Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF.

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.

Discovery and synthesis of leaderless bacteriocins from the Actinomycetota.

Leaderless bacteriocins are a unique class of bacteriocins that possess antimicrobial activity after translation and have few cases of documented resistance. Aureocin A53 and lacticin Q are considered two of the most well-studied leaderless bacteriocins. Here, we used in silico genome mining to search for novel aureocin A53-like leaderless bacteriocins in GenBank and MGnify. We identified 757 core peptides across 430 genomes with 75 species found currently without characterized leaderless bacteriocin production. These include putative novel species containing bacteriocin gene clusters (BGCs) from the genera Streptomyces (sp. NBC_00237) and Agrococcus (sp. SL85). To date, all characterized leaderless bacteriocins have been found within the phylum Bacillota, but this study identified 97 core peptides within the phylum Actinomycetota. Members of this phylum are traditionally associated with the production of antibiotics, such is the case with the genus Streptomyces. Actinomycetota is an underexplored phylum in terms of bacteriocin production with no characterized leaderless bacteriocin production to date. The two novel leaderless bacteriocins arcanocin and arachnicin from Actinomycetota members Arcanobacterium sp. and Arachnia sp., respectively, were chemically synthesized and antimicrobial activity was verified. These peptides were encoded in human gut (PRJNA485056) and oral (PRJEB43277) microbiomes, respectively. This research highlights the biosynthetic potential of Actinomycetota in terms of leaderless bacteriocin production and describes the first antimicrobial peptides encoded in the genera Arcanobacterium and Arachnia.IMPORTANCEBacteriocins are gathering attention as alternatives to current antibiotics given the increasing incidence of antimicrobial resistance. Leaderless bacteriocins are considered a commercially attractive subclass of bacteriocins due to the ability to synthesize active peptide and low levels of documented resistance. Therefore, in this work, we mined publicly available data to determine how widespread and diverse leaderless bacteriocins are within the domain of bacteria. Actinomycetota, known for its antibiotic producers but lacking described and characterized bacteriocins, proved to be a rich source of leaderless bacteriocins-97 in total. Two such peptides, arcanocin and arachnicin, were chemically synthesized and have antimicrobial activity. These bacteriocins may provide a novel source of novel antimicrobials that could aid in the development of future alternative antimicrobials and highlight that the Actinomycetota are an underexplored resource of bacteriocin peptides.

Opposing genetic polymorphisms of two ABC transporters contribute to the variation of nukacin resistance in Streptococcus mutans.

Streptococcus mutans is a cariogenic bacterium that produces a variety of bacteriocins and retains resistance to these bacteriocins. In this study, we investigated the susceptibility of 127 S. mutans strains to nukacins produced by Staphylococcus spp., which are commensal bacteria in humans. We detected diverse susceptibilities among strains. Nineteen strains had a disrupted LctF (type I), which is responsible for nukacin susceptibility, whereas the remaining 108 strains had an intact LctF (type II) and displayed resistance to nukacins. However, the type I strains still showed resistance to nukacins to some extent. Interestingly, 18/19 (94.7%) type I strains carried a mukA-T locus, which is related to the synthesis of mutacin K8, and mukFEG, an ABC transporter. In contrast, among type II strains, only 6/108 strains (5.6%) had both the mukA-T locus and mukFEG, 19/108 strains (17.6%) carried only mukFEG, and 83/108 strains (76.9%) harbored neither mukA-T nor mukFEG. We also found that MukF had two variants: 305 amino acids (type α) and 302 amino acids (type ß). All type I strains showed a type α (MukFα), whereas most type II strains with mukFEG (22/25 strains) had a type ß (MukFß). Then, we constructed a mukFEG-deletion mutant complemented with MukFαEG or MukFßEG and found that only MukFαEG was involved in nukacin resistance. The nukacin resistance capability of type II-LctFEG was stronger than that of MukFαEG. In conclusion, we identified a novel nukacin resistance factor, MukFEG, and either LctFEG or MukFEG was active in most strains via genetic polymorphisms depending on mukA-T genes. IMPORTANCE: Streptococcus mutans is an important pathogenic bacterium not only for dental caries but also for systemic diseases. S. mutans is known to produce a variety of bacteriocins and to retain resistance these bacteriocins. In this study, two ABC transporters, LctFEG and MukFEG, were implicated in nukacin resistance and each ABC transporter has two subtypes, active and inactive. Of the two ABC transporters, only one ABC transporter was always resistant, while the other ABC transporter was inactivated by genetic mutation. Interestingly, this phenomenon was defined by the presence or absence of the mutacin K8 synthesis gene region, one of the bacteriocins of S. mutans. This suggests that the resistance acquisition is tightly controlled in each strain. This study provides important evidence that the insertion of bacteriocin synthesis genes is involved in the induction of genetic polymorphisms and suggests that bacteriocin synthesis genes may play an important role in bacterial evolution.

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.

Bacteriocin distribution patterns in Enterococcus faecium and Enterococcus lactis: bioinformatic analysis using a tailored genomics framework.

Multidrug-resistant Enterococcus faecium strains represent a major concern due to their ability to thrive in diverse environments and cause life-threatening infections. While antimicrobial resistance and virulence mechanisms have been extensively studied, the contribution of bacteriocins to E. faecium's adaptability remains poorly explored. E. faecium, within the Bacillota phylum, is a prominent bacteriocin producer. Here, we developed a tailored database of 76 Bacillota bacteriocins (217 sequences, including 40 novel bacteriocins) and applied it to uncover bacteriocin distribution patterns in 997 quality-filtered E. faecium and Enterococcus lactis (former E. faecium clade B) genomes. Curated using computational pipelines and literature mining, our database demonstrates superior precision versus leading public tools in identifying diverse bacteriocins. Distinct bacteriocin profiles emerged between E. faecium and E. lactis, highlighting species-specific adaptations. E. faecium strains from hospitalized patients were significantly enriched in bacteriocins as enterocin A and bacteriocins 43 (or T8), AS5, and AS11. These bacteriocin genes were strongly associated with antibiotic resistance, particularly vancomycin and ampicillin, and Inc18 rep2_pRE25-derivative plasmids, classically associated with vancomycin resistance transposons. Such bacteriocin arsenal likely enhances the adaptability and competitive fitness of E. faecium in the nosocomial environment. By combining a novel tailored database, whole-genome sequencing, and epidemiological data, our work elucidates meaningful connections between bacteriocin determinants, antimicrobial resistance, mobile genetic elements, and ecological origins in E. faecium and provides a framework for elucidating bacteriocin landscapes in other organisms. Characterizing species- and strain-level differences in bacteriocin profiles may reveal determinants of ecological adaptation, and translating these discoveries could further inform strategies to exploit bacteriocins against high-risk clones. IMPORTANCE: This work significantly expands the knowledge on the understudied bacteriocin diversity in opportunistic enterococci, revealing their contribution in the adaptation to different environments. It underscores the importance of placing increased emphasis on genetic platforms carrying bacteriocins as well as on cryptic plasmids that often exclusively harbor bacteriocins since bacteriocin production can significantly contribute to plasmid maintenance, potentially facilitating their stable transmission across generations. Further characterization of strain-level bacteriocin landscapes could inform strategies to combat high-risk clones. Overall, these insights provide a framework for unraveling the therapeutic and biotechnological potential of bacteriocins.

Promiscuous, persistent and problematic: insights into current enterococcal genomics to guide therapeutic strategy.

Vancomycin-resistant enterococci (VRE) are major opportunistic pathogens and the causative agents of serious diseases, such as urinary tract infections and endocarditis. VRE strains mainly include species of Enterococcus faecium and E. faecalis which can colonise the gastrointestinal tract (GIT) of patients and, following growth and persistence in the gut, can transfer to blood resulting in systemic dissemination in the body. Advancements in genomics have revealed that hospital-associated VRE strains are characterised by increased numbers of mobile genetic elements, higher numbers of antibiotic resistance genes and often lack active CRISPR-Cas systems. Additionally, comparative genomics have increased our understanding of dissemination routes among patients and healthcare workers. Since the efficiency of currently available antibiotics is rapidly declining, new measures to control infection and dissemination of these persistent pathogens are urgently needed. These approaches include combinatory administration of antibiotics, strengthening colonisation resistance of the gut microbiota to reduce VRE proliferation through commensals or probiotic bacteria, or switching to non-antibiotic bacterial killers, such as bacteriophages or bacteriocins. In this review, we discuss the current knowledge of the genomics of VRE isolates and state-of-the-art therapeutic advances against VRE infections.

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.

The clinical praxis of bacteriocins as natural anti-microbial therapeutics.

In recent decades, the excessive use of antibiotics has resulted in a rise in antimicrobial drug resistance (ADR). Annually, a significant number of human lives are lost due to resistant infectious diseases, leading to around 700,000 deaths, and it is estimated that by 2050, there could be up to 10 million casualties. Apart from their possible application as preservatives in the food sector, bacteriocins are gaining acknowledgment as potential clinical treatments. Not only this, these antimicrobial peptides have revealed in modulating the host immune system producing anti-inflammatory and anti-modulatory responses. At the same time, due to the ever-increasing global threat of antibiotic resistance, bacteriocins have gained attraction among researchers due to their potential clinical applications. Bacteriocins as antimicrobial peptides, represent one of the most important natural defense mechanisms among bacterial species, particularly lactic acid bacteria (LAB), that can fight against infection-causing pathogens. In this review, we are highlighting the potential of bacteriocins as novel therapeutics for inhibiting a wide range of clinically relevant and multi-drug-resistant pathogens (MDR). We also highlight the effectiveness and potential applications of current bacteriocin treatments in combating antimicrobial resistance (AMR), thereby promoting human health.

Natural preservatives used in foods: a review.

Food quality is adversely affected by physical, chemical, enzymatic, and microbiological reactions, leading to it becoming inedible. Thus, finding alternative methods to preserve foods effectively and extend their shelf life is important. While chemical preservatives have been effective in preventing the growth of harmful pathogens in foods and extending their shelf life, they can also adversely affect consumers' health. For example, nitrites commonly used as preservatives in processed meats have been linked to the development of cancer. This is why researchers, and the food industry are exploring various options to find nontoxic and safe biopreservatives that can be used to preserve food. One such promising option is biopreservatives because they are derived from natural sources, such as plants and insects. This review explores the antimicrobial properties of various biopreservatives, including bacteriocins, polymers, bacteriophages, enzymes, and natural oils, and how they work together to create a synergistic effect in food preservation.

Automated workflow for characterization of bacteriocin production in natural producers Lactococcus lactis and Latilactobacillus sakei.

BACKGROUND: Lactic acid bacteria are commonly used as protective starter cultures in food products. Among their beneficial effects is the production of ribosomally synthesized peptides termed bacteriocins that kill or inhibit food-spoiling bacteria and pathogens, e.g., members of the Listeria species. As new bacteriocins and producer strains are being discovered rapidly, modern automated methods for strain evaluation and bioprocess development are required to accelerate screening and development processes. RESULTS: In this study, we developed an automated workflow for screening and bioprocess optimization for bacteriocin producing lactic acid bacteria, consisting of microcultivation, sample processing and automated antimicrobial activity assay. We implemented sample processing workflows to minimize bacteriocin adsorption to producer cells via addition of Tween 80 and divalent cations to the cultivation media as well as acidification of culture broth prior to cell separation. Moreover, we demonstrated the applicability of the automated workflow to analyze influence of media components such as MES buffer or yeast extract for bacteriocin producers Lactococcus lactis B1629 and Latilactobacillus sakei A1608. CONCLUSIONS: Our automated workflow provides advanced possibilities to accelerate screening and bioprocess optimization for natural bacteriocin producers. Based on its modular concept, adaptations for other strains, bacteriocin products and applications are easily carried out and a unique tool to support bacteriocin research and bioprocess development is provided.

Innovative perspectives on bacteriocins: advances in classification, synthesis, mode of action, and food industry applications.

Bacteriocins, natural antimicrobial peptides produced by bacteria, present eco-friendly, non-toxic, and cost-effective alternatives to traditional chemical antimicrobial agents in the food industry. This review provides a comprehensive update on the classification of bacteriocins in food preservation. It highlights the significant industrial potential of pediocin-like and two-peptide bacteriocins, emphasizing chemical synthesis methods like Fmoc-SPPS to meet the demand for bioactive bacteriocins. The review details the mode of action, focusing on mechanisms such as transmembrane potential disruption and pH-dependent effects. Furthermore, it addresses the limitations of bacteriocins in food preservation and explores the potential of nanotechnology-based encapsulation to enhance their antimicrobial efficacy. The benefits of nanoencapsulation, including improved stability, extended antimicrobial spectrum, and enhanced functionality, are underscored. This understanding is crucial for advancing the application of bacteriocins to ensure food safety and quality.

Bacteriocinogenic anti-listerial properties and safety assessment of Enterococcus faecium and Lactococcus garvieae strains isolated from Brazilian artisanal cheesemaking environment.

AIMS: This study aimed to prospect and isolate lactic acid bacteria (LAB) from an artisanal cheese production environment, to assess their safety, and to explore their bacteriocinogenic potential against Listeria monocytogenes. METHODS AND RESULTS: Samples were collected from surfaces of an artisanal-cheese production facility and after rep-PCR and 16S rRNA sequencing analysis, selected strains were identified as to be belonging to Lactococcus garvieae (1 strain) and Enterococcus faecium (14 isolates, grouped into three clusters) associated with different environments (worktables, cheese mold, ripening wooden shelves). All of them presented bacteriocinogenic potential against L. monocytogenes ATCC 7644 and were confirmed as safe (γ-hemolytic, not presenting antibiotic resistance, no mucus degradation properties, and no proteolytic or gelatinase enzyme activity). Additionally, cell growth, acidification and bacteriocins production kinetics, bacteriocin stability in relation to different temperatures, pH, and chemicals were evaluated. According to performed PCR analysis all studied strains generated positive evidence for the presence of entA and entP genes (for production of enterocins A and enterocins P, respectively). However, pediocin PA-1 associated gene was recorded only in DNA obtained from E. faecium ST02JL and Lc. garvieae ST04JL. CONCLUSIONS: It is worth considering the application of these safe LAB or their bacteriocins in situ as an alternative means of controlling L. monocytogenes in cheese production environments, either alone or in combination with other antimicrobials.

Study of an Enterococcus faecium strain isolated from an artisanal Mexican cheese, whole-genome sequencing, comparative genomics, and bacteriocin expression.

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.

Competitiveness of reutericyclin producing and nonproducing Limosilactobacillus reuteri in food and intestinal ecosystems: a game of rock, paper, and scissors?

The ecological relationships among antimicrobial producing, resistant, and sensitive strains have been proposed to follow rock-paper-scissors dynamics, but evidence is mainly based on Gram-negative bacteriocins in vitro. The ecological relevance of antimicrobials in vivo or in situ has not been systematically studied. This study therefore aimed to analyze binary and ternary competitions among reutericyclin-producing strain Limosilactobacillus reuteri TMW1.656, its reutericyclin-resistant, nonproducing isogenic derivative L. reuteri TMW1.656∆rtcN, and the reutericyclin-sensitive, nonproducing L. reuteri TMW1.656∆rtcN∆rtcT in vitro (liquid culture and static plate), in situ (sourdough fermentation), and in vivo (gut of germ-free mice). In liquid culture, L. reuteri TMW1.656 had a higher fitness than TMW1.656∆rtcN and TMW1.656∆rtcN∆rtcT. Limosilactobacillus reuteri TMW1.656∆rtcN∆rtcT had a higher fitness than TMW1.656∆rtcN. On agar plates, L. reuteri TMW1.656 had a higher fitness than TMW1.656∆rtcN∆rtcT. In situ, reutericyclin production and resistance had no influence on the fitness of the strains. In vivo, TMW1.656 had an advantage over TMW1.656∆rtcN and TMW1.656∆rtcN∆rtcT. Ternary competitions showed reutericyclin production was ecologically beneficial in all ecosystems. The findings support the ecological importance of reutericyclin in a variety of environments/niches, providing an explanation for the acquisition of the reutericyclin gene cluster in L. reuteri and its contribution to the ecological fitness of Streptococcus mutans.

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.

Lactococcus lactis MA5 is a potential autochthonous probiotic for nutrient digestibility enhancement and bacterial pathogen inhibition in hybrid catfish (Ictalurus punctatus × I. furcatus).

With the emergence of diseases, the U.S. catfish industry is under challenge. Current trends prefer autochthonous bacteria as potential probiotic candidates owing to their adaptability and capacity to effectively colonize the host's intestine, which can enhance production performance and bolster disease resistance. The objective of this study was to isolate an autochthonous bacterium as probiotic for hybrid catfish. Initially, an analysis of the intestinal microbiota of hybrid catfish reared in earthen ponds was conducted for subsequent probiotic development. Twenty lactic acid bacteria were isolated from the digesta of overperforming catfish, and most of the candidates demonstrated probiotic traits, including proteolytic and lipolytic abilities; antagonistic inhibition of catfish enteric bacterial pathogens, negative haemolytic activity and antibiotic susceptibility. Subsequent to this screening process, an isolate of Lactococcus lactis (MA5) was deemed the most promising probiotic candidate. In silico analyses were conducted, and several potential probiotic functions were predicted, including essential amino acids and vitamin synthesis. Moreover, genes for three bacteriocins, lactococcin A, enterolysin A and sactipeptide BmbF, were identified. Lastly, various protectant media for lyophilization of MA5 were assessed. These findings suggest that Lactococcus lactis MA5 can be an autochthonous probiotic from hybrid catfish, holding promise to be further tested in feeding trials.

Staphylococcus aureus in Dairy Industry: Enterotoxin Production, Biofilm Formation, and Use of Lactic Acid Bacteria for Its Biocontrol.

Staphylococcus aureus is a well-known pathogen capable of producing enterotoxins during bacterial growth in contaminated food, and the ingestion of such preformed toxins is one of the major causes of food poisoning around the world. Nowadays 33 staphylococcal enterotoxins (SEs) and SE-like toxins have been described, but nearly 95% of confirmed foodborne outbreaks are attributed to classical enterotoxins SEA, SEB, SEC, SED, and SEE. The natural habitat of S. aureus includes the skin and mucous membranes of both humans and animals, allowing the contamination of milk, its derivatives, and the processing facilities. S. aureus is well known for the ability to form biofilms in food processing environments, which contributes to its persistence and cross-contamination in food. The biocontrol of S. aureus in foods by lactic acid bacteria (LAB) and their bacteriocins has been studied for many years. Recently, LAB and their metabolites have also been explored for controlling S. aureus biofilms. LAB are used in fermented foods since in ancient times and nowadays characterized strains (or their purified bacteriocin) can be intentionally added to prolong food shelf-life and to control the growth of potentially pathogenic bacteria. Regarding the use of these microorganism and their metabolites (such as organic acids and bacteriocins) to prevent biofilm development or for biofilm removal, it is possible to conclude that a complex network behind the antagonistic activity remains poorly understood at the molecular level. The use of approaches that allow the characterization of these interactions is necessary to enhance our understanding of the mechanisms that govern the inhibitory activity of LAB against S. aureus biofilms in food processing environments.

Antimicrobial Peptides Derived from Bacteria: Classification, Sources, and Mechanism of Action against Multidrug-Resistant Bacteria.

Antimicrobial peptides (AMPs) are short, usually cationic peptides with an amphiphilic structure, which allows them to easily bind and interact with the cellular membranes of viruses, bacteria, fungi, and other pathogens. Bacterial AMPs, or bacteriocins, can be produced from Gram-negative and Gram-positive bacteria via ribosomal synthesis to eliminate competing organisms. Bacterial AMPs are vital in addressing the increasing antibiotic resistance of various pathogens, potentially serving as an alternative to ineffective antibiotics. Bacteriocins have a narrow spectrum of action, making them highly specific antibacterial compounds that target particular bacterial pathogens. This review covers the two main groups of bacteriocins produced by Gram-negative and Gram-positive bacteria, their modes of action, classification, sources of positive effects they can play on the human body, and their limitations and future perspectives as an alternative to antibiotics.

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.

Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer.

The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively.