Microcin Research Articles

Bacteriocin Microcin J25’s antibacterial infection effects and novel non-microbial regulatory mechanisms: differential regulation of dopaminergic receptors

BackgroundThe antibacterial and immunomodulatory activities of bacteriocins make them attractive targets for development as anti-infective drugs. Although the importance of the enteric nervous system (ENS) in the struggle against infections of the intestine has been demonstrated, whether it is involved in bacteriocins anti-infective mechanisms is poorly defined.ResultsHere, we demonstrated that the bacteriocin Microcin J25 (J25) significantly alleviated diarrhea and intestinal inflammation in piglets caused by enterotoxigenic Escherichia coli (ETEC) infection. Mechanistically, macrophage levels were significantly downregulated after J25 treatment, and this was replicated in a mouse model. Omics analysis and validation screening revealed that J25 treatment induced significant changes in the dopaminergic neuron pathway, but little change in microbial structure. The alleviation of inflammation may occur by down-regulating dopamine receptor (DR) D1 and the downstream DAG-PKC pathway, thus inhibiting arachidonic acid decomposition, and the inhibition of macrophages may occur through the up-regulation of DRD5 and the downstream cAMP-PKA pathway, thus inhibiting NF-κB.ConclusionsOur studies’ findings provide insight into the changes and possible roles of the ENS in J25 treatment of ETEC infection, providing a more sophisticated foundational understanding for developing the application potential of J25.

Structure Prediction and Protein Engineering Yield New Insights into Microcin J25 Precursor Recognition.

Microcin J25 (MccJ25), a lasso peptide antibiotic with a unique structure that resembles the lariat knot, has been a topic of intense interest since its discovery in 1992. The precursor (McjA) contains a leader and a core segment. McjB is a protease activated upon binding to the leader, and McjC converts the core segment into the mature MccJ25. Previous studies suggested that these biosynthetic steps likely proceed in a (nearly) concerted fashion; however, there is only limited information regarding the structural and molecular intricacies of MccJ25 biosynthesis. To close this knowledge gap, we used AlphaFold2 to predict the structure of the precursor (McjA) in complex with its biosynthetic enzymes (McjB and McjC) and queried the critical predicted features by protein engineering. Based on the predicted structure, we designed protein variants to show that McjB can still be functional and form a proficient biosynthetic complex with McjC when its recognition and protease domains were circularly permutated or split into separate proteins. Specific residues important for McjA recognition were also identified, which permitted us to pinpoint a compensatory mutation (McjBM108T) to restore McjA/McjB interaction that rescued an otherwise nearly nonproductive precursor variant (McjAT-2M). Studies of McjA, McjB, and McjC have long been mired by them being extremely difficult to handle experimentally, and our results suggest that the AF2 predicted ternary complex structure may serve as a reasonable starting point for understanding MccJ25 biosynthesis. The prediction-validation workflow presented herein combined artificial intelligence and laboratory experiments constructively to gain new insights.

Merging Flow Synthesis and Enzymatic Maturation to Expand the Chemical Space of Lasso Peptides.

Many peptidic natural products, such as lasso peptides, cyclic peptides, and cyclotides, are conformationally constrained and show biological stability, making them attractive scaffolds for drug development. Although many peptides can be synthesized and modified through chemical methods, knot-like lasso peptides such as microcin J25 (MccJ25) and their analogues remain elusive. As the chemical space of MccJ25 analogues accessible through purely biological methods is also limited, we proposed a hybrid approach: flow-based chemical synthesis of non-natural precursor peptides, followed by in vitro transformation with recombinant maturation enzymes, to yield a more diverse array of lasso peptides. Herein, we established the rapid, flow-based synthesis of chemically modified MccJ25 precursor peptides (57 amino acids). Heterologous expression of enzymes McjB and McjC was extensively optimized to improve yields and facilitate the synthesis of multiple analogues of MccJ25, including the incorporation of non-canonical tyrosine and histidine derivatives into the lasso scaffold. Finally, using our chemoenzymatic strategy, we produced a biologically active analogue containing three d-amino acids in the loop region and incorporated backbone N-methylations. Our method provides rapid access to chemically modified lasso peptides that could be used to investigate structure-activity relationships, epitope grafting, and the improvement of therapeutic properties.

Effects of antibacterial peptide microcin J25 on growth performance, antioxidant capacity, intestinal barrier function and intestinal microbiota in pigeon squabs

The present study aims to evaluate the effects of the antibacterial peptide Microcin J25 (ABP MccJ25) on growth performance, intestinal barrier function, antioxidant capacity and ileal microbiota in pigeon squabs. The 80 pairs of American Silver King pigeon parents and 240 healthy day 1 squabs were randomly divided into four groups, including 4 parent pairs and 12 pigeon squabs per group with 5 replicates. Parent pigeons were fed a basal diet (CON) or the addition of 100, 200, 300 mg/kg of ABP MccJ25 (ABP100, ABP200, and ABP300, respectively) for 4 weeks. Results showed that the group ABP200 remarkably reduced the feed conversion ratio and increased average daily gain in pigeon squabs from 7 to 21 d (p < 0.05), and improved serum antioxidant capacity in pigeon squabs from 14 to 28 d (p < 0.05). Villus height and the ratio of villus height to crypt depth in the duodenum, jejunum, and ileum were significantly increased in groups ABP200 and ABP300 (p < 0.05). In addition, group ABP200 obviously up-regulated the relative mRNA expression of intestinal antioxidant-related genes and barrier-related genes (p < 0.05). Moreover, the proportion of beneficial bacteria including Firmicutes and Lactobacillales, which were favourable to the intestinal health of pigeon squabs increased noticeably (p < 0.05). Taken together, these results indicated that group ABP200 enhanced antioxidant capacity, strengthened intestinal barrier function, and facilitated the regulation of intestinal microbiota, leading to the improvement in growth performance of pigeon squabs. Therefore, this study provides support for the application of 200 mg/kg ABP MccJ25 in pigeon production.

Exploring the genetic basis of natural resistance to microcins.

Enterobacteriaceae produce an arsenal of antimicrobial compounds including microcins, ribosomally produced antimicrobial peptides showing diverse structures and mechanisms of action. Microcins target close relatives of the producing strain to promote its survival. Their narrow spectrum of antibacterial activity makes them a promising alternative to conventional antibiotics, as it should decrease the probability of resistance dissemination and collateral damage to the host's microbiota. To assess the therapeutic potential of microcins, there is a need to understand the mechanisms of resistance to these molecules. In this study, we performed genomic analyses of the resistance to four microcins [microcin C, a nucleotide peptide; microcin J25, a lasso peptide; microcin B17, a linear azol(in)e-containing peptide; and microcin E492, a siderophore peptide] on a collection of 54 Enterobacteriaceae from three species: Escherichia coli, Salmonella enterica and Klebsiella pneumoniae. A gene-targeted analysis revealed that about half of the microcin-resistant strains presented mutations of genes involved in the microcin mechanism of action, especially those involved in their uptake (fhuA, fepA, cirA and ompF). A genome-wide association study did not reveal any significant correlations, yet relevant genetic elements were associated with microcin resistance. These were involved in stress responses, biofilm formation, transport systems and acquisition of immunity genes. Additionally, microcin-resistant strains exhibited several mutations within genes involved in specific metabolic pathways, especially for S. enterica and K. pneumoniae.

Lasso Peptides: Exploring the Folding Landscape of Nature's Smallest Interlocked Motifs.

Lasso peptides make up a class of natural products characterized by a threaded structure. Given their small size and stability, chemical synthesis would offer tremendous potential for the development of novel therapeutics. However, the accessibility of the pre-folded lasso architecture has limited this advance. To better understand the folding process de novo, simulations are used herein to characterize the folding propensity of microcin J25 (MccJ25), a lasso peptide known for its antimicrobial properties. New algorithms are developed to unambiguously distinguish threaded from nonthreaded precursors and determine handedness, a key feature in natural lasso peptides. We find that MccJ25 indeed forms right-handed pre-lassos, in contrast to past predictions but consistent with all natural lasso peptides. Additionally, the native pre-lasso structure is shown to be metastable prior to ring formation but to readily transition to entropically favored unfolded and nonthreaded structures, suggesting that de novo lasso folding is rare. However, by altering the ring forming residues and appending thiol and thioester functionalities, we are able to increase the stability of pre-lasso conformations. Furthermore, conditions leading to protonation of a histidine imidazole side chain further stabilize the modified pre-lasso ensemble. This work highlights the use of computational methods to characterize lasso folding and demonstrates that de novo access to lasso structures can be facilitated by optimizing sequence, unnatural modifications, and reaction conditions like pH.

The pearl jubilee of microcin J25: thirty years of research on an exceptional lasso peptide.

Covering: 1992 up to 2023Since their discovery, lasso peptides went from peculiarities to be recognized as a major family of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products that were shown to be spread throughout the bacterial kingdom. Microcin J25 was first described in 1992, making it one of the earliest known lasso peptides. No other lasso peptide has since then been studied to such an extent as microcin J25, yet, previous review articles merely skimmed over all the research done on this exceptional lasso peptide. Therefore, to commemorate the 30th anniversary of its first report, we give a comprehensive overview of all literature related to microcin J25. This review article spans the early work towards the discovery of microcin J25, its biosynthetic gene cluster, and the elucidation of its three-dimensional, threaded lasso structure. Furthermore, the current knowledge about the biosynthesis of microcin J25 and lasso peptides in general is summarized and a detailed overview is given on the biological activities associated with microcin J25, including means of self-immunity, uptake into target bacteria, inhibition of the Gram-negative RNA polymerase, and the effects of microcin J25 on mitochondria. The in vitro and in vivo models used to study the potential utility of microcin J25 in a (veterinary) medicine context are discussed and the efforts that went into employing the microcin J25 scaffold in bioengineering contexts are summed up.

PepDRED: De Novo Peptide Design with Strong Binding Affinity for Target Protein.

De novo design of peptides that bind specifically to functional proteins is beneficial for diagnostics and therapeutics. However, complex permutations and combinations of amino acids pose significant challenges to the rational design of peptides with desirable stability and affinity. Herein, we develop a computational-based evolution method, namely, peptidomimetics-driven recognition elements design (PepDRED), to derive hemoglobin-inspired peptidomimetics. PepDRED mimics the natural evolutionism pipeline to generate stable apovariant (AVs) structures for wild-type counterparts via automated point mutations and validates their efficiency through free binding energy analysis and per residue energy decomposition analysis. For application demonstration, we applied PepDRED to design de novo peptides to bind FhuA, a typical TonB-dependent transporter (TBDT). TBDTs are Gram-negative bacterial outer membrane proteins responsible for iron transport and vital for bacterial resistance. PepDRED generated a pool of AVs and proceeded to reach an optimized peptide, AV440, with a remarkable binding affinity of -21 kcal/mol. AV440 is ∼2.5-fold stronger than the existing FhuA inhibitor Microcin J25. Network energy analysis further unveils that incorporating methionine (M42) in the N-terminal region significantly enhances inter-residue contacts and binding affinity. PepDRED offers a prompt and efficient in silico approach to develop potent peptide candidates for target proteins.

Establishing Heterologous Production of Microcins J25 and Y in Bacillus subtilis

Microcin J25 (MccJ25) and microcin Y (MccY) are lasso peptides and considered potential alternatives to antibiotics and harmful preservatives. The combination of these two microcins can provide a wide antimicrobial spectrum against food-borne Salmonella. Currently, MccJ25 and MccY are produced using Escherichia coli expression systems; however, the entire production process is accompanied by negative effects from endotoxins. In this study, we identified Bacillus subtilis as a suitable host for MccJ25 and MccY production. High-level production of microcins was achieved by promoter optimization, host strain selection, and recombinant expression. The engineered strains produced maximum yields of 2.827 μM MccJ25 and 1.481 μM MccY. This is the first study to demonstrate the expression of MccJ25 and MccY in B. subtilis, and it offers a few engineered strains that are without antibiotic resistance markers, inducer-free, sporulation-deficient, and free of the negative effects of endotoxins for antibacterial therapy and food preservation.

Lasso peptide microcin J25 variant containing RGD motif as a PET probe for integrin a v ß 3 in tumor imaging

Microcin J25 (MccJ25), a lasso peptide, has a unique 3-D interlocked structure that provides high stability under acidic conditions, at high temperatures, and in the presence of proteases. In this study, we generated a positron emission tomography (PET) probe based on MccJ25 analog with an RGD motif and investigated their pharmacokinetics and utility for integrin αvβ3 imaging in tumors. The MccJ25 variant with an RGD motif in the loop region and a lysine substitution at the C-terminus (MccJ25(RGDF)GtoK) was produced in E. coli transfected with plasmid DNA containing the MccJ25 biosynthetic gene cluster (mcjABCD). [64Cu]Cu-MccJ25(RGDF)GtoK was synthesized using the C-terminal lysine labeled with copper-64 (t1/2 = 12.7 h) via a bifunctional chelator; it showed stability in 90% mouse plasma for 45 min. Using PET imaging for integrin αvβ3 positive U87MG tumor bearing mice, [64Cu]Cu-MccJ25(RGDF)GtoK could clearly distinguish the tumor, and its accumulation was significantly higher than that of MccJ25(GIGT)GtoK without the binding motif for integrin αvβ3. Furthermore, MccJ25(RGDF)GtoK enabled visualization of only U87MG tumors but not MCF-7 tumors with low integrin αvβ3 expression in double tumor-bearing mice. In ex vivo biodistribution analysis, the integrin αvβ3 non-specific accumulation of [64Cu]Cu-MccJ25(RGDF)GtoK was significantly lower in various tissues, except for the kidneys, as compared to the control probe ([64Cu]Cu-cyclic RGD peptide). These results of the present study indicate that 64Cu-labeling methods are appropriate for the synthesis of MccJ25-based PET probes, and [64Cu]Cu-MccJ25 variants are useful tools for cancer molecular imaging.

Effects of bacterial-derived antimicrobial solutions on shelf-life, microbiota and sensory attributes of raw chicken legs under refrigerated storage condition

In the present study, bacterial-derived antimicrobial agents included 5 mM reuterin combined with either 103.91 mM lactic acid (RL) or 0.08 μM microcin J25 (RJ) were evaluated for their effects on the microbiota and sensory attributes of raw chicken legs. Peracetic acid (13.67 mM), a conventional chemical commonly used in the poultry industry, was used as a positive control to compare efficacy. The chicken legs were sprayed with antimicrobial solutions and aerobically stored at 4 °C for 10 days. The RL treatment maintained the total viable count below the limit of 7 log CFU/g until the 8th day. Therefore, compared to the nontreated group, shelf-life was extended by 3 days in the RL treated group. The RJ treatment extended the shelf-life to 7 days, which is similar to what was achieved with the use of peracetic acid. Based on culture-independent amplicon sequencing, the RL and RJ treatments affected the microbial community on the chicken legs, inducing a delay in the increase of Pseudomonas, Psychrobacter and Carnobacterium while decreasing of Shigella. Significant decreases in sensory scores occurred in the nontreated group, while slight changes occurred in the combinations treated groups over the same period. Overall, sensory property scores for chicken legs treated with RL and RJ remained higher (P < 0.05) than those treated with peracetic acid or without antimicrobial agents. The antimicrobial combinations delayed the deterioration of sensory attributes throughout the storage period. These results suggest that RL and RJ provide a promising natural-sourced antimicrobial approach to control the growth of spoilage microorganisms on chicken legs.

Impact of microcin J25 on the porcine microbiome in a continuous culture model.

The increased prevalence of Salmonella spp. resistance in swine spurs the search for alternatives to antibiotics. Microcin J25 (MccJ25), a bacteriocin produced by Escherichia coli, is a potent inhibitor of several pathogenic bacteria including Salmonella enterica. In this study, we aimed to evaluate in vitro the impact of MccJ25 on the composition and the metabolic activity of the swine colonic microbiota. The PolyFermS in vitro continuous fermentation model was used here with modified Macfarlane medium to simulate the porcine proximal colon. During 35 days of fermentation, a first-stage reactor containing immobilized swine fecal microbiota fed two second-stage control and test reactors operated in parallel and used to test the effects of MccJ25 on the composition and the metabolic activity of the microbiota. Reuterin, a broad-spectrum antimicrobial compound produced by Limosilactobacillus reuteri, a lactic acid bacterium naturally present in the gastro-intestinal tract of human and animals, and the antibiotic rifampicin were tested for comparison. Sequencing of 16S rRNA was performed using the Illumina MiSeq technology to evaluate microbial diversity, and liquid chromatography coupled to mass spectrometry (LC-MS) followed by multivariate analysis was used to assess the bacteriocin/antibiotic degradation products and to monitor changes in the swine colonic microbiota metabolome. The results show that MccJ25 or reuterin treatments only induce subtle changes of both the microbial diversity and the metabolome of the swine colon microbiota, while rifampicin induces significant modification in amino acid levels. Although these findings need being validated in vivo, this study affords a first proof of concept for considering MccJ25 as a possible alternative to antibiotics for veterinary and farming applications, taking into account its pathogen-selective and potent inhibitory activity.

Evaluating the Potential and Synergetic Effects of Microcins against Multidrug-Resistant Enterobacteriaceae.

ABSTRACTThe advent of multidrug-resistant bacteria has hampered the development of new antibiotics, exacerbating their morbidity and mortality. In this context, the gastrointestinal tract reveals a valuable source of novel antimicrobials. Microcins are bacteriocins produced by members of the family Enterobacteriaceae, which are endowed with a wide diversity of structures and mechanisms of action, and exert potent antibacterial activity against closely related bacteria. In this study, we investigated the antibacterial activities of four microcins against 54 Enterobacteriaceae isolates from three species (Escherichia coli, Klebsiella pneumoniae, and Salmonella enterica). The selected microcins, microcin C (McC, nucleotide peptide), microcin J25 (MccJ25, lasso peptide), microcin B17 (MccB17, linear azol(in)e-containing peptide), and microcin E492 (MccE492, siderophore peptide) carry different post-translational modifications and have distinct mechanisms of action. MICs and minimal bactericidal concentrations (MBC) of the microcins were measured and the efficacy of combinations of the microcins together or with antibiotics was assessed to identify potential synergies. Every isolate showed sensitivity to at least one microcin with MIC values ranging between 0.02 μM and 42.5 μM. Among the microcins tested, McC exhibited the broadest spectrum of inhibition with 46 strains inhibited, closely followed by MccE492 with 38 strains inhibited, while MccJ25 showed the highest activity. In general, microcin activity was observed to be independent of antibiotic resistance profile and strain genus. Of the 42 tested combinations, 20 provided enhanced activity (18 out of 20 being microcin–antibiotic combinations), with two being synergetic.IMPORTANCE With their wide range of structures and mechanisms of action, microcins are shown to exert antibacterial activities against Enterobacteriaceae resistant to antibiotics together with synergies with antibiotics and in particular colistin.

Antibacterial Activity of the Peptide Microcin J25 Produced by Escherichia coli

Antibacterial Activity of the Peptide Microcin J25 Produced by Escherichia coli

In vitro assessment of skin sensitization, irritability and toxicity of bacteriocins and reuterin for possible topical applications

Bacteriocins and reuterin are promising antimicrobials for application in food, veterinary, and medical sectors. In the light of their high potential for application in hand sanitizer, we investigated the skin toxicity of reuterin, microcin J25, pediocin PA-1, bactofencin A, and nisin Z in vitro using neutral red and LDH release assays on NHEK cells. We determined their skin sensitization potential using the human cell line activation test (h-CLAT). Their skin irritation potential was measured on human epidermal model EpiDerm™. We showed that the viability and membrane integrity of NHEK cells remained unaltered after exposure to bacteriocins and reuterin at concentrations up to 400 µg/mL and 80 mg/mL, respectively. Furthermore, microcin J25 and reuterin showed no skin sensitization at concentrations up to 100 µg/mL and 40 mg/mL, respectively, while pediocin PA-1, bactofencin A, and nisin Z caused sensitization at concentrations higher than 100 µg/mL. Tissue viability was unaffected in presence of bacteriocins and reuterin at concentrations up to 200 µg/mL and 40 mg/mL, respectively, which was confirmed by measuring cytokine IL-1α and IL-8 levels and by histological analysis. In conclusion, the current study provides scientific evidence that some bacteriocins and reuterin, could be safely applied topically as sanitizers at recommended concentrations.

Disrupting Transcription and Folate Biosynthesis Leads to Synergistic Suppression of Escherichia coli Growth

The use of synergistic antibiotic combinations has emerged as a viable approach to contain the rapid spread of antibiotic‐resistant pathogens. Here we report the discovery of a new strongly synergistic pair – microcin J25 and sulfamonomethoxine. The former is a lasso peptide that inhibits the function of RNA polymerase and the latter is a sulfonamide antibacterial agent that disrupts the folate pathway. Key to our discovery was a screening strategy that focuses on an antibiotic (microcin J25) that targets a hub (transcription) in the densely interconnected network of cellular pathways. The rationale was that disrupting such a hub likely weakens the entire network, generating weak links that potentiate the growth inhibitory effect of other antibiotics. We found that MccJ25 potentiates five other antibiotics as well. These results showcase the merit of taking a more targeted approach in the search and study of synergistic antibiotic pairs.

Effects of drinking water supplementation with Lactobacillus reuteri, and a mixture of reuterin and microcin J25 on the growth performance, caecal microbiota and selected metabolites of broiler chickens

BackgroundSince the overuse of antibiotics in animal production has led to a selection of antibiotic-resistant pathogens that affect humans and animals as well. Scientists are therefore searching for novel natural alternatives to antibiotics. In this study Lactobacillus reuteri and a combination of reuterin and microcin J25 (RJ) were evaluated as promoters of growth and modulators of the cecal microbiota and metabolite profiles in broiler chickens. One-day-old Cobb 500 male broilers were distributed to 8 treatments: negative control (without antibiotic), positive control (bacitracin), three concentrations of RJ and three doses of L. reuteri plus glycerol. The birds (2176, 34 per pen, 8 pens per treatment) were reared for 35 d.ResultsThe body weight of the bacitracin and 5 mmol/L reuterin combined with 0.08 μmol/L microcin J25 (10RJ) treatment group was significantly higher than that of the negative control group (P < 0.05). L. reuteri had no significant effect on broiler growth. MiSeq high-throughput sequencing of 16S rRNA showed clustering of cecal microbial operational taxonomic unit diversity according to treatment. The influence of bacitracin and 10RJ on bacterial community overall structure was similar. They promoted Ruminococcaceae, Lachnospiraceae and Lactobacillaceae, increased the relative abundance of Faecalibacterium and decreased the abundance of Bacteroides and Alistipes, while the negative control condition favored Bacteroidaceae and Rikenellaceae. Furthermore, 10RJ increased the concentration of short-chain fatty acid in the cecum and changed the metabolome overall.ConclusionsThese overall suggest that 10RJ can promote a host-friendly gut environment by changing the cecal microbiome and metabolome. This combination of natural antimicrobial agents in the drinking water had a positive effect on broiler growth and may be suitable as an alternative to antibiotic growth promoters.

Biosynthetic Microcin J25 Exerts Strong Antibacterial, Anti-Inflammatory Activities, Low Cytotoxicity Without Increasing Drug-Resistance to Bacteria Target.

Multidrug resistant (MDR) bacterial infection has emerged, raising concerns about untreatable infections, and posing the highest health risks. Antimicrobial peptides (AMPs) are thought to be the best remedy for this problem. Here, we showed biosynthetic microcin J25 (MccJ25) exhibited excellent bactericidal activity against standard and clinically relevant veterinary MDR strains with high stability, no cytotoxicity, and no increase in drug resistance. Analysis of antimicrobial mechanism possessed by sensitive enterotoxigenic Escherichia coli (ETEC) based on electron microscopy and Sytox Green methods was carried out. Results showed excellent activity against ETEC was due to permeabilizing bacterial membranes and strong affinity. MccJ25 exhibited high endotoxin-neutralizing activity in both in vivo and in vitro environments, and mice exposed to lipopolysaccharide (LPS) showed decreased plasma LPS levels and improved survival after administration of MccJ25. In an LPS-treated mouse septicemia model, MccJ25 treatment significantly alleviated inflammatory responses by inhibiting proinflammatory factor secretion and expression. In a mouse E. coli infection model, administration of MccJ25 effectively improved host defense against clinically source cocktail of multidrug-resistant E. coli strains induced intestinal inflammation and bacteria dissemination. Results of studies on anti-inflammatory mechanisms showed that MccJ25 downregulated nuclear factor kappa B kinase and mitogen-activated protein kinase, thereby reducing the production of toll-like receptor 4, myeloid differentiation factor 88 and decreasing the key proinflammatory cytokines. These findings clarify MccJ25 may be an ideal antibacterial/antiendotoxic drug candidate that has the potential to further guide the development of anti-inflammatory and/or antimicrobial agents in the war against MDR bacterial infection.

Bacteriocin-Based Synergetic Consortia: a Promising Strategy to Enhance Antimicrobial Activity and Broaden the Spectrum of Inhibition

ABSTRACTBacteria-derived natural antimicrobial compounds such as bacteriocins, reruterin, and organic acids have recently received substantial attention as food preservatives or therapeutic alternatives in human or animal sectors. This study aimed to evaluate the antimicrobial activity of different bacteria-derived antimicrobials, alone or in combination, against a large panel of Gram-negative and Gram-positive bacteria. Bacteriocins, including microcin J25, pediocin PA-1, nisin Z, and reuterin, were investigated alone or in combination with lactic acid and citric acid, using a checkerboard assay. Concentrations were selected based on predetermined MICs against Salmonella enterica subsp. enterica serovar Newport ATCC 6962 and Listeria ivanovii HPB28 as Gram-negative and Gram-positive indicator strains, respectively. The results demonstrated that the combination of microcin J25 + citric acid + lactic acid; microcin J25 + reuterin + citric acid; and microcin J25 + reuterin + lactic acid tested against S. Newport ATCC 6962 showed synergistic effects (FIC index = 0.5). Moreover, a combination of pediocin PA-1 + citric acid + lactic acid; and reuterin + citric acid + lactic acid against L. ivanovii HPB28 showed a partially synergistic interactions (FIC index = 0.75). Nisin Z exerted a partially synergistic effect in combination with acids (FIC index = 0.625 -0.75), whereas when it was combined with reuterin or pediocin PA-1, it showed additive effects (FIC index = 1) against L. ivanovii HPB28. The inhibitory activity of synergetic consortia were tested against a large panel of Gram-positive and Gram-negative bacteria. According to our results, combining different antimicrobials with different mechanisms of action led to higher potency and a broad spectrum of inhibition, including multidrug-resistance pathogens.IMPORTANCE Reuterin and bacteriocins, including microcin J25, pediocin PA-1, nisin were produced and purified with >90% purity. Using the broth-based checkerboard assay the interaction between these compounds (synergetic, additive, or antagonistic) was assessed. By combining different natural antimicrobials with different modes of action and structure (reuteirn, microcin J25, pediocin PA-1, and organic acids), we successfully developed five different synergetic consortia with improved antimicrobial activity and a broad spectrum of inhibition. These consortia were shown to be effective against a large panel of pathogenic and spoilage microorganisms as well as clinically important multidrug-resistance bacteria. Moreover, because the lower concentrations of bacteriocins and reuterin are used in the synergetic consortia, there is a limited risk of toxicity and resistance development for these compounds.

Gastrointestinal Stability and Cytotoxicity of Bacteriocins From Gram-Positive and Gram-Negative Bacteria: A Comparative in vitro Study.

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