Enterococci are ubiquitous microorganisms in almost all environments, from the soil we step on to the food we eat. They are frequently found in naturally fermented foods, contributing to ripening through protein, lipid, and sugar metabolism. On the other hand, these organisms are also leading the current antibiotic resistance crisis. In this study, we performed whole-genome sequencing and comparative genomics of an Enterococcus faecium strain isolated from an artisanal Mexican Cotija cheese, namely QD-2. We found clear genomic differences between commensal and pathogenic strains, particularly in their carbohydrate metabolic pathways, resistance to vancomycin and other antibiotics, bacteriocin production, and bacteriophage and CRISPR content. Furthermore, a bacteriocin transcription analysis performed by RT-qPCR revealed that, at the end of the log phase, besides enterocins A and X, two putative bacteriocins not reported previously are also transcribed as a bicistronic operon in E. faecium QD-2, and are expressed 1.5 times higher than enterocin A when cultured in MRS broth.

Food preservation is a schematic and scientific procedure employed for the maintenance and improvement of food\'s quality, shelf life, and nutritional value. Although, on one hand, ancient conventional methods such as freezing, pasteurization, canning, and chemical methods have the potential to lengthen the shelf life of edible substances, but on the other hand, they can deteriorate its nutritional value as well. Present research focuses on the identification of promising bacteriocins against Pseudomonas fragi via subtractive proteomics pipeline as an alternative approach for food preservation. Bacteriocins are small peptides produced by certain microbes to naturally defend themselves by destroying other closely related bacteria residing in their neighborhood. P. fragi lies among the most notable microbes responsible for the elicitation of food spoilage. Due to increasing emergence and prevalence of multidrug resistance bacteria, there is a need to unravel novel drug targets, crucially involved in food decay process. Based on subtractive scrutinization, UDP-N-acetylglucosamine O-acyltransferase (LpxA) was chosen as promising therapeutic protein target that could play a significant role in progression of food spoilage. Subtilosin A, thuricin-CD, and mutacin B-NY266 were found as the most robust inhibitors of LpxA according to the molecular docking assay results. Molecular dynamic simulations and binding energy calculations via MM/PBSA method of LpxA and three top hit docked complexes, i.e., LpxA-subtilosin A, LpxA-thuricin-CD, and LpxA-mutacin B-NY266, revealed stability throughout simulations and ensured that shortlisted bacteriocins had strong affinity for LpxA.

Phage tail-like bacteriocins (PTLBs) are large proteomic structures similar to the tail phages. These structures function in bacterial competition by making pores in the membrane of their competitors. The PTLBs identified in Pseudomonas aeruginosa are known as R-type and F-type pyocins, which have a narrow spectrum of action. Their specificity is determined by the tail fiber and is closely related to the lipopolysaccharide type of the target competitor strain. In this study, the genome sequences of 32 clinical of P. aeruginosa clinical isolates were analysed to investigate the presence of R-type and F-type pyocins, and one was detected in all strains tested. The pyocins were classified into 4 groups on the basis of the tail fiber and also the homology, phylogeny and structure of the cluster components. A relationship was established between these groups and the sequence type and serotype of the strain of origin and finally the killing spectrum of the representative pyocins was determined showing a variable range of activity between 0 and 37.5%. The findings showed that these pyocins could potentially be used for typing of P. aeruginosa clinical isolates, on the basis of their genomic sequence and cluster structure, and also as antimicrobial agents.

The antibiotic-resistant bacteria are emerging as a great threat worldwide. For this reason it is important to develop new antibiotic substances. Bacillus is considered as a factory of a wide range of chemical compounds with a variety of activities. Among these substances are bacteriocins which are small peptides showing stability in a wide range of pH and temperatures and having a potent antibacterial activity. Bacillus species can be grouped into families such as B. cereus group based on their genetic similarity. It can be helpful to study the bacteriocins presented in these related species identifying the differences and similarities between them to relate the presence of a given bacteriocin with the producer specie. The aim of this study was to isolate the bacteriocins from three related species of B. cereus group such as B. mycoides, B. weihenstephanensis and B. toyonensis and compare among them and with the bacteriocins isolated from B. velezensis. Besides it was analyzed the bactericidal activity of each isolated bacteriocin. Five different bacteriocins of similar molecular mass and specific against foodborne pathogens were isolated from three Bacillus species related to B. cereus group, that were quite different both in molecular mass and bactericidal activity from that was isolated from B. velezensis. The results indicated that bacteriocins can be distinguished according to Bacillus specie from it has been isolated.

Most bacteria in nature exist in multispecies communities known as biofilms. In the natural habitat where resources (nutrient, space, etc.) are usually limited, individual species must compete or collaborate with other neighboring species in order to perpetuate in the multispecies community. The human oral cavity is colonized by >700 microbial species known as the indigenous microbiota. This indigenous flora normally maintains an ecological balance through antagonistic as well as mutualistic interspecies interactions. However, environmental perturbation may disrupt this balance, leading to overgrowth of pathogenic species which could in turn initiate diseases such as dental caries (tooth decay) and periodontitis (gum disease). Understanding the mechanisms of diversity maintenance may help developing novel approaches to manage these \"polymicrobial diseases\". In this chapter, we will focus on a well-characterized form of biochemical warfare: bacteriocins produced by Streptococcus mutans, a primary dental caries pathogen, and hydrogen peroxide (H2O2) produced by several oral commensal streptococci. We will describe detailed methodologies on the competition assay, isolation, purification, and characterization of bacteriocins.

The present work aimed to assess the general antagonistic activity against opportunistic pathogens and to compare antagonistic action spectra of lactic acid bacteria (LAB) strains, isolated from Ukrainian traditional fermented foods. Overall, 161 profiles of the antagonistic activity spectrum were obtained from 1056 LAB strains. Among them, 114 profiles were genus-specific and 47 spectra of antagonistic activity were found in LAB strains of different genera. Furthermore, 19 LAB strains were active only against Gram-negative indicator strains and 149 LAB strains only against Gram-positive indicator strains. The size of growth inhibition zones of indicator strains by LAB strains of each genus did not correlate with the level of acidification. Zones of growth inhibition of indicator strains appeared after 6-8 h of incubation and in most cases decreased with further incubation, up to absence after 24 h. The difference in the antagonistic activity of 16-h-old and 24-h-old hours LAB cultures also was found. Among LAB tested, 241 strains are the most promising for further practical use, they have antagonistic action towards 10 indicator strains. The cross-streaking method can be used for rapid screening of bacteriocinogenic LAB strains and has advantages over the well-diffusion assay. To the best of our knowledge, this is the first report on a comparative characteristic of spectra of antagonistic activity against opportunistic pathogens of LAB strains belonging to different genera.

Bacteriocins are receiving increased attention as potent candidates in food preservation and medicine. Although the inhibitory activity of bacteriocins has been studied widely, little is known about their gastrointestinal stability and toxicity toward normal human cell lines. The aim of this study was to evaluate the gastrointestinal stability and activity of microcin J25, pediocin PA-1, bactofencin A and nisin using in vitro models. In addition cytotoxicity and hemolytic activity of these bacteriocins were investigated on human epithelial colorectal adenocarcinoma cells (Caco-2) and rat erythrocytes, respectively. Pediocin PA-1, bactofencin A, and nisin were observed to lose their stability while passing through the gastrointestinal tract, while microcin J25 is only partially degraded. Besides, selected bacteriocins were not toxic to Caco-2 cells, and integrity of cell membrane was observed to remain unaffected in presence of these bacteriocins at concentrations up to 400 μg/mL. In hemolysis study, pediocin PA-1, bactofencin A, and nisin were observed to lyse rat erythrocytes at concentrations higher than 50 μg/mL, while microcin J25 showed no effect on these cells. According to data indicating gastrointestinal degradation and the absence of toxicity of pediocin PA-1, bactofencin A, and microcin J25 they could potentially be used in food or clinical applications.

Staphylococcus aureus colonizes patients with atopic dermatitis (AD) and exacerbates disease by promoting inflammation. The present study investigated the safety and mechanisms of action of Staphylococcus hominis A9 (ShA9), a bacterium isolated from healthy human skin, as a topical therapy for AD. ShA9 killed S. aureus on the skin of mice and inhibited expression of a toxin from S. aureus (psmα) that promotes inflammation. A first-in-human, phase 1, double-blinded, randomized 1-week trial of topical ShA9 or vehicle on the forearm skin of 54 adults with S. aureus-positive AD (NCT03151148) met its primary endpoint of safety, and participants receiving ShA9 had fewer adverse events associated with AD. Eczema severity was not significantly different when evaluated in all participants treated with ShA9 but a significant decrease in S. aureus and increased ShA9 DNA were seen and met secondary endpoints. Some S. aureus strains on participants were not directly killed by ShA9, but expression of mRNA for psmα was inhibited in all strains. Improvement in local eczema severity was suggested by post-hoc analysis of participants with S. aureus directly killed by ShA9. These observations demonstrate the safety and potential benefits of bacteriotherapy for AD.

Applying a circular economy approach, this research explores the use of cheese whey permeate (CWP), by-product of whey ultrafiltration, as cheap substrate for the production of bacterial cellulose (BC) and Sakacin-A, to be used in an antimicrobial packaging material. BC from the acetic acid bacterium Komagataeibacter xylinus was boosted up to 6.77 g/L by supplementing CWP with β-galactosidase. BC was then reduced to nanocrystals (BCNCs, 70% conversion yield), which were then conjugated with Sakacin-A, an anti-Listeria bacteriocin produced by Lactobacillus sakei in a CWP based broth. Active conjugates (75 Activity Units (AU)/mg), an innovative solution for bacteriocin delivery, were then included in a coating mixture applied onto paper sheets at 25 AU/cm2. The obtained antimicrobial food package was found effective in reducing Listeria population in storage trials carried out on a fresh Italian soft cheese (named \"stracchino\") intentionally inoculated with Listeria. Production costs of the active material have been mainly found to be associated (90%) to the purification steps. Setting a maximum prudential 50% cost reduction during process up-scaling, conjugates coating formulation would cost around 0.89 €/A4 sheet. Results represent a practical example of a circular economy production procedure by using a food industry by-product to produce antimicrobials for food preservation.

BACKGROUND: Plantaricin IIA-1A5 is a bacteriocin produced by Lactobacillus plantarum IIA-1A5, a locally isolated probiotic from Indonesia. Plantaricin IIA-1A5 exhibits antibacterial activity against wide spectrum of pathogenic bacteria, thus promising to be applied in various food products. Nevertheless, thermal stability of this bacteriocin remains to be fully investigated.
OBJECTIVE: This study aims to determine thermal stability of plantaricin IIA-1A5 through kinetic and thermodynamic parameters.
METHODS: To address, plantaricin IIA-1A5 was purified from Lactobacillus plantarum IIA-1A5, which was growth under whey media, using ammonium sulfate precipitation followed by ionexchange chromatography. Purified plantaricin IIA-IA5 was then subjected to analysis of its bacteriocin activity. The thermal inactivation of bacteriocin from L. plantarum IIA-1A5 was calculated by incubating the bacteriocin at different temperatures ranging from 60-80 °C for 30 to 90 min, which was then used to calculate its kinetic and thermodynamic parameters.
RESULTS: The result showed the inactivation rates (k-value) were ranging from 0.008 to 0.013 min-1. Heat resistance of plantaricin IIA-1A5 (D-value) at constant heating temperature of 60, 65, 70, 75, and 80 °C were 311.6, 305.9, 294.5, 198.9, and 180.2 min, which indicated a faster inactivation at higher temperatures. D-value sensitivity for temperature changes (z-value) was calculated to be 75.76 °C. Further, thermodynamic analysis suggested that plantaricin IIA-1A5 is thermostable, with activation energy (Ea) of 29.02 kJ mol-1.
CONCLUSIONS: This result showed that plantaricin IIA-1A5 is considerably more heat-stable than plantaricin members and promises to be applied in food industries where heat treatments are applied. Furthermore, a possible mechanism by which plantaricin IIA-1A5 maintains its stability was also discussed by referring to its thermodynamic parameters.