Nanocomposites: silver nanoparticles and bacteriocins obtained from lactic acid bacteria against multidrug-resistant Escherichia coli and Staphylococcus aureus.

Drug-resistant bacteria such as Escherichia coli and Staphylococcus aureus represent a global health problem that requires priority attention. Due to the current situation, there is an urgent need to develop new, more effective and safe antimicrobial agents. Biotechnological approaches can provide a possible alternative control through the production of new generation antimicrobial agents, such as silver nanoparticles (AgNPs) and bacteriocins. AgNPs stand out for their antimicrobial potential by employing several mechanisms of action that can act simultaneously on the target cell such as the production of reactive oxygen species and cell wall rupture. On the other hand, bacteriocins are natural peptides synthesized ribosomally that have antimicrobial activity and are produced, among others, by lactic acid bacteria (LAB), whose main mechanism of action is to produce pores at the level of the cell membrane of bacterial cells. However, these agents have disadvantages. Nanoparticles also have limitations such as the tendency to form aggregates, which decreases their antibacterial activity and possible cytotoxic effects, and bacteriocins have a narrow spectrum of action, require high doses to be effective, and can be degraded by proteases. Given these limitations, nanoconjugates of these two agents have been developed that can act synergistically in the control of pathogenic bacteria resistant to antibiotics. This review focuses on knowing relevant aspects of the antibiotic resistance of E. coli and S. aureus, the characteristics of these new generation antibacterial agents, and their effect alone or forming nanoconjugates that are more effective against the multiresistant mentioned bacteria.

Acidocin A and Acidocin 8912 Belong to a Distinct Subfamily of Class II Bacteriocins with a Broad Spectrum of Antimicrobial Activity.

Within class II bacteriocins, we assume the presence of a separate subfamily of antimicrobial peptides possessing a broad spectrum of antimicrobial activity. Although these peptides are structurally related to the subclass IIa (pediocin-like) bacteriocins, they have significant differences in biological activities and, probably, a mechanism of their antimicrobial action. A representative of this subfamily is acidocin A from Lactobacillus acidophilus TK9201. We discovered the similarity between acidocin A and acidocin 8912 from Lactobacillus acidophilus TK8912 when analyzing plasmids from lactic acid bacteria and suggested the presence of a single evolutionary predecessor of these peptides. We obtained the C-terminally extended homolog of acidocin 8912, named acidocin 8912A, a possible intermediate form in the evolution of the former. The study of secondary structures and biological activities of these peptides showed their structural similarity to acidocin A; however, the antimicrobial activities of acidocin 8912 and acidocin 8912A were lower than that of acidocin A. In addition, these peptides demonstrated stronger cytotoxic and membranotropic effects. Building upon what we previously discovered about the immunomodulatory properties of acidocin A, we studied its proteolytic stability under conditions simulating those in the digestive tract and also assessed its ability to permeate intestinal epithelium using the Caco-2 cells monolayer model. In addition, we found a pronounced effect of acidocin A against fungi of the genus Candida, which might also expand the therapeutic potential of this bacterial antimicrobial peptide.

ParalichenysinDY4, a novel bacteriocin-like substance, is employed to control Clostridium perfringens.

Clostridium perfringens (C. perfringens) is an important pathogen that contributes to human and animal disease. At present, antibiotic therapy is one of the most effective strategies for C. perfringens. However, with the rise of antibacterial resistance, new agents with novel mechanisms of action are urgently needed. Bacteriocins are recognized as a viable alternative to antibiotics. In this study, the bacteriocin-like substance ParalichenysinDY4, derived from the Bacillus paralicheniformis (B. paralicheniformis) DY4 strain, is investigated as a potential alternative for combating Clostridium perfringens. The substance was isolated from B. paralicheniformis DY4 fermentation broth through a series of purification steps including methanol extraction, gel filtration, and high-performance liquid chromatography. Mass spectrometry analysis of ParalichenysinDY4 revealed that the detected peptide sequences did not match any previously known bacteriocins, indicating it is a novel bacteriocin-like substance. The novel bacteriocin-like substance exhibits effective antibacterial activity and broad antimicrobial spectrum against C. perfringens. Subsequent analyses utilizing methodologies including flow cytometry and scanning electron microscopy suggest that its mechanism of action is linked to its effects on the cell membrane. At the same time, due to its exceptional stability, safety, and efficient ability to remove pathogens both in vitro and in vivo, ParalichenysinDY4 holds promise as a valuable natural antimicrobial agent.

Antibacterial Activity and Cytotoxicity of the Novel Bacteriocin Pkmh.

Bacteriocins are a class of proteins produced by bacteria that are toxic to other bacteria. These bacteriocins play a role in bacterial competition by helping to inhibit potential competitors. In this study, we isolated and purified a novel bacteriocin Pkmh, different from the previously reported bacteriocin PA166, from Pseudomonas sp. strain 166 by ammonium sulfate precipitation, dialysis membrane method, ion exchange chromatography, and gel filtration chromatography. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) revealed that the molecular weight of Pkmh is approximately 35 kDa. Pkmh exhibited potent antimicrobial activity against bovine Mannheimia haemolytica (M. haemolytica) with low cytotoxicity, and lower hemolytic activity was observed. In addition, Pkmh retained antimicrobial activity at different pH ranges (2-10) and temperature conditions (40, 60, 80, 100 °C). Our analysis of its antimicrobial mechanism showed that Pkmh acts on bacterial cell membranes, increasing their permeability and leading to cell membrane rupture and death. In conclusion, Pkmh exhibited low hemolytic activity, low toxicity, and potent antibacterial effects, suggesting its potential as a promising candidate for clinical therapeutic drugs.

Marine Bacteriocins: An Evolutionary Gold Mine to Payoff Antibiotic Resistance.

The rapid evolution of drug resistance is one of the greatest health issues of the 21st century. There is an alarming situation to find new therapeutic strategies or candidate drugs to tackle ongoing multi-drug resistance development. The marine environment is one of the prime natural ecosystems on Earth, the majority of which is still unexplored, especially when it comes to the microbes. A wide variety of bioactive compounds have been obtained from a varied range of marine organisms; however, marine bacteria-produced bacteriocins are still undermined. Owing to the distinct environmental stresses that marine bacterial communities encounter, their bioactive compounds frequently undergo distinct adaptations that confer on them a variety of shapes and functions, setting them apart from their terrestrial counterparts. Bacterially produced ribosomally synthesized and posttranslationally modified peptides (RiPPs), known as bacteriocins, are one of the special interests to be considered as an alternative to conventional antibiotics because of their variety in structure and diverse potential biological activities. Additionally, the gut microbiome of marine creatures are a largely unexplored source of new bacteriocins with promising activities. There is a huge possibility of novel bacteriocins from marine bacterial communities that might come out as efficient candidates to fight against antibiotic resistance, especially in light of the growing pressure from antibiotic-resistant diseases and industrial desire for innovative treatments. The present review summarizes known and fully characterized marine bacteriocins, their evolutionary aspects, challenges, and the huge possibilities of unexplored novel bacteriocins from marine bacterial communities present in diverse marine ecosystems.

Agents Targeting the Bacterial Cell Wall as Tools to Combat Gram-Positive Pathogens.

The cell wall is an indispensable element of bacterial cells and a long-known target of many antibiotics. Penicillin, the first discovered beta-lactam antibiotic inhibiting the synthesis of cell walls, was successfully used to cure many bacterial infections. Unfortunately, pathogens eventually developed resistance to it. This started an arms race, and while novel beta-lactams, either natural or (semi)synthetic, were discovered, soon upon their application, bacteria were developing resistance. Currently, we are facing the threat of losing the race since more and more multidrug-resistant (MDR) pathogens are emerging. Therefore, there is an urgent need for developing novel approaches to combat MDR bacteria. The cell wall is a reasonable candidate for a target as it differentiates not only bacterial and human cells but also has a specific composition unique to various groups of bacteria. This ensures the safety and specificity of novel antibacterial agents that target this structure. Due to the shortage of low-molecular-weight candidates for novel antibiotics, attention was focused on peptides and proteins that possess antibacterial activity. Here, we describe proteinaceous agents of various origins that target bacterial cell wall, including bacteriocins and phage and bacterial lysins, as alternatives to classic antibiotic candidates for antimicrobial drugs. Moreover, advancements in protein chemistry and engineering currently allow for the production of stable, specific, and effective drugs. Finally, we introduce the concept of selective targeting of dangerous pathogens, exemplified by staphylococci, by agents specifically disrupting their cell walls.

Class IIb Microcin MccM Interferes with Oxidative Phosphorylation in Escherichia coli.

Dysbiosis of the human gut microbiota is linked to numerous diseases. Understanding the molecular mechanisms by which microbes interact and compete with one another is required for developing successful strategies to modulate the microbiome. The natural product Microcin M (MccM) consists of a 77-residue bioactive peptide conjugated to a siderophore and is a class II microcin involved in microbial competition with an enigmatic mode-of-action. In this work, we investigated the basis for MccM activity and leveraged bioinformatics to expand the known chemical diversity of class II microcins. We applied automated fast-flow solid phase peptide synthesis coupled with chemoenzymatic chemistry to acquire MccM and demonstrated that its activity was bacteriostatic. We then used our synthetic molecule to ascertain that catecholate siderophore transporters in Escherichia coli K-12 are necessary for MccM import. Once inside the cell, we found that MccM treatment decreased the levels of intracellular ATP and interfered with gene expression. These effects were ameliorated in genetic mutants lacking ATP synthase or in conditions that support substrate-level phosphorylation. Further, we showed that MccM elevated the levels of reactive oxygen species within the target cell. We propose that MccM effects its bacteriostatic activity by decreasing the total energy level of the cell through inhibition of oxidative phosphorylation. Lastly, using genome mining, we bioinformatically identified 171 novel putative class II microcins. Our investigation sheds light on the natural processes involved in microbial competition and provides inspiration, in the form of new molecules, for future therapeutic endeavors.

Pseudocin 196, a novel lantibiotic produced by Bifidobacterium pseudocatenulatum elicits antimicrobial activity against clinically relevant pathogens.

Bacteriocins are broad or narrow-spectrum antimicrobial compounds that have received significant scientific attention due to their potential to treat infections caused by antibiotic-resistant pathogenic bacteria. The genome of Bifidobacterium pseudocatenulatum MM0196, an antimicrobial-producing, fecal isolate from a healthy pregnant woman, was shown to contain a gene cluster predicted to encode Pseudocin 196, a novel lantibiotic, in addition to proteins involved in its processing, transport and immunity. Following antimicrobial assessment against various indicator strains, protease-sensitive Pseudocin 196 was purified to homogeneity from cell-free supernatant. MALDI TOF mass spectrometry confirmed that the purified antimicrobial compound corresponds to a molecular mass of 2679 Da, which is consistent with that deduced from its genetic origin. Pseudocin 196 is classified as a lantibiotic based on its similarity to lacticin 481, a lanthionine ring-containing lantibiotic produced by Lactococcus lactis. Pseudocin 196, the first reported bacteriocin produced by a B. pseudocatenulatum species of human origin, was shown to inhibit clinically relevant pathogens, such as Clostridium spp. and Streptococcus spp. thereby highlighting the potential application of this strain as a probiotic to treat and prevent bacterial infections.

Raffinocyclicin is a novel plasmid-encoded circular bacteriocin produced by Lactococcus raffinolactis with broad-spectrum activity against many gram-positive food pathogens.

This study describes the discovery and characterization of raffinocyclicin, a novel plasmid-encoded circular bacteriocin, produced by the raw milk isolate Lactococcus raffinolactis APC 3967. This bacteriocin has a molecular mass of 6,092 Da and contains 61 amino acids with a three-amino acid leader peptide. It shows the highest identity to the circular bacteriocins bacicyclicin XIN-1 (42.62%), aureocyclicin 4185 (42.62%), and garvicin ML (41.53%). A broad inhibitory spectrum includes strains from Staphylococcus, Enterococcus, Streptococcus, Micrococcus, Lactobacillus, Leuconostoc, and Listeria, in addition to a pronounced inhibitory effect against Lactococcus and Clostridium. It displays low sensitivity to trypsin, most likely as a result of its circular nature. The raffinocyclicin gene cluster is composed of 10 genes: 6 core genes, genes encoding an accessory three-component ABC transporter (rafCDE), and a putative transcriptional regulator related to the MutR family. A lack of inhibitory activity in the cell-free supernatant combined with the pronounced activity of cell extracts suggests that the majority of raffinocyclicin is associated with the cell rather than being released to the extracellular environment. This is the first report of a bacteriocin produced by the L. raffinolactis species.IMPORTANCEThe present study aimed to characterize raffinocyclicin, a novel circular bacteriocin produced by the lactic acid bacteria Lactococcus raffinolactis APC 3967. Bacteriocins are generally cationic and hydrophobic peptides with antimicrobial activity, which present diverse biotechnological properties of interest for the food industry. Raffinocyclicin inhibits a wide range of bacteria, including foodborne pathogens, and is stable against different treatments which suggest its potential as a natural biopreservative. Whole-genome sequencing and the genetic analysis of the raffinocyclicin gene cluster showed that it is encoded by plasmid that could be used in the future to transfer the ability to produce the bacteriocin to other lactic acid bacteria for industrial applications. These results together highlight the potential of this novel antimicrobial as a biopreservative to be used by the food industry.

An insight into structure-activity relationships in subclass IIb bacteriocins: Plantaricin EvF.

This study reports the structure-activity relationships of a unique subclass IIb bacteriocin, plantaricin EvF, which consists of two peptide chains and possesses potent antimicrobial activity. Because the plantaricin Ev peptide chain lacks an α-helix structure, plantaricin EvF is unable to exert its antimicrobial activity through helix-helix interactions like typical subclass IIb bacteriocins. We have shown by various structural evaluation methods that plantaricin Ev can be stabilized by hydrogen bonding at amino acid residues R3, V12, and R13 to the N-terminal region of plantaricin F. This binding gives plantaricin EvF a special spade-shaped structure that exerts antimicrobial activity. In addition, the root-mean-square deviations (RMSDs) of the amino acid residues Y6, F8, and R13 of plantaricin Ev pre- and post-binding were 1.512, 1.723, and 1.369, respectively, indicating that they underwent large structural changes. The alanine scanning experiments demonstrated the important role of the above key amino acids in maintaining the structural integrity of plantaricin EvF. This study not only reveals the unique structural features of plantaricin EvF, but also provides an insight into the structure-activity relationships of subclass IIb bacteriocins.

Biochemical Characterization of Recombinant Enterococcus faecalis EntV Peptide to Elucidate Its Antihyphal and Antifungal Mechanisms against Candida albicans.

Candida albicans is a common opportunistic fungus in humans, whose morphological switch between yeast and hyphae forms represents a key virulence trait. Developing strategies to inhibit C. albicans hyphal growth may provide insights into designs of novel antivirulent therapeutics. Importantly, the gut commensal bacterium, Enterococcus faecalis, secretes a bacteriocin EntV which has potent antivirulent and antifungal effects against C. albicans in infection models; however, hampered by the challenges to access large quantities of bioactive EntV, the detailed understanding of its mechanisms on C. albicans has remained elusive. In this work, we biochemically reconstituted the proteolytic cleavage reaction to obtain recombinant EntV88-His6 on a large preparative scale, providing facile access to the C-terminal EntV construct. Under in vitro C. albicans hyphal assay with specific inducers, we demonstrated that EntV88-His6 exhibits potent bioactivity against GlcNAc-triggered hyphal growth. Moreover, with fluorescent FITC-EntV88-His6, we revealed that EntV88-His6 enters C. albicans via endocytosis and perturbs the proper localization of the polarisome scaffolding Spa2 protein. Our findings provide important clues on EntV's mechanism of action. Surprisingly, we showed that EntV88-His6 does not affect C. albicans yeast cell growth but potently exerts cytotoxicity against C. albicans under hyphal-inducing conditions in vitro. The combination of EntV88-His6 and GlcNAc displays rapid killing of C. albicans, rendering it a promising antivirulent and antifungal agent.

Atomic structures of a bacteriocin targeting Gram-positive bacteria.

Due to envelope differences between Gram-positive and Gram-negative bacteria, engineering precision bactericidal contractile nanomachines requires atomic-level understanding of their structures; however, only those killing Gram-negative bacteria are currently known. Here, we report the atomic structures of an engineered diffocin, a contractile syringe-like molecular machine that kills the Gram-positive bacterium Clostridioides difficile. Captured in one pre-contraction and two post-contraction states, each structure fashions six proteins in the bacteria-targeting baseplate, two proteins in the energy-storing trunk, and a collar linking the sheath with the membrane-penetrating tube. Compared to contractile machines targeting Gram-negative bacteria, major differences reside in the baseplate and contraction magnitude, consistent with target envelope differences. The multifunctional hub-hydrolase protein connects the tube and baseplate and is positioned to degrade peptidoglycan during penetration. The full-length tape measure protein forms a coiled-coil helix bundle homotrimer spanning the entire diffocin. Our study offers mechanical insights and principles for designing potent protein-based precision antibiotics.

Antibacterial mode of action of garviecin LG34 against Gram-negative bacterium Salmonella typhimurium.

Garviecin LG34 produced by Lactococcus garvieae LG34 exhibits wide-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria. This work aimed at clarifying the antibacterial mode of action of garviecin LG34 against Gram-negative bacterium Salmonella typhimurium. To determine the concentration for the bacteriocin antimicrobial mode experiments, the minimum inhibitory concentration of garviecin LG34 against S. typhimurium CICC21484 was determined as 0.25 mg/ml. Garviecin LG34 decreased the viable count of S. typhimurium CICC21484 and its antibacterial activity was the dose and time dependant. Garviecin LG34 led to the dissipation of transmembrane potential, the rise in the extracellular conductivity, UV-absorbing material at 260 nm, and LDH level of S. typhimurium CICC21484. Scanning electron micrographs results shown that garviecin LG34 cause dramatic deformation and fragmentation including the flagellum shedding, pores formation in surface, and even completely breakage of S. typhimurium cell. Moreover, garviecin LG34 decreased the intracellular ATP level. The results of this study demonstrated that garviecin LG34 can destroy cell structure, increase membrane permeability of S. typhimurium, thereby might be used as biopreservative for treating food borne and salmonellosis resulting from Gram-negative bacterium S. typhimurium.

Biosynthesis of bacteriocin BacZY05-silver nanoconjugates and evaluation of their antibacterial properties.

Bacteriocins are antimicrobial peptides produced by bacteria to prevent the growth of pathogens. Combining bacteriocins with metal nanoparticles, like silver nanoparticles (AgNPs), has developed into a viable strategy to get over bacteriocin limitations. In this study, bacteriocin BacZY05 was extracted from Bacillus subtilis ZY05 and purified using various techniques. The resulting purified bacteriocin was then combined with silver nanoparticles to form bacteriocin silver nanoconjugates (BacZY05-AgNPs). The physicochemical properties of the BacZY05-AgNPs were characterized using various analytical techniques. The mean diameter of the synthesized AgNPs was approximately 20-60 nm with an oval or spherical shape. The antimicrobial activity of the BacZY05-AgNPs was evaluated against several indicator strains by their zone of inhibition (ZOI), using the agar well diffusion method. Compared to bacteriocin (ZOI- 13 to 20 mm) and AgNPs (ZOI- 10-22 mm) alone, the antibacterial activity data demonstrated a 1.3-1.5-fold increase in the activity of bacteriocin-nanoconjugates (ZOI- 22 to 26 mm). For Staphylococcus aureus MTCC3103 and Klebsiella pneumoniae MTCC109, BacZY05-capped AgNPs exhibited the lowest minimum inhibitory concentration (MIC), measuring 10.93 µg/mL. For Salmonella typhi NCIM2501, the MIC was 28.75 µg/mL. The highest MIC value was 57.5 µg/mL for Escherichia coli DH5α and Vibrio cholerae MTCC3909. With BacZY05-capped AgNPs, the lowest minimum bactericidal concentration (MBC) of 28.75 µg/mL was observed for Staphylococcus aureus MTCC31003. In the cases of Salmonella typhi NCIM2501 and Klebsiella pneumoniae MTCC109 concentration was 57.5 µg/mL. Vibrio cholerae MTCC3909 and Escherichia coli DH5α had the highest MBC values at 115 µg/mL.

Bactofencin YH, a novel bacteriocin with high inhibitory activity against clinical Streptococcus species.

Strain Lactiplantibacillus plantarum D1 with bacteriocin producing ability was found in the intestine of Gambusia affinis. The bacteriocin was found to have high inhibitory activity against multiple Streptococcus species and several other Gram-positive and Gram-negative bacteria. Bacteriocin was purified from culture supernatant by ion-exchange chromatography, Sep-Pak C18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectral analysis determined that purified bacteriocin has a molecular mass of 2,731 Da. A partial N-terminal sequence KRKKHKXQIYNNGM was obtained from the Edman analysis. The N-terminal sequence was employed to search against a translation of the draft genome of strain D1. The translated full amino acid sequence of the mature peptide is as follows: NH2- KRKKHKCQIYNNGMPTGQYRWC, which has a molecular weight of 2738 Da. A BLAST search revealed that this bacteriocin was most similar to bactofencin A but differed from it with three amino acid residues. No identical peptide or protein has been previously reported, and this peptide, termed bactofencin YH, was therefore considered to be a new bacteriocin produced by Lactiplantibacillus plantarum D1.

Chemical optimization and derivatization of micrococcin p2 to target multiple bacterial infections: new antibiotics from thiopeptides.

Antimicrobial resistance poses a significant threat to humanity, and the development of new antibiotics is urgently needed. Our research has focused on thiopeptide antibiotics such as micrococcin P2 (MP2) and derivatives thereof as new anti-infective agents. Thiopeptides are sulfur-rich, structurally complex substances that exhibit potent activity against Gram-positive pathogens and Mycobacteria species, including clinically resistant strains. The clinical development of thiopeptides has been hampered by the lack of efficient synthetic platforms to conduct detailed structure-activity relationship studies of these natural products. The present contribution touches upon efficient synthetic routes to MP2 that laid the groundwork for clinical translation. The medicinal chemistry campaign on MP2 has been guided by computational molecular dynamic simulations and parallel investigations to improve drug-like properties, such as enhancing the aqueous solubility and optimizing antibacterial activity. Such endeavors have enabled identification of promising lead compounds, AJ-037 and AJ-206, against Mycobacterium avium complex (MAC). Extensive in vitro studies revealed that these compounds exert potent activity against MAC species, a subspecies of non-tuberculous mycobacteria (NTM) that proliferate inside macrophages. Two additional pre-clinical candidates have been identified: AJ-024, for the treatment of Clostridioides difficile infections, and AJ-147, for methicillin-resistant Staphylococcus aureus impetigo. Both compounds compare quite favorably with current first-line treatments. In particular, the ability of AJ-147 to downregulate pro-inflammatory cytokines adds a valuable dimension to its clinical use. In light of above, these new thiopeptide derivatives are well-poised for further clinical development.

Microcin C7 as a Potential Antibacterial-Immunomodulatory Agent in the Postantibiotic Era: Overview of Its Bioactivity Aspects and Applications.

In the postantibiotic era, the pathogenicity and resistance of pathogens have increased, leading to an increase in intestinal inflammatory disease. Bacterial infections remain the leading cause of animal mortality. With increasing resistance to antibiotics, there has been a significant decrease in resistance to both inflammation and disease in animals, thus decreasing production efficiency and increasing production costs. These side effects have serious consequences and have detracted from the development of China's pig industry. Microcin C7 (McC7) demonstrates potent antibacterial activity against a broad spectrum of pathogens, stable physicochemical properties, and low toxicity, reducing the likelihood of resistance development. Thus, McC7 has received increasing attention as a potential clinical antibacterial and immunomodulatory agent. McC7 has the potential to serve as a new generation of antibiotic substitutes; however, its commercial applications in the livestock and poultry industry have been limited. In this review, we summarize and discuss the biosynthesis, biochemical properties, structural characteristics, mechanism of action, and immune strategies of McC7. We also describe the ability of McC7 to improve intestinal health. Our aim in this study was to provide a theoretical basis for the application of McC7 as a new feed additive or new veterinary drug in the livestock and poultry breeding industry, thus providing a new strategy for alleviating resistance through feed and mitigating drug resistance. Furthermore, this review provides insight into the new functions and anti-infection mechanisms of bacteriocin peptides and proposes crucial ideas for the research, product development, and application of bacteriocin peptides in different fields, such as the food and medical industries.

A bacteriocin expression platform for targeting pathogenic bacterial species.

Bacteriocins are antimicrobial peptides that are naturally produced by many bacteria. They hold great potential in the fight against antibiotic resistant bacteria, including ESKAPE pathogens. Engineered live biotherapeutic products (eLBPs) that secrete bacteriocins can be created to deliver targeted bacteriocin production. Here we develop a modular bacteriocin secretion platform that can be used to express and secrete multiple bacteriocins from non-pathogenic Escherichia coli host strains. As a proof of concept we create Enterocin A (EntA) and Enterocin B (EntB) secreting strains that show strong antimicrobial activity against Enterococcus faecalis and Enterococcus faecium in vitro, and characterise this activity in both solid culture and liquid co-culture. We then develop a Lotka-Volterra model that can be used to capture the interactions of these competitor strains. We show that simultaneous exposure to EntA and EntB can delay Enterococcus growth. Our system has the potential to be used as an eLBP to secrete additional bacteriocins for the targeted killing of pathogenic bacteria.

Dual bacteriocin and extracellular vesicle-mediated inhibition of Campylobacter jejuni by the potential probiotic candidate Ligilactobacillus salivarius UO.C249.

Campylobacter jejuni (C. jejuni) is one of the most common causes of foodborne infections worldwide and a major contributor to diarrheal diseases. This study aimed to explore the ability of commensal gut bacteria to control C. jejuni infection. Bacterial strains from the intestinal mucosa of broilers were screened in vitro against C. jejuni ATCC BAA1153. The cell-free supernatant (CFS) of Ligilactobacillus salivarius UO.C249 showed potent dose-dependent antimicrobial activity against the pathogen, likely due to the presence of bacteriocin-like moieties, as confirmed by protease treatment. Genome and exoproteome analyses revealed the presence of known bacteriocins, including Abp118. The genome of Lg. salivarius UO.C249 harbors a 1.8-Mb chromosome and a 203-kb megaplasmid. The strain was susceptible to several antibiotics and had a high survival rate in the simulated chicken gastrointestinal tract (GIT). Post-protease treatment revealed residual inhibitory activity, suggesting alternative antimicrobial mechanisms. Short-chain fatty acid (SCFA) quantification confirmed non-inhibitory levels of acetic (24.4 ± 1.2 mM), isovaleric (34 ± 1.0 µM), and butyric (32 ± 2.5 µM) acids. Interestingly, extracellular vesicles (EVs) isolated from the CFS of Lg. salivarius UO.C249 were found to inhibit C. jejuni ATCC BAA-1153. Proteome profiling of these EVs revealed the presence of unique proteins distinct from bacteriocins identified in CFS. The majority of the identified proteins in EVs are located in the membrane and play roles in transmembrane transport and peptidoglycan degradation, peptidase, proteolysis, and hydrolysis. These findings suggest that although bacteriocins are a primary antimicrobial mechanism, EV production also contributes to the inhibitory activity of Lg. salivarius UO.C249 against C. jejuni. IMPORTANCE: Campylobacter jejuni (C. jejuni) is a major cause of gastroenteritis and a global public health concern. The increasing antibiotic resistance and lack of effective alternatives in livestock production pose serious challenges for controlling C. jejuni infections. Therefore, alternative strategies are needed to control this pathogen, especially in the poultry industry where it is prevalent and can be transmitted to humans through contaminated food products. In this study, Ligilactobacillus salivarius UO.C249 isolated from broiler intestinal mucosa inhibited C. jejuni and exhibited important probiotic features. Beyond bacteriocins, Lg. salivarius UO.C249 secretes antimicrobial extracellular vesicles (EVs) with a unique protein set distinct from bacteriocins that are involved in transmembrane transport and peptidoglycan degradation. Our findings suggest that beyond bacteriocins, EV production is also a distinct inhibitory signaling mechanism used by Lg. salivarius UO.C249 to control C. jejuni. These findings hold promise for the application of probiotic EVs for pathogen control.

Production and SERS characterization of bacteriocin-like inhibitory substances by latilactobacillus sakei in whey permeate powder: exploring natural antibacterial potential.

Bacteriocins are antimicrobial compounds that have awakened interest across several industries due to their effectiveness. However, their large-scale production often becomes unfeasible on an industrial scale, primarily because of high process costs. Addressing this challenge, this work analyzes the potential of using low-cost whey permeate powder, without any supplementation, to produce bacteriocin-like inhibitory substances (BLIS) through the fermentation of Latilactobacillus sakei. For this purpose, different concentrations of whey permeate powder (55.15 gL-1, 41.3 gL-1 and 27.5 gL-1) were used. The ability of L. sakei to produce BLIS was evaluated, as well as the potential of crude cell-free supernatant to act as a preservative. Raman spectroscopy and surface-enhanced Raman scattering (SERS) provided detailed insights into the composition and changes occurring during fermentation. SERS, in particular, enhanced peak definition significantly, allowing for the identification of key components, such as lactose, proteins, and phenylalanine, which are crucial in understanding the fermentation process and BLIS characteristics. The results revealed that the concentration of 55.15 gL-1 of whey permeate powder, in flasks without agitation and a culture temperature of 32.5 °C, presented the highest biological activity of BLIS, reaching 99% of inhibition of Escherichia coli and Staphylococcus aureus with minimum inhibitory concentration of 36-45%, respectively. BLIS production began within 60 h of cultivation and was associated with class II bacteriocins. The results demonstrate a promising approach for producing BLIS in an economical and environmentally sustainable manner, with potential implications for various industries.