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.

Screening canine sources for novel antimicrobials reveals the circular broad-spectrum bacteriocin, caledonicin, produced by Staphylococcus caledonicus.

Introduction: Antimicrobial-resistant pathogens present an ongoing threat to human and animal health, with deaths linked to antimicrobial resistance (AMR) predicted to increase annually. While the misuse and overuse of antibiotics in humans undoubtedly contribute to this escalation, antibiotic use in the veterinary field, including companion animals, also plays a contributing role. Pet owners' desire to improve the quality of life of their pets is likely to support antibiotic use in this field. Consequently, there is a need for antibiotic alternatives to treat bacterial infections. This study set out to screen for antimicrobial peptides known as bacteriocins from bacterial isolates of aerobic/microaerophilic environments of canine sources and determine their potential as antibiotic alternatives against clinically relevant pathogens. Methods: Following a laboratory-based protocol, 22 bacterial isolates were subjected to whole-genome sequencing (WGS), and a total of 14 putative novel bacteriocins were identified from both class I and II bacteriocin classes. One particular bacteriocin, herein named caledonicin, was identified via in silico analysis from a Staphylococcus caledonicus strain and partially purified for further in vitro evaluation. Results: Caledonicin is a 64-amino acid (IAANLGVSSGTAYS MANALNNISNVATA LTIIGTFTGVGTIGSGIA ATILAILKKKGVAAAAAF) novel circular bacteriocin most closely related to enterocin_NKR-5-3B based on core peptide alignment (39.1%), with a molecular weight of 6077.1 Da. Caledonicin exhibits a broad-spectrum of activity against a range of pathogenic bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), methicillin-resistant Staphylococcus pseudintermedius (MRSP), and Listeria monocytogenes; and the gut-related bacterium associated with Crohn's disease, Mediterraneibacter gnavus ATCC 29149 (previously Ruminococcus gnavus ATCC 29149). Discussion: This represents the first bacteriocin screening study involving bacteria from canine sources and confirms this is a rich environment for bacteriocin-producing strains. This study also identifies and characterises the first novel bacteriocin from the staphylococcal species, Staphylococcus caledonicus.

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.

Inhibition of Clinical MRSA Isolates by Coagulase Negative Staphylococci of Human Origin.

Staphylococcus aureus is frequently highlighted as a priority for novel drug research due to its pathogenicity and ability to develop antibiotic resistance. Coagulase-negative staphylococci (CoNS) are resident flora of the skin and nares. Previous studies have confirmed their ability to kill and prevent colonization by S. aureus through the production of bioactive substances. This study screened a bank of 37 CoNS for their ability to inhibit the growth of methicillin-resistant S. aureus (MRSA). Deferred antagonism assays, growth curves, and antibiofilm testing performed with the cell-free supernatant derived from overnight CoNS cultures indicated antimicrobial and antibiofilm effects against MRSA indicators. Whole genome sequencing and BAGEL4 analysis of 11 CoNS isolates shortlisted for the inhibitory effects they displayed against MRSA led to the identification of two strains possessing complete putative bacteriocin operons. The operons were predicted to encode a nukacin variant and a novel epilancin variant. From this point, strains Staphylococcus hominis C14 and Staphylococcus epidermidis C33 became the focus of the investigation. Through HPLC, a peptide identical to previously characterized nukacin KQU-131 and a novel epilancin variant were isolated from cultures of C14 and C33, respectively. Mass spectrometry confirmed the presence of each peptide in the active fractions. Spot-on-lawn assays demonstrated both bacteriocins could inhibit the growth of an MRSA indicator. The identification of natural products with clinically relevant activity is important in today's climate of escalating antimicrobial resistance and a depleting antibiotic pipeline. These findings also highlight the prospective role CoNS may play as a source of bioactive substances with activity against critical pathogens.

Anionic liposome formulation for oral delivery of thuricin CD, a potential antimicrobial peptide therapeutic.

Thuricin CD is a two-peptide antimicrobial produced by Bacillus thuringiensis. Unlike previous antibiotics, it has shown narrow spectrum activity against Clostridioides difficile, a bacterium capable of causing infectious disease in the colon. However, peptide antibiotics have stability, solubility, and permeability problems that can affect their performance in vivo. This work focuses on the bioactivity and bioavailability of thuricin CD with a view to developing a formulation for delivery of active thuricin CD peptides through the gastrointestinal tract (GIT) for local delivery in the colon. The results indicate that thuricin CD is active at low concentrations only when both peptides are present. While thuricin CD was degraded by proteases and was unstable and poorly soluble in gastric fluid, it showed increased solubility in intestinal fluid, probably due to micelle encapsulation. Based on this, thuricin CD was encapsulated in anionic liposomes, which showed increased activity compared to the free peptide, maintained activity after exposure to pepsin in gastric fluid and intestinal fluid, was stable in suspension for over 21 days at room temperature and for 60 days at 4 °C, and exhibited no toxicity to epithelial intestinal cells. These findings suggest that an anionic lipid-based nano formulation may be a promising approach for local oral delivery of thuricin CD.

Impact of bacteriocin-producing strains on bacterial community composition in a simplified human intestinal microbiota.

Bacteriocins are antimicrobial peptides that have been studied for decades as food bio-preservatives or as alternatives to antibiotics. They also have potential as modulators of the gut microbiome, which has been linked to human health. However, it is difficult to predict a priori how bacteriocins will impact complex microbial communities through direct and indirect effects. Here we assess the effect of different bacteriocin-producing strains on a Simplified Human Intestinal Microbiota (SIHUMI) model, using a set of bacteriocin-producing strains (Bac+) and otherwise isogenic non-producers (Bac-). Bacteriocins from different classes and with different activity spectra were selected, including lantibiotics such as lacticin 3147 and nisin A, and pediocin-like bacteriocins such as pediocin PA-1 among other peptides. SIHUMI is a bacterial consortium of seven diverse human gut species that assembles to a predictable final composition in a particular growth medium. Each member can be individually tracked by qPCR. Bac+ and Bac- strains were superimposed on the SIHUMI system, and samples were taken at intervals up to 48 h. The genome copy number of each SIHUMI member was evaluated using specific primers. We establish that the composition of the community changes in response to the presence of either broad- or narrow-spectrum bacteriocin producers and confirm that there are significant off-target effects. These effects were analyzed considering antagonistic inter-species interactions within the SIHUMI community, providing a comprehensive insight into the possible mechanisms by which complex communities can be shaped by bacteriocins.

Selection of lactic acid bacteria as biopreservation agents and optimization of their mode of application for the control of Listeria monocytogenes in ready-to-eat cooked meat products.

In order to meet consumers´ demands for more natural foods and to find new methods to control foodborne pathogens in them, research is currently being focused on alternative preservation approaches, such as biopreservation with lactic acid bacteria (LAB). Here, a collection of lactic acid bacteria (LAB) isolates was characterized to identify potential biopreservative agents. Six isolates (one Lactococcus lactis, one Lacticaseibacillus paracasei and four Lactiplantibacillus plantarum) were selected based on their antimicrobial activity in in vitro assays. Whole genome sequencing showed that none of the six LAB isolates carried known virulence factors or acquired antimicrobial resistance genes, and that the L. lactis isolate was potentially a nisin Z producer. Growth of L. monocytogenes was successfully limited by L. lactis ULE383, L. paracasei ULE721 and L. plantarum ULE1599 throughout the shelf-life of cooked ham, meatloaf and roasted pork shoulder. These LAB isolates were also applied individually or as a cocktail at different inoculum concentrations (4, 6 and 8 log10 CFU/g) in challenge test studies involving cooked ham, showing a stronger anti-Listerial activity when a cocktail was used at 8 log10 CFU/g. Thus, a reduction of up to ~5.0 log10 CFU/g in L. monocytogenes growth potential was attained in cooked ham packaged under vacuum, modified atmosphere packaging or vacuum followed by high pressure processing (HPP). Only minor changes in color and texture were induced, although there was a significant acidification of the product when the LAB cultures were applied. Remarkably, this acidification was delayed when HPP was applied to the LAB inoculated batches. Metataxonomic analyses showed that the LAB cocktail was able to grow in the cooked ham and outcompete the indigenous microbiota, including spoilage microorganisms such as Brochothrix. Moreover, none of the batches were considered unacceptable in a sensory evaluation. Overall, this study shows the favourable antilisterial activity of the cocktail of LAB employed, with the combination of HPP and LAB achieving a complete inhibition of the pathogen with no detrimental effects in physico-chemical or sensorial evaluations, highlighting the usefulness of biopreservation approaches involving LAB for enhancing the safety of cooked meat products.

Modulation of the gut microbiome with nisin.

Nisin is a broad spectrum bacteriocin used extensively as a food preservative that was identified in Lactococcus lactis nearly a century ago. We show that orally-ingested nisin survives transit through the porcine gastrointestinal tract intact (as evidenced by activity and molecular weight determination) where it impacts both the composition and functioning of the microbiota. Specifically, nisin treatment caused a reversible decrease in Gram positive bacteria, resulting in a reshaping of the Firmicutes and a corresponding relative increase in Gram negative Proteobacteria. These changes were mirrored by the modification in relative abundance of pathways involved in acetate, butyrate (decreased) and propionate (increased) synthesis which correlated with overall reductions in short chain fatty acid levels in stool. These reversible changes that occur as a result of nisin ingestion demonstrate the potential of bacteriocins like nisin to shape mammalian microbiomes and impact on the functionality of the community.

The development of a solid lipid nanoparticle (SLN)-based lacticin 3147 hydrogel for the treatment of wound infections.

Chronic wounds affect millions of people globally. This number is set to rise with the increasing incidence of antimicrobial-resistant bacterial infections, such as methicillin-resistant Staphylococcus aureus (MRSA), which impair the healing of chronic wounds. Lacticin 3147 is a two-peptide chain bacteriocin produced by Lactococcus lactis that is active against S. aureus including MRSA strains. Previously, poor physicochemical properties of the peptides were overcome by the encapsulation of lacticin 3147 into solid lipid nanoparticles. Here, a lacticin 3147 solid lipid nanoparticle gel is proposed as a topical treatment for S. aureus and MRSA wound infections. Initially, lacticin 3147's antimicrobial activity against S. aureus was determined before encapsulation into solid lipid nanoparticles. An optimised gel formulation with the desired physicochemical properties for topical application was developed, and the lacticin-loaded solid lipid nanoparticles and free lacticin 3147 aqueous solution were incorporated into separate gels. The release of lacticin 3147 from both the solid lipid nanoparticle and free lacticin gels was measured where the solid lipid nanoparticle gel exhibited increased activity for a longer period (11 days) compared to the free lacticin gel (9 days). Both gels displayed potent activity ex vivo against S. aureus-infected pig skin with significant bacterial eradication (> 75%) after 1 h. Thus, a long-acting potent lacticin 3147 solid lipid nanoparticle gel with the required physicochemical properties for topical delivery of lacticin 3147 to the skin for the potential treatment of S. aureus-infected chronic wounds was developed.

Nisin E Is a Novel Nisin Variant Produced by Multiple Streptococcus equinus Strains.

Nisin A, the prototypical lantibiotic, is an antimicrobial peptide currently utilised as a food preservative, with potential for therapeutic applications. Here, we describe nisin E, a novel nisin variant produced by two Streptococcus equinus strains, APC4007 and APC4008, isolated from sheep milk. Shotgun whole genome sequencing and analysis revealed biosynthetic gene clusters similar to nisin U, with a unique rearrangement of the core peptide encoding gene within the cluster. The 3100.8 Da peptide by MALDI-TOF mass spectrometry, is 75% identical to nisin A, with 10 differences, including 2 deletions: Ser29 and Ile30, and 8 substitutions: Ile4Lys, Gly18Thr, Asn20Pro, Met21Ile, His27Gly, Val32Phe, Ser33Gly, and Lys34Asn. Nisin E producing strains inhibited species of Lactobacillus, Bacillus, and Clostridiodes and were immune to nisin U. Sequence alignment identified putative promoter sequences across the nisin producer genera, allowing for the prediction of genes in Streptococcus to be potentially regulated by nisin. S. equinus pangenome BLAST analyses detected 6 nisin E operons across 44 publicly available genomes. An additional 20 genomes contained a subset of nisin E transport/immunity and regulatory genes (nseFEGRK), without adjacent peptide production genes. These genes suggest that nisin E response mechanisms, distinct from the canonical nisin immunity and resistance operons, are widespread across the S. equinus species. The discovery of this new nisin variant and its immunity determinants in S. equinus suggests a central role for nisin in the competitive nature of the species.

Exploring the antibacterial potential of Lactococcus lactis subsp. lactis bv. diacetylactis BGBU1-4 by genome mining, bacteriocin gene overexpression, and chemical protein synthesis of lactolisterin BU variants.

Lactic acid bacterium Lactococcus lactis BGBU1-4 produces 43 amino acids (aa) long bacteriocin, lactolisterin BU (LBU), a 5.161 kDa peptide with potent antibacterial activity against many Gram-positive pathogens. In addition, BGBU1-4 produces an additional unknown product of 3.642 kDa with antibacterial activity. Here, we determined that the significant amount of naturally produced LBU breaks down to create a 3.642 kDa truncated form of LBU bacteriocin consisting of 31 N-terminal aa (LBU1-31) that exhibits 12.5% the antibacterial activity of the full-length LBU. We showed that chemically synthesized LBU is stable and 50% less active than native LBU, and so we used the synthetic peptides of LBU and its variants to further study their activities and antibacterial potential. Deletion analysis of LBU revealed that the 24 N-terminal aa of LBU (LBU1-24) are responsible for antibacterial activity, while downstream aa (25-43) determine the species-specific effectiveness of LBU. Although LBU1-31 contains aa 1-24, the truncation at position 31 is predicted to change the structure within aa 15-31 and might impact on antibacterial activity. Intriguingly, whole genome sequencing and genome mining established that BGBU1-4 is abundant in genes that encode potential antibacterials, but produces LBU and its breakdown product LBU1-31 exclusively.

Effect of a bacteriocin-producing Streptococcus salivarius on the pathogen Fusobacterium nucleatum in a model of the human distal colon.

The gut microbiome is a vast reservoir of microbes, some of which produce antimicrobial peptides called bacteriocins that may inhibit specific bacteria associated with disease. Fusobacterium nucleatum is an emerging human bacterial pathogen associated with gastrointestinal diseases including colorectal cancer (CRC). In this study, fecal samples of healthy donors were screened for potential bacteriocin-producing probiotics with antimicrobial activity against F. nucleatum. A novel isolate, designated as Streptococcus salivarius DPC6993 demonstrated a narrow-spectrum of antimicrobial activity against F. nucleatum in vitro. In silico analysis of the S. salivarius DPC6993 genome revealed the presence of genes involved in the production of the bacteriocins salivaricin A5 and salivaricin B. After 6 h in a colon fermentation model, there was a significant drop in the number of F. nucleatum in samples that had been simultaneously inoculated with S. salivarius DPC6993 + F. nucleatum DSM15643 compared to those inoculated with F. nucleatum DSM15643 alone (mean ± SD: 9243.3 ± 3408.4 vs 29688.9 ± 4993.9 copies/µl). Furthermore, 16S rRNA amplicon analysis revealed a significant difference in the mean relative abundances of Fusobacterium between samples inoculated with both S. salivarius DPC6993 and F. nucleatum DSM15643 (0.05%) and F. nucleatum DSM15643 only (0.32%). Diversity analysis indicated minimal impact exerted by S. salivarius DPC6993 on the surrounding microbiota. Overall, this study highlights the ability of a natural gut bacterium to target a bacterial pathogen associated with CRC. The specific targeting of CRC-associated pathogens by biotherapeutics may ultimately reduce the risk of CRC development and positively impact CRC outcomes.

Single versus double occupancy solid lipid nanoparticles for delivery of the dual-acting bacteriocin, lacticin 3147.

The bacteriocin lacticin 3147 (lacticin) has shown activity against clinically relevant and antimicrobial-resistant bacteria such as Listeria monocytogenes and Clostridioides difficile. It is composed of two peptides, Ltnα and Ltnß, which work together to form pores in the membrane of Gram-positive bacteria. Lacticin possesses poor aqueous solubility and is degraded by intestinal proteases. In a previous study, peptides encapsulated into solid lipid nanoparticles (SLNs) displayed activity in aqueous media and were protected from enzyme degradation but showed a low encapsulation efficiency (EE%) for Ltnα. In this study, however, lacticin was encapsulated into SLNs both individually (single occupancy, SLNα + SLNß) and together (double occupancy SLNαß) via a nanoprecipitation technique. This achieved SLNs of uniform size with an EE% above 87% for both peptides at loadings of 9 or 18 mg/g of lipid under single occupancy or double occupancy respectively. SLNαß dispersions displayed more potent activity at 3.13 and 1.56 µg/ml lacticin than SLNα + SLNß dispersions. Thus, the SLNαß dispersion was chosen for further analysis. SLNαß dispersions showed no cytotoxicity to endothelial cells. The SLN release media (fasted state simulated intestinal fluid; FaSSIF) retained activity at 1 h and 3 h indicating that lacticin may be sufficiently protected from proteases present in the duodenum. Finally, a reconstituted freeze-dried SLNαß dispersion was stable and achieved 99.99% bacterial killing at 3.125 µg/ml lacticin. Thus, an SLN based lacticin delivery system was developed, potentially enabling oral administration of the bacteriocin to the colon to treat local infections such as C. difficile.

In Vitro and In Silico Based Approaches to Identify Potential Novel Bacteriocins from the Athlete Gut Microbiome of an Elite Athlete Cohort.

Exercise reduces inflammation, fatigue, and aids overall health. Additionally, physical fitness has been associated with desirable changes in the community composition of the athlete gut microbiome, with health-associated taxa being shown to be increased in active individuals. Here, using a combination of in silico and in vitro methods, we investigate the antimicrobial activity of the athlete gut microbiome. In vitro approaches resulted in the generation of 284 gut isolates with inhibitory activity against Clostridioides difficile and/or Fusobacterium nucleatum, and the most potent isolates were further characterized, and potential bacteriocins were predicted using both MALDI-TOF MS and whole-genome sequencing. Additionally, metagenomic reads from the faecal samples were used to recover 770 Metagenome Assembled Genomes (MAGs), of which 148 were assigned to be high-quality MAGs and screened for the presence of putative bacteriocin gene clusters using BAGEL4 software, with 339 gene clusters of interest being identified. Class I was the most abundant bacteriocin class predicted, accounting for 91.3% of predictions, Class III had a predicted abundance of 7.5%, and Class II was represented by just 1% of all predictions.

An oxidation resistant pediocin PA-1 derivative and penocin A display effective anti-Listeria activity in a model human gut environment.

Pediocin PA-1 is a class IIa bacteriocin that is particularly effective against the foodborne pathogen Listeria monocytogenes. The loss of activity of PA-1 pediocin due to methionine oxidation is one of the challenges that limit the wider application of the bacteriocin. In this study, we heterologously expressed an oxidation resistant form of pediocin PA-1, i.e., pediocin M31L, and compared its activity to that of native pediocin PA-1 and to penocin A, a pediocin-like bacteriocin that displays a narrower antimicrobial spectrum. Minimal inhibitory concentration assays revealed that pediocin M31L was as effective as PA-1 and more effective than synthetic penocin A against Listeria with negligible activity against a range of obligate anaerobic commensal gut bacterial species. The anti-Listeria activity of these pediocins was also assessed in a simulated human distal colon model assay using the L. monocytogenes, spiked at 6.5 ± 0.13 Log CFU/mL, as a bioindicator. At 24 h, pediocin M31L and penocin A (2.6 µM) reduced Listeria counts to 3.5 ± 0.4 and 3.64 ± 0.62 Log CFU/mL, respectively, whereas Listeria counts were considerably higher, i.e. 7.75 ± 0.43 Log CFU/mL, in the non-bacteriocin-containing control. Ultimately, it was established that synthetic penocin A and the stable pediocin M31L derivative, heterologously produced, display effective anti-Listeria activity in a human gut environment.

Impact of nisin on Clostridioides difficile and microbiota composition in a faecal fermentation model of the human colon.

AIMS: Nisin is a bacteriocin with a broad spectrum of activity against Gram-positive bacteria. The aims were to assess nisin activity against Clostridioides difficile in a complex microbial environment and determine the minimum inhibitory concentration at which C. difficile growth is suppressed whilst having minimal impact on the faecal microbiota. METHODS AND RESULTS: Faecal slurries were prepared from fresh faecal samples and spiked with C. difficile (106  CFU per ml). Nisin was added to each fermentation at a range of concentrations from 0 to 500 µM. Following 24 h, 16S rRNA gene sequencing was performed, and the presence of viable C. difficile was assessed. There was no viable C. difficile detected in the presence of 50-500 µM nisin. There was a decrease in the diversity of the microbiota in a nisin dose-dependent manner. Nisin predominantly depleted the relative abundance of the Gram-positive bacteria whilst the relative abundance of Gram-negative bacteria such as Escherichia Shigella and Bacteroides increased. CONCLUSIONS: Using an ex vivo model of the colon, this study demonstrates the ability of purified nisin to selectively deplete C. difficile in a faecal microbial environment and establishes the minimum concentration at which this occurs whilst having a minimal impact on the composition of the microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: This study opens up the potential to use nisin as a therapeutic for clostridial gut infections.

Simultaneous Production of Multiple Antimicrobial Compounds by Bacillus velezensis ML122-2 Isolated From Assam Tea Leaf [Camellia sinensis var. assamica (J.W.Mast.) Kitam.].

Bacillus velezensis ML122-2 is an antimicrobial-producing strain isolated from the leaf of Assam tea or Miang [Camellia sinensis var. assamica (J.W.Mast.) Kitam.]. The cell-free supernatant (CFS) of strain ML122-2 exhibits a broad-spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria as well as the mold Penicillium expansum. The genome of B. velezensis ML122-2 was sequenced and in silico analysis identified three potential bacteriocin-associated gene clusters, that is, those involved in the production of mersacidin, amylocyclicin, and LCI. Furthermore, six gene clusters exhibiting homology (75-100% DNA sequence identity) to those associated with the secondary metabolites bacilysin, bacillibactin, surfactin, macrolactin H, bacillaene, and plipastatin were identified. Individual antimicrobial activities produced by B. velezensis ML122-2 were purified and characterized by Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis, revealing three antimicrobial peptides with molecular masses corresponding to surfactin, plipastatin, and amylocyclicin. Transcriptional analysis of specific genes associated with mersacidin (mrsA), amylocyclicin (acnA), plipastatin (ppsA), and surfactin (srfAA) production by B. velezensis ML122-2 showed that the first was not transcribed under the conditions tested, while the latter three were consistent with the presence of the associated peptides as determined by mass spectrometry analysis. These findings demonstrate that B. velezensis ML122-2 has the genetic capacity to produce a wide range of antimicrobial activities that may support a specific community structure and highlight the biotechnological properties of Assam tea.

C-protein α-antigen modulates the lantibiotic thusin resistance in Streptococcus agalactiae.

Screening for producers of potent antimicrobial peptides, resulted in the isolation of Bacillus cereus BGNM1 with strong antimicrobial activity against Listeria monocytogenes. Genome sequence analysis revealed that BGNM1 contains the gene cluster associated with the production of the lantibiotic, thusin, previously identified in B. thuringiensis. Purification of the antimicrobial activity confirmed that strain BGMN1 produces thusin. Both thusin sensitive and resistant strains were detected among clinical isolates of Streptococcus agalactiae. Random mutagenesis of a thusin sensitive strain, S. agalactiae B782, was performed in an attempt to identify the receptor protein for thusin. Three independent thusin resistant mutants were selected and their complete genomes sequenced. Comparative sequence analysis of these mutants with the WT strain revealed that duplication of a region encoding a 79 amino acids repeat in a C-protein α-antigen was a common difference, suggesting it to be responsible for increased resistance to thusin. Since induced thusin resistant mutants showed higher level of resistance than the naturally resistant B761 strain, complete genome sequencing of strain B761 was performed to check the integrity of the C-protein α-antigen-encoding gene. This analysis revealed that this gene is deleted in B761, providing further evidence that this protein promotes interaction of the thusin with receptor.

Pharmaceutical design of a delivery system for the bacteriocin lacticin 3147.

Lacticin 3147 is a dual-acting two-peptide bacteriocin which is generally active against Gram-positive bacteria, including Listeria monocytogenes and antimicrobial-resistant bacteria such as Closteroides difficile in the colon. L. monocytogenes infections can cause life-long effects in the elderly and vulnerable and can cause severe complications in pregnant women. C. difficile causes one of the most common healthcare-associated infections and can be fatal in vulnerable groups such as the elderly. Although lacticin 3147 is degraded by intestinal proteases and has poor aqueous solubility, encapsulation of the bacteriocin could enable its use as an antimicrobial for treating these bacterial infections locally in the gastrointestinal tract. Lacticin 3147 displayed activity in aqueous solutions at a range of pH values and in gastric and intestinal fluids. Exposure to trypsin and α-chymotrypsin resulted in complete inactivation, implying that lacticin 3147 should be protected from these enzymes to achieve successful local delivery to the gastrointestinal tract. The amount of lacticin 3147 dissolved, i.e. its solution concentration, in water or buffered solutions at pH 1.6 and 7.4 was low and varied with time but increased and was stabilized in gastrointestinal fluids by the phospholipid and bile salt components present. Thus, the feasibility of a solid lipid nanoparticle (SLN) delivery system for local administration of lacticin 3147 was investigated. Bacteriocin activity was observed after encapsulation and release from a lipid matrix. Moreover, activity was seen after exposure to degrading enzymes. Further optimization of SLN delivery systems could enable the successful pharmaceutical development of active lacticin 3147 as an alternative to traditional antibiotics.