, we reported a positive effect the probiotic formulation, Lactobacillus acidophilus INMIA 9602 Er-2 strain 317/402 (Narine strain), had on the blood characteristics of patients with familial Mediterranean fever disease (FMF). The aim of this investigation was to evaluate the effect of the Narine probiotic on growth characteristics in the predominant commensal Escherichia coli isolates from the gut microbiota in FMF-positive study participants. Bacterial growth of 192 prevalent commensal E. coli isolates found in the volunteer participants' guts was evaluated using Verhulst's logistic function. This study showed that the duration of the preparatory growth phase for the E. coli isolates collected from FMF-positive volunteers was significantly shorter, whereas the duration of the logarithmic growth phase was significantly longer (P < 0·03) than that of the isolates collected from healthy participants. The Narine probiotic formulation caused a significant extension (P < 0·001) of the preparatory growth phase in the commensal E. coli isolated from FMF subjects a month after the Narine probiotic administration was terminated. The data suggest that the mathematical model characterizes the growth of commensal E. coli isolates from FMF-positive participants and it can be useful in a decision-making process on the practical use of probiotics during FMF. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the effects of Narine, containing the probiotic Lactobacillus acidophilus, on the growth of gut commensal Escherichia coli from study participants with familial Mediterranean fever disease (FMF). Verhulst's logistic function was demonstrated to act as a possible tool for the evaluation and quantification of effects produced by the probiotic formulation in FMF participants.

DNA-protection and antioxidant properties of fermentates from Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933.

UNLABELLED: DNA protective and antioxidant activity of Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933 were evaluated by Escherichia coli-based Lux biosensors. Two biosensor strains of E. coli, MG1655 (pColD-lux) and MG1655 (pSoxS-lux), which react on DNA damage and superoxide-anion radical activity, were used. SOS-response and Sox-response were stimulated by addition of dioxidine (2,3-Quinoxalinedimethanol,1,4-dioxide) and paraquat (N,N'-dimethyl-4,4'-bipyridinium dichloride) respectively. Preparations of both Bacillus fermentates demonstrated DNA protective and antioxidant (superoxide scavenging) activity (up to 60·19%). The strain К1933 is, in general, characterized by higher DNA protective activity (28·85%), with parameters of antioxidant activity of both bacilli strains being statistically not significantly different. Sporogenous potential probiotic micro-organisms with antioxidant and DNA protective activities can become an effective tool for compensation of various negative oxidative stress processes in humans. SIGNIFICANCE AND IMPACT OF THE STUDY: In humans, oxidative stress is a cause or an important component of many serious diseases, as well as being one of the age influencing factors. Environmental stresses lead to the increase in levels of reactive oxygen species (ROS). Oxidative DNA damage is a side effect of nonspecific inflammation. These human health challenging factors trigger the search for health-promoting bacteria capable of production of antioxidants and DNA-protectors. In this study, two Bacillus strains of interest were shown to produce noticeable DNA protective and antioxidant activities.

Isolation and characterization of large spectrum and multiple bacteriocin-producing Enterococcus faecium strain from raw bovine milk.

AIMS: To assess the antimicrobial properties of lactic acid bacteria from Tunisian raw bovine milk. METHODS AND RESULTS: A bacteriocin-producing Enterococcus faecium strain was isolated from raw cow milk with activity against Gram-positive and Gram-negative bacteria. Antimicrobial substances produced by this strain were sensitive to proteolytic enzymes and were thermostable and resistant to a broad range of pH (2-10). Mode of action of antimicrobial substances was determined as bactericidal. Maximum activity was reached at the end of the exponential growth phase when checked against Listeria ivanovii BUG 496 (2366.62 AU ml(-1)). However, maximum antimicrobial activity against Pseudomonas aeruginosa 28753 was recorded at the beginning of the exponential growth phase. Enterococcus faecium GGN7 was characterized as free from virulence factors and was susceptible to tested antibiotics. PCR analysis of the micro-organism's genome revealed the presence of genes coding for enterocins A and B. Mass spectrometry analysis of RP-HPLC active fractions showed molecular masses corresponding to enterocins A (4835.77 Da) and B (5471.56 Da), and a peptide with a molecular mass of 3215.5 Da active only against Gram-negative indicator strains. The latter was unique in the databases. CONCLUSIONS: Enterococcus faecium GGN7 produces three bacteriocins with different inhibitory spectra. Based on its antimicrobial properties and safety, Ent. faecium GGN7 is potentially useful for food biopreservation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest the bacteriocins from GGN7 strain could be useful for food biopreservation.

Antiherpes simplex virus type 2 activity of the antimicrobial peptide subtilosin.

AIMS: In this study, we evaluated the antiviral activity of subtilosin, a cyclical peptide isolated from Bacillus amyloliquefaciens, against herpes simplex virus type 2 (HSV-2) in cell cultures and we investigated subtilosin mode of action. METHODS AND RESULTS: We determined, using a virus yield inhibition assay, that noncytotoxic concentrations of subtilosin inhibit HSV-2 replication in Vero cell cultures. Subtilosin strongly inhibited extracellular and total virus production even when it was added at 8 h postinfection indicating that not only virus release but also viral particle formation is impeded by the antiviral peptide. Although viral glycoprotein gD level of expression is not affected by the bacteriocin, an altered pattern of gD intracellular localization was detected by immunofluorescence assay in subtilosin-treated culture. On the other hand, at high concentrations, subtilosin displays virucidal action. CONCLUSIONS: Subtilosin displays antiviral and virucidal actions against HSV-2. The target of subtilosin inhibitory effect would be late stages of the viral replicative cycle such as viral glycoprotein intracellular transport. SIGNIFICANCE AND IMPACT OF THE STUDY: Given its antimicrobial activity and its safety for human tissues, subtilosin could represent a valuable alternative to be considered in the development of new microbicide formulations.

Non-dairy probiotic beverages: the next step into human health.

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. The two main genera of microorganisms indicated as sources of probiotic bacteria are Lactobacillus and Bifidobacterium. Historically used to produce fermented dairy products, certain strains of both genera are increasingly utilised to formulate other functional foods. As the consumers' understanding of the role of probiotics in health grows, so does the popularity of food containing them. The result of this phenomenon is an increase in the number of probiotic foods available for public consumption, including a rapidly-emerging variety of probiotic-containing non-dairy beverages, which provide a convenient way to improve and maintain health. However, the composition of non-dairy probiotic beverages can pose specific challenges to the survival of the health conferring microorganisms. To overcome these challenges, strain selection and protection techniques play an integral part in formulating a stable product. This review discusses non-dairy probiotic beverages, characteristics of an optimal beverage, and commonly used probiotic strains, including spore-forming bacteria. It also examines the most recent developments in probiotic encapsulation technology with focus on nano-fibre formation as a means of protecting viable cells. Utilising bacteria's natural armour or creating barrier mechanisms via encapsulation technology will fuel development of stable non-dairy probiotic beverages.

Mode of action and safety of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050.

AIMS: To determine the mechanism of action of antimicrobial protein, lactosporin, against Gardnerella vaginalis and to evaluate its safety in vitro. METHODS AND RESULTS: Bacillus coagulans ATCC 7050 was grown at 37°C for 18 h. The cell-free supernatant was concentrated 10-fold and screened for antimicrobial activity against indicator strain Micrococcus luteus. The mode of action of lactosporin was determined by measuring the potassium release and monitoring the changes in transmembrane potential (Δψ) and transmembrane pH (ΔpH) of the sensitive cells. Lactosporin caused the efflux of potassium ions from M. luteus cells and dissipation of ΔpH in G. vaginalis, while it had no effect on the Δψ. The safety of lactosporin was evaluated by using EpiVaginal(™) ectocervical (VEC-100) tissue model. Over 80% of the cells in the vaginal tissue remained viable after exposure to lactosporin for 24 h. CONCLUSIONS: Lactosporin potentially exerts its antimicrobial activity by selective dissipation of ΔpH and/or by causing leakage of ions from the sensitive cells. Safety studies suggest that lactosporin is a noncytotoxic antimicrobial for vaginal application. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that lactosporin is an effective and safe antimicrobial preparation with potential application for the control of bacterial vaginosis.

The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A.

AIMS: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. METHODS AND RESULTS: Subtilosin was purified from a culture of Bacillus amyloliquefaciens. The minimal inhibitory concentration of subtilosin against L. monocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (ΔΨ) and pH gradient (ΔpH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogenes growth at a concentration of 19 µg ml(-1) . Subtilosin caused a partial depletion of the ΔΨ and had a similar minor effect on the ΔpH. There was no significant efflux of intracellular ATP. CONCLUSION: Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin's mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species-specific mode of action.

The aetiology of bacterial vaginosis.

Bacterial vaginosis (BV) is the most common vaginal infection among women of childbearing age. This condition is notorious for causing severe complications related to the reproductive health of women. Five decades of intense research established many risk factors for acquisition of BV; however, because of the complexity of BV and lack of a reliable animal model for this condition, its exact aetiology remains elusive. In this manuscript, we use a historical perspective to critically review the development of major theories on the aetiology of BV, ultimately implicating BV-related pathogens, healthy vaginal microbiota, bacteriophages and the immune response of the host. None of these theories on their own can reliably explain the epidemiological data. Instead, BV is caused by a complex interaction of multiple factors, which include the numerous components of the vaginal microbial ecosystem and their human host. Many of these factors are yet to be characterized because a clear understanding of their relative contribution to the aetiology of BV is pivotal to the formulation of an effective treatment for and prophylaxis of this condition.

Heterologous Processing and Export of the Bacteriocins Pediocin PA-1 and Lactococcin A in Lactococcus Lactis: A Study with Leader Exchange.

The bacteriocins pediocin PA-1 and lactococcin A are synthesized as precursors carrying N-terminal extensions with a conserved cleavage site preceded by two glycine residues in positions -2 and -1. Each bacteriocin is translocated through the cytoplasmic membrane by an integral membrane protein of the ABC cassette superfamily which, in the case of pediocin PA-1, has been shown to possess peptidase activity responsible for proteolytic cleavage of the pre-bacteriocin. In each case, another integral membrane protein is essential for bacteriocin production. In this study, a two-step PCR approach was used to permutate the leaders of pediocin PA-1 and lactococcin A. Wild-type and chimeric pre-bacteriocins were assayed for maturation by the processing/export machinery of pediocin PA-1 and lactococcin A. The results show that pediocin PA-1 can be efficiently exported by the lactococcin machinery whether it carries the lactococcin or the pediocin leader. It can also compete with wild-type lactococcin A for the lactococcin machinery. Pediocin PA-1 carrying the lactococcin A leader or lactococcin A carrying that of pediocin PA-1 was poorly secreted when complemented with the pediocin PA-1 machinery, showing that the pediocin machinery is more specific for its bacteriocin substrate. Wild-type pre-pediocin and chimeric pre-pediocin were shown to be processed by the lactococcin machinery at or near the double-glycine cleavage site. These results show the potential of the lactococcin LcnC/LcnD machinery as a maturation system for peptides carrying double-glycine-type amino-terminal leaders.

Characterization of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050.

AIMS: To characterize the antimicrobial protein produced by Bacillus coagulans used in the probiotic dietary supplement (Lactospore) Probiotic, Sabinsa Corp., Piscataway, NJ, USA). METHODS AND RESULTS: Bacillus coagulans ATCC 7050 was grown at 37 degrees C for 18 h. The cell free supernatant was concentrated 10-fold (lactosporin preparation, LP). The antimicrobial activity of LP was confirmed against Micrococcus luteus ATCC 10420 in a well diffusion assay. The proteinaceous nature of LP was determined following exposure to different enzymes. The activity of LP was pH-dependent but stable to heat. The isoelectric point of LP was determined to be 3.5-4.0. PCR analyses showed no similarity between lactosporin and known antimicrobial proteins produced by the Bacillus spp. CONCLUSIONS: Lactosporin is a novel antimicrobial protein. Initial characterization indicates that it may fall outside of the conventional classification of class I and II bacteriocins. Loss of activity after exposure to a number of proteolytic enzymes and lipase suggest that lactosporin may posses a lipid moiety which contributes to its inhibitory activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The unique characteristics of lactosporin, including its antimicrobial activity against pathogenic micro-organisms, indicate that it may have potential for application in foods and personal care products.

Salicylic acid-based poly(anhydride esters) for control of biofilm formation in Salmonella enterica serovar Typhimurium.

AIMS: Bacterial biofilms generally are more resistant to stresses as compared with free planktonic cells. Therefore, the discovery of antimicrobial stress factors that have strong inhibitory effects on bacterial biofilm formation would have great impact on the food, personal care, and medical industries. METHODS AND RESULTS: Salicylate-based poly(anhydride esters) (PAE) have previously been shown to inhibit biofilm formation, possibly by affecting surface attachment. Our research evaluated the effect of salicylate-based PAE on biofilm-forming Salmonella enterica serovar Typhimurium. To remove factors associated with surface physical and chemical parameters, we utilized a strain that forms biofilms at the air-liquid interface. Surface properties can influence biofilm characteristics, so the lack of attachment to a solid surface eliminates those constraints. The results indicate that the salicylic acid-based polymers do interfere with biofilm formation, as a clear difference was seen between bacterial strains that form biofilms at the air-liquid interface (top-forming) and those that form at the surface-liquid interface (bottom-forming). CONCLUSION: These results lead to the conclusion that the polymers may not interfere with attachment; rather, the polymers likely affect another mechanism essential for biofilm formation in Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm formation can be prevented through controlled release of nature-derived antimicrobials formulated into polymer systems.

NATURAL ANTIMICROBIALS AND THEIR ROLE IN VAGINAL HEALTH: A SHORT REVIEW.

Lactobacillus species maintain the vaginal ecosystem in a healthy condition by production of antimicrobial substances. Depletion of lactobacilli in the vagina results in bacterial vaginosis (BV), where the normal flora is replaced by several bacterial pathogens, usually Gardnerella vaginalis and obligate anaerobes. BV may cause complications such as premature labor, low birth weight and increased risk of HIV acquisition. The currently recommended antibiotic treatments for BV are not always effective and often lead to reoccurrence of the infection. In many cases, this is due to the antibiotic-resistant forms of the pathogens. Therefore, there is an interest in the development of treatments using antimicrobials derived primarily from Lactobacillus spp., such as ribosomally produced antimicrobial peptides (bacteriocins) and lactic acid. These substances effectively inhibit pathogenic bacteria, are safe and do not pose any threat to healthy vaginal Lactobacillus spp. It may be possible to find an effective treatment against BV while reducing the infection's reoccurrence and the treatment-related complications through hurdle technology. This would be achieved by combining antimicrobials produced by Lactobacillus spp. with different natural antimicrobials obtained from plants or other non-pathogenic organisms.