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Clostridium perfringens (C. perfringens) is an important pathogen that contributes to human and animal disease. At present, antibiotic therapy is one of the most effective strategies for C. perfringens. However, with the rise of antibacterial resistance, new agents with novel mechanisms of action are urgently needed. Bacteriocins are recognized as a viable alternative to antibiotics. In this study, the bacteriocin-like substance ParalichenysinDY4, derived from the Bacillus paralicheniformis (B. paralicheniformis) DY4 strain, is investigated as a potential alternative for combating Clostridium perfringens. The substance was isolated from B. paralicheniformis DY4 fermentation broth through a series of purification steps including methanol extraction, gel filtration, and high-performance liquid chromatography. Mass spectrometry analysis of ParalichenysinDY4 revealed that the detected peptide sequences did not match any previously known bacteriocins, indicating it is a novel bacteriocin-like substance. The novel bacteriocin-like substance exhibits effective antibacterial activity and broad antimicrobial spectrum against C. perfringens. Subsequent analyses utilizing methodologies including flow cytometry and scanning electron microscopy suggest that its mechanism of action is linked to its effects on the cell membrane. At the same time, due to its exceptional stability, safety, and efficient ability to remove pathogens both in vitro and in vivo, ParalichenysinDY4 holds promise as a valuable natural antimicrobial agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

E50-52, a class IIa-peptidic bacteriocin produced by a strain of Enterococcus faecium , has broad-spectrum antimicrobial activity against various foodborne pathogens. However, effective utilization of the E50-52 has been limited by low production yields and challenges associated with separation and purification of this 39-amino acid antimicrobial peptide. In this study, we have successfully produced a biologically active recombinant form of E50-52 by fusing it with the 16-kDa catalytic domain of lysostaphin-class III bacteriocin (LssCAT), which resulted in high-yield production. Initially, the LssCAT-E50-52 chimeric protein was insoluble upon over-expression in Escherichia coli , but it became soluble using phosphate buffer (pH 7.4) supplemented with 8 M urea. Purification using immobilized-Ni 2+ affinity chromatography under urea denaturing conditions resulted in consistent production a homogenous products (LssCAT-E50-52) with >95% purity. The purified protein was refolded using an optimized stepwise dialysis process. The resulting refolded LssCAT-E50-52 protein exhibited dose-dependent inhibitory activity against Helicobacter pylori , a Gram-negative, flagellated, helical bacterium that is associated with gastric cancer. Overall, the optimized protocol described in this study effectively produced large quantities of high-purity recombinant LssCAT-E50-52 protein, yielding approximately 100 mg per liter of culture. To the best of our knowledge, this is the first report on the impact of LssCAT-E50-52 on H. pylori . This finding could pave the way for further research into bactericidal mechanism and potential applications of this bacteriocin in biomedical industry.

A novel bacteriocin, plantaricin ZFM9, was purified from Lactiplantibacillus plantarum ZFM9 using a combination of ammonium sulfate precipitation, XAD-2 macroporous resin, Sephadex G-50, Sephadex LH-20, and reversed-phase high performance liquid chromatography. The molecular mass of plantaricin ZFM9 was 1151.606 Da, and the purity was 98.3%. Plantaricin ZFM9 has thermal stability (95.6% retention at 120 °C for 30 min), pH stability (pH ≤ 5), and sensitivity to the pepsin, trypsin, papain, and proteinase K. Plantaricin ZFM9 exhibited broad-spectrum antimicrobial activity and notably inhibit methicillin-resistant Staphylococcus aureus D48 (MRSA). According to the results of electron microscopy and fluorescence leakage assay, it was found that plantaricin ZFM9 caused damage to the cells membrane and leakage of the contents of S. aureus D48. In addition, Lipid II was not the anti-MRSA target of plantaricin ZFM9. This study underscores the potential of plantaricin ZFM9 for applications in the food field and biopharmaceuticals against MRSA infection., Competing Interests: Declaration of competing interest All author declares that there are no potential commercial or financial conflicts of interest in this study., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Strain Lactiplantibacillus plantarum D1 with bacteriocin producing ability was found in the intestine of Gambusia affinis. The bacteriocin was found to have high inhibitory activity against multiple Streptococcus species and several other Gram-positive and Gram-negative bacteria. Bacteriocin was purified from culture supernatant by ion-exchange chromatography, Sep-Pak C 18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectral analysis determined that purified bacteriocin has a molecular mass of 2,731 Da. A partial N-terminal sequence KRKKHKXQIYNNGM was obtained from the Edman analysis. The N-terminal sequence was employed to search against a translation of the draft genome of strain D1. The translated full amino acid sequence of the mature peptide is as follows: NH 2 - KRKKHKCQIYNNGMPTGQYRWC, which has a molecular weight of 2738 Da. A BLAST search revealed that this bacteriocin was most similar to bactofencin A but differed from it with three amino acid residues. No identical peptide or protein has been previously reported, and this peptide, termed bactofencin YH, was therefore considered to be a new bacteriocin produced by Lactiplantibacillus plantarum D1., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Antimicrobial peptides or bacteriocins are excellent candidates for alternative antimicrobials, but high manufacturing costs limit their applications. Recombinant gene expression offers the potential to produce these peptides more cost-effectively at a larger scale. Saccharomyces cerevisiae is a popular host for recombinant protein production, but with limited success reported on antimicrobial peptides. Individual recombinant S. cerevisiae strains were constructed to secrete two class IIa bacteriocins, plantaricin 423 (PlaX) and mundticin ST4SA (MunX). The native and codon-optimised variants of the plaA and munST4SA genes were cloned into episomal expression vectors containing either the S. cerevisiae alpha mating factor (MFα1) or the Trichoderma reesei xylanase 2 (XYNSEC) secretion signal sequences. The recombinant peptides retained their activity and stability, with the MFα1 secretion signal superior to the XYNSEC secretion signal for both bacteriocins. An eight-fold increase in activity against Listeria monocytogenes was observed for MunX after codon optimisation, but not for PlaX-producing strains. After HPLC-purification, the codon-optimised genes yielded 20.9 mg/L of MunX and 18.4 mg/L of PlaX, which displayed minimum inhibitory concentrations (MICs) of 108.52 nM and 1.18 µM, respectively, against L. monocytogenes. The yields represent a marked improvement relative to an Escherichia coli expression system previously reported for PlaX and MunX. The results demonstrated that S. cerevisiae is a promising host for recombinant bacteriocin production that requires a simple purification process, but the efficacy is sensitive to codon usage and secretion signals., (© 2023. The Author(s).)

Bacteriocins are a kind of antimicrobial peptides that kill or inhibit the growth of bacterial strains. The purpose of this study was to investigate the antibacterial effect of Serratia marcescens on several pathogenic bacterial strains. Bacteriocin produced by S. marcescens was purified by chromatography with Sephadex G-75 column, and its antibacterial effect on gram-negative bacteria, including Escherichia coli ATCC 700928, Pseudomonas aeruginosa PTCC 1707, S. marcescens PTCC 1621, Vibrio fischeri PTCC 1693, and Vibrio harveyi PTCC 1755, were evaluated by the disk diffusion method. The structure of bacteriocin was determined by nuclear magnetic resonance spectroscopy. The interaction of bacteriocin with the antigen 43 (Ag43) of E. coli was evaluated by the molecular docking method. Bacteriocin extracted from bacterial isolates had antibacterial activity on E. coli strains but not on other studied strains. Bioinformatics analysis also showed bacteriocin docking with Ag43 with an energy of -159.968 kJ/mol. Natural compounds, such as bacteriocin, can be an alternative to common chemical compounds and antibiotics. To reach a definite conclusion in this regard, there is a need for further research and understanding of their mechanism of action., Competing Interests: The authors declare that they have no conflict of interest.

The rapid spread of multidrug-resistant pathogens with the low efficacy of common antibiotics for humans and animals in its clinical therapeutics are a global health concern. Therefore, there is a need to develop new treatment strategies to control them clinically. The study aimed to evaluate the effects of Plantaricin Bio-LP1 bacteriocin produced from Lactiplantibacillus plantarum NWAFU-BIO-BS29 to alleviate the inflammation caused by multidrug-resistance Escherichia Coli (MDR-E. coli) infection in BALB/c mice-model. The focus was given on aspects linked to the mechanism of the immune response. Results indicated that Bio-LP1 had highly promising effects on partially ameliorating MDR-E. coli infection by reducing the inflammatory response through inhibiting the overexpression of proinflammatory-cytokines such as secretion of tumor necrosis factor (TNF-α) and interleukin (IL-6 and IL-β) and strongly regulated theTLR4 signaling-pathway. Additionally, avoided the villous destruct, colon length shortening, loss of intestinal barrier integrity, and increased disease activity index. Furthermore, significantly increased the relative abundance of beneficial-intestinal-bacteria including Ligilactobacillus, Enterorhabdus, Pervotellaceae, etc. Finally, improved the intestinal mucosal barrier to alleviate the pathological damages and promote the production of short-chain fatty acids (SCFAs) a source of energy for the proliferation. In conclusion, plantaricin Bio-LP1 bacteriocin can be considered a safe alternative to antibiotics against MDR-E. coli-induced intestinal inflammation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Traditionally fermented foods and beverages are still produced and consumed at a large scale in Romania. They are rich sources for novel lactic acid bacteria with functional properties and with potential application in food industry or health. Lactobacillus helveticus 34.9, isolated from a home-made fermented milk is able to inhibit the growth of other bacteria, such as other lactic acid bacteria, but also strains of Bacillus subtilis, Bacillus cereus, Staphylococcus aureus, and Halobacillus hunanensis, a halobacterium isolated from the degraded wall of a Romanian monastery. L. helveticus 34.9 produces a large bacteriocin (35 KDa), active in a wide pH range, but inactivated by heat and proteinase K treatment. It shares about 20% sequence coverage with helveticin J, as determined by LC-MS analysis. Bacteriocin production was enhanced under stress conditions, especially when combined stresses were applied. Its mode of action and degree of inhibition depended on the concentration and on the indicator strain that was used; L. delbrueckii subsp. bulgaricus LMG 6901 T cells from a suspension were killed, but the viability of H. hunanensis 5Hum cells was only reduced to 60%, within 8 h. However, the bacteriocin was able to prevent the bacterial growth of both indicator strains when added to the cultivation medium prior inoculation. Scanning electron microscopy images revealed morphological changes induced by the bacteriocin treatment in both sensitive strains, but more severe in the case of L. delbrueckii subsp. bulgaricus. Due to the broad antibacterial spectrum and its production under various stress conditions, the bacteriocin or the producing strain may find application in health, food and non-food related fields, including in the restoration of historical buildings., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Epibiotic bacteria associated with the filamentous marine cyanobacterium Moorea producens were explored as a novel source of antibiotics and to establish whether they can produce cyclodepsipeptides on their own. Here, we report the isolation of micrococcin P1 ( 1 ) (C 48 H 49 N 13 O 9 S 6 ; obs. m / z 1144.21930/572.60381) and micrococcin P2 ( 2 ) (C 48 H 47 N 13 O 9 S 6 ; obs. m / z 1142.20446/571.60370) from a strain of Bacillus marisflavi isolated from M. producens ' filaments. Interestingly, most bacteria isolated from M. producens ' filaments were found to be human pathogens. Stalked diatoms on the filaments suggested a possible terrestrial origin of some epibionts. CuSO 4 ·5H 2 O assisted differential genomic DNA isolation and phylogenetic analysis showed that a Kenyan strain of M. producens differed from L. majuscula strain CCAP 1446/4 and L. majuscula clones. Organic extracts of the epibiotic bacteria Pseudoalteromonas carrageenovora and Ochrobactrum anthropi did not produce cyclodepsipeptides. Further characterization of 24 Firmicutes strains from M. producens identified extracts of B. marisflavi as most active. Our results showed that the genetic basis for synthesizing micrococcin P1 ( 1 ), discovered in Bacillus cereus ATCC 14579, is species/strain-dependent and this reinforces the need for molecular identification of M. producens species worldwide and their epibionts. These findings indicate that M. producens -associated bacteria are an overlooked source of antimicrobial compounds.

Lantibiotics are a promising class of natural antimicrobial peptides. Lichenicidin is a two-peptide lantibiotic in which two mature peptides act synergistically to exhibit full bioactivity. Considering the two-peptide lantibiotics described so far, only cytolysin has been deeply characterized in terms of toxicity towards eukaryotic cells and it was found to be hemolytic and cytotoxic. This work aimed to improve the production of lichenicidin in vivo and characterize its antibacterial activity and toxicity against human cells. Peptides were purified and minimal inhibitory concentration (MIC) was determined against several strains; a time-kill assay was performed with Staphylococcus aureus. The hemolytic effect of lichenicidin was evaluated on blood samples from healthy donors and its toxicity towards human fibroblasts. The quantity of purified peptides was 1 mg/l Bliα and 0.4 mg/l Bliβ. MIC for methicillin-sensitive and resistant S. aureus (MSSA and MRSA) strains were 16-32 µg/ml and 64-128 µg/ml, respectively. At the MIC, lichenicidin took less than 3 h to eliminate MSSA, indicating a strong bactericidal effect. It induces cell lysis at the highest concentration, an effect that might be potentiated by Bliβ. Lichenicidin was not cytotoxic to human erythrocytes and fibroblasts. In this work, we evaluated the therapeutic potential of lichenicidin as a possible antimicrobial alternative., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The biopreservation strategy allows extending the shelf life and food safety through the use of indigenous or controlled microbiota and their antimicrobial compounds. The aim of this work was to characterize an inhibitory substance with bacteriocin-like activity (Sak-59) produced by the potentially probiotic L. sakei strain from artisanal traditional Kazakh horse meat product Kazy. The maximum production of Sak-59 occurred at the stationary phase of the L. sakei growth. Sak-59 showed inhibitory activity against gram-positive meat spoilage bacteria strains of Listeria monocytogenes, Staphylococcus aureus, and pathogenic gram-negative bacteria strains of Serratia marcescens and Escherichia coli, but not against the tested Lactobacilli strains. Sak-59 activity, as measured by diffusion assay in agar wells, was completely suppressed after treatment with proteolytic enzymes and remained stable after treatment with α-amylase and lipase, indicating that Sak-59 is a peptide and most likely not glycosylated or lipidated. It was concluded that Sak-59 is a potential new bacteriocin with a characteristic activity spectrum, which can be useful in the food and feed industries., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The interaction of antimicrobial peptides with membrane lipids plays a major role in numerous physiological processes. In this study, polydiacetylene (PDA) vesicles were synthesized using 10, 12-tricosadiynoic acid (TRCDA) and 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). These vesicles were applied as artificial membrane biosensor for the detection of plantaricin LD1 purified from Lactobacillus plantarum LD1. Plantaricin LD1 (200 μg/mL) was able to interact with PDA vesicles by changing the color from blue to red with colorimetric response 30.26 ± 0.59. Nisin (200 μg/mL), used as control, also changed the color of the vesicles with CR% 50.56 ± 0.98 validating the assay. The vesicles treated with nisin and plantaricin LD1 showed increased infrared absorbance at 1411.46 and 1000-1150 cm -1 indicated the interaction of bacteriocins with phospholipids and fatty acids, respectively suggesting membrane-acting nature of these bacteriocins. Further, microscopic observation of bacteriocin-treated vesicles showed several damages indicating the interaction of bacteriocins. These findings suggest that the PDA vesicles may be used as bio-mimetic sensor for the detection of bacteriocins produced by several probiotics in food and therapeutic applications., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bifidocin LHA, a novel bacteriocin, was extracted from bee honey B. adolescentis and purified. Bifidocin LHA was characterized as a protein in nature, without lipid or carbohydrate moieties, the molecular weight was 16,000 Da protein, heat-stable and active at a wide range of pH values, bactericidal effect, detergent, and solvents did not affect bifidocin activity and can be classified as type II bacteriocin. In vitro, the antibacterial activity of purified bifidocin LHA was significantly higher than crude bifidocin LHA (P < 0.05) against Pseudomonas aeruginosa (P. aeruginosa). The antibiofilm activity of bifidocin LHA was significantly higher than the antibiofilm activity of Amikacin (P < 0.05). In vivo, bifidocin LHA demonstrates a significant decreased in the number of P. aeruginosa in the eye, while complete clearance of P. aeruginosa comparing with the control (P < 0.05) when treating with Bifidobacterium adolescentis and bifidocin LHA together. Bifidobacterium adolescentis and bifidocin LHA treatment together induced substantial elevation of IL10 and IL-12 concentrations (P < 0.01) that helped to prevent damage caused by the inflammatory response. Succeeded to eradicate P. aeruginosa infection improved by histological patterns of the eye tissues. This study indicated Bifidobacterium adolescentis and bifidocin LHA consider as crucial strategies for the practical treatment of eye infection in the future., (Copyright © 2021. Published by Elsevier B.V.)

Bacteriocins are gaining utmost importance in antimicrobial and chemotherapy due to their diverse structure and activity. This study centres on magainin-2 like bacteriocin with anticancer action, produced by Bacillus safensis strain SDG14 isolated from gut of marine fish Sardinella longiceps. The purified bacteriocin designated as BpSl14 was thermostable and pH tolerant. The molecular weight of BpS114 was estimated to be 6061.2 Da using MALDI-ToF MS. The partial primary sequence was elucidated by peptide mass fingerprinting using MALDI MS/MS. The tertiary structure of the partial sequence was similar to that of two magainin-2 α-helices joined together by extended indolicidin. The BpSl14 protein inhibited the cells of lung carcinoma, one of the deadliest cancers. Docking studies conducted with DR5 and TGF-β, two of the most prominent apoptotic receptors in adenocarcinoma, also proved the anti-apoptotic action of BpSl14., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A bacteriocin from Bacillus subtilis (MK733983) originated from ethnomedicinal plant was purified using Preparative RP-HPLC. The HPLC fraction eluted with 65% acetonitrile showed the highest antimicrobial activity with Mycobacterium smegmatis as an indicator. Its specific activity and purification fold increased by 70.5% and 44%, respectively, compared to the crude bacteriocin. The bacteriocin showed stability over a wide range of pH (3.0-8.0) and preservation (- 20 °C and 4 °C), also thermal stability up to 80 °C for 20 min. Its proteinaceous nature was confirmed with complete loss of activity on its treatment with Trypsin, Proteinase K, and α-Chymotrypsin. Nevertheless, the bacteriocin retained up to 45% activity with Papainase treatment and was unaffected by salivary Amylase. It maintained ~ 95% activity on UV exposure up to 3 h and its activity was augmented by ethyl alcohol and metal ions like Fe 2+ and Mn 2+ . Most of the common organic solvents, general surfactants, preservatives, and detergents like Sulfobetaine-14, Deoxy-cholic-acid did not affect the bacteriocin's action. Its molecular weight was estimated to be 3.4KDa by LC-ESI-MS/MS analysis. The bacteriocin is non-hemolytic and exhibited a broad inhibition spectrum with standard strains of Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli and Chromobacterium violaceum with MICs ranging 0.225 ± 0.02-0.55 ± 0.05 mg/mL. Scanning Electron Microscopy showed cell annihilation with pores in cell membranes of S. aureus and P. aeruginosa treated with the bacteriocin, implicating bactericidal mode of action. These promising results suggest that the bacteriocin is significant and has wide-ranging application prospects.

Bacteriocins have attracted increasing interest because of their potential as natural preservatives. Recent studies showed that the Bacillus cereus group is a prominent producer of bacteriocins. Using a laboratory-based screening strategy, we identified a strain in the B. cereus group, Bacillus toyonensis XIN-YC13, with antimicrobial activity against B. cereus. A novel, 70-amino-acid-long leaderless bacteriocin, toyoncin, was purified from the culture supernatant of strain XIN-YC13, and its molecular mass was found to be 7,817.1012 Da. Toyoncin shares no similarity with any other known bacteriocins, and its N-terminal amino acid is formylmethionine rather than methionine. Toyoncin shows good pH and heat stability and exhibits specific antimicrobial activity against two important foodborne pathogens, B. cereus and Listeria monocytogenes. Additionally, toyoncin exerts bactericidal activity and induces cell membrane damage. Toyoncin can also inhibit the outgrowth of B. cereus spores. Preservation assays showed that toyoncin effectively suppressed or eradicated B. cereus and L. monocytogenes in pasteurized skim milk. These results suggest that toyoncin can be used as a new biopreservative against B. cereus and L. monocytogenes in the food industry. IMPORTANCE We identified a novel leaderless bacteriocin, toyoncin, produced by B. toyonensis XIN-YC13. Toyoncin shows good pH and heat stability, and it has specific antimicrobial activity against B. cereus and L. monocytogenes (two important foodborne pathogens), likely by destroying their cell membrane integrity. Toyoncin inhibited the outgrowth of B. cereus spores and effectively inhibited or eliminated B. cereus and L. monocytogenes in a milk model system. These results indicate the potential of toyoncin as a food preservative.

Aim: Bacteriocins with antimicrobial activity are considered as potential natural bio-preservatives to control the growth of food spoilage bacteria. The aim of this work was to characterize a novel bacteriocin BM1029 discovered from Lactobacillus crustorum MN047 and evaluate its antibacterial mechanism., Methods and Results: Bacteriocin BM1029 was purified by cation-exchange chromatography and reversed-phase chromatography. Antibacterial activity assay showed that BM1029 is antagonistic against both Gram-positive and Gram-negative bacteria. Furthermore, it was found that BM1029 showed low haemolysis with high stability to the pretreatment with different temperatures, pH and surfactants. Moreover electron microscopy and flow cytometry suggested that BM1029 inhibit indicator strains by damaging the cell envelope integrity. Cell cycle assay suggested that BM1029 arrested cell cycle in R-phase., Conclusion: The novel bacteriocin BM1029 showed high bactericidal activity against Escherichia coli and Staphylococcus aureus through a cell envelope-associated mechanism., Significance and Impact of the Study: Application of BM1029 inhibited the growth of indicator strains on beef meat storage at 4°C suggesting that this bacteriocin is promising to be used as a novel preservative in food processing and preservation., (© 2020 The Society for Applied Microbiology.)

Introduction: The emergence and spread of antibiotic resistance among pathogenic bacteria drives the search for alternative antimicrobial therapies. Bacteriocins represent a potential alternative to antibiotic treatment. In contrast to antibiotics, bacteriocins are peptides or proteins that have relatively narrow spectra of antibacterial activities and are produced by a wide range of bacterial species. Bacteriocins of Escherichia coli are historically classified as microcins and colicins, and, until now, more than 30 different bacteriocin types have been identified and characterized., Areas Covered: We performed bibliographical searches of online databases to review the literature regarding bacteriocins produced by E. coli with respect to their occurrence, bacteriocin role in bacterial colonization and pathogenicity, and application of their antimicrobial effect., Expert Opinion: The potential use of bacteriocins for applications in human and animal medicine and the food industry includes (i) the use of bacteriocin-producing probiotic strains, (ii) recombinant production in plants and application in food, and (iii) application of purified bacteriocins.

Nosocomial infections caused by enterococci are an ongoing global threat. Thus, finding therapeutic agents for the treatment of such infections are crucial. Some Enterococcus faecalis strains are able to produce antimicrobial peptides called bacteriocins. We analyzed 65 E. faecalis isolates from 43 food samples and 22 clinical samples in Egypt for 17 common bacteriocin-encoding genes of Enterococcus spp. These genes were absent in 11 isolates that showed antimicrobial activity putatively due to bacteriocins (three from food, including isolate OS13, and eight from clinical isolates). The food-isolated E. faecalis OS13 produced bacteriocin-like inhibitory substances (BLIS) named enterocin OS13, which comprised two peptides (enterocin OS13α OS13β) that inhibited the growth of antibiotic-resistant nosocomial E. faecalis and E. faecium isolates. The molecular weights of enterocin OS13α and OS13β were determined as 8079 Da and 7859 Da, respectively, and both were heat-labile. Enterocin OS13α was sensitive to proteinase K, while enterocin OS13β was resistant. Characterization of E. faecalis OS13 isolate revealed that it belonged to sequence type 116. It was non-hemolytic, bile salt hydrolase-negative, gelatinase-positive, and sensitive to ampicillin, penicillin, vancomycin, erythromycin, kanamycin, and gentamicin. In conclusion, BLIS as enterocin OS13α and OS13β represent antimicrobial agents with activities against antibiotic-resistant enterococcal isolates.

There is an increasing demand for dairy products, but the presence of food-spoilage bacteria seriously affects the development of the dairy industry. Bacteriocins are considered to be a potential antibacterial or antibiofilm agent that can be applied as a preservative. In this study, bacteriocin BM173 was successfully expressed in the Escherichia coli expression system and purified by a 2-step method. Furthermore, it exhibited a broad-spectrum antibacterial activity, high thermal stability (121°C, 20 min), and broad pH stability (pH 3-11). Moreover, the minimum inhibitory concentration values of BM173 against E. coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were 14.8 μg/mL and 29.6 μg/mL, respectively. Growth and time-kill curves showed that BM173 exhibited antibacterial and bactericidal activity. The results of scanning electron microscopy and transmission electron microscopy demonstrated that BM173 increased membrane permeability, facilitated pore formation, and even promoted cell lysis. The disruption of cell membrane integrity was further verified by propidium iodide uptake and lactic dehydrogenase release. In addition, BM173 exhibited high efficiency in inhibiting biofilm formation. Therefore, BM173 has promising potential as a preservative used in the dairy industry., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Extensive usage of antibiotics has led to the emergence of drug-resistant strains of pathogens and hence, there is an urgent need for alternative antimicrobial agents. Antimicrobial Peptides (AMPs) of bacterial origin have shown the potential to replace some conventional antibiotics. In the present study, an AMP was isolated from Bacillus subtilis subsp. spizizenii strain Ba49 present on the Allium cepa, the common onion and named as peptide-Ba49. The isolated AMP was purified and characterized. The purified peptide-Ba49, having a molecular weight of ~ 3.3 kDa as determined using mass spectroscopy, was stable up to 121 °C and in the pH range of 5-10. Its interaction with protein degrading enzymes confirmed the peptide nature of the molecule. The peptide exhibited low minimum inhibitory concentration (MIC) against Staphylococcus aureus and its (Methicillin-resistant Staphylococcus aureus) MRSA strains (MIC, 2-16 µM/mL). Further, time kill kinetic assay was performed and analysis of the results of membrane depolarization and permeabilization assays (TEM, DiBAC 4 (3) and PI) suggested peptide-Ba49 to be acting through the change in membrane potential leading to disruption of S. aureus membrane. Additionally, cytotoxicity studies of peptide-Ba49, carried out using three mammalian cell lines viz. HEK 293T, RAW 264.7, and L929, showed limited cytotoxicity on these cell lines at a concentration much higher than its MIC values. All these studies suggested that the AMP isolated from strain Ba49 (peptide-Ba49) has the potential to be an alternative to antibiotics in terms of eradicating the pathogenic as well as drug-resistant microorganisms.

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

Drug resistance has become a major threat due to the frequent use of commercial antibiotics and there is an urgent need to combat this problem. Having this in mind, the present research was aimed at developing a novel P. aeruginosa puBac bacteriocin molecule. The bacteriocin was purified by ammonium sulfate precipitation followed by Sepharose FF and Sephadex G15 column purification and the purified bacteriocin has been reported to have the molar mass of 43 kDa. The findings of the optimization showed that 3500 AU/mL of bacteriocin was obtained at 37 °C, 3410 AU/mL of bacteriocin at 6.5 pH and 3780 AU/mL of bacteriocin at 48 h of incubation time. In addition, 3863 AU/mL of bacteriocin activity was obtained with Tween-80 followed by 3789 AU/mL with a concentration of 2% NaCl and 4200 AU/mL for Fe 2+ . PuBac bacteriocin has been shown to have a significant effect on test pathogens. For example, E. coli was found to have 3.6 μM of MIC, followed by Staphylococcus sp. with 6.15 μM of MIC and Bacillus sp. with a 7.5 μM of MIC. The remarkable properties of bacteriocin suggest that it could be used in various pharmaceutical and food industries., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chitosan films loaded with bacteriocin were examined by FTIR spectroscopy, tested for color, puncture strength, water vapor permeability, and as antimicrobials of Listeria innocua HPB13. Divergicin M35, a bacteriocin produced by Carnobacterium divergens, was incorporated into films made with chitosan of molecular mass 2 kDa, 20 kDa, or 100 kDa and de-acetylated either 87% or 95%. Only 100 kDa chitosan yielded films that could be peeled and handled easily. The higher degree of de-acetylation increased the total color factor (ΔE) of bacteriocin-loaded films, their permeability, and puncture strength. Incorporation of divergicin M35 into the films increased amide I peak intensity but otherwise did not induce significant structural change. The FTIR spectra of divergicin M35 shed from the films did not differ from those of the original free bacteriocin, except in overall peak intensity. The release of active divergicin M35 from the film was faster into the buffer than into tryptic soy broth and peaked at 10-12 h in both cases. Chitosan 95% de-acetylated and loaded with divergicin M35 was the most active, producing a six-log drop in Listeria innocua HPB13 viable count within 24 h. These results suggest that the biocompatible and biodegradable films developed here have the potential for application as antimicrobials of Listeria spp. in foods, especially ready-to-eat, minimally processed products.

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 μg of amikacin and 5 μg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize ("Rojo Criollo") and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and β-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants., Competing Interests: The authors have declared that no competing interests exist.

A new thiopeptide (micrococcin P3, 1 ) and a known one (micrococcin P1, 2 ) were isolated from the culture broth of a marine-derived strain of Bacillus stratosphericus . The structures of both compounds were elucidated using spectroscopic methods, including extensive 1D and 2D NMR analysis, high resolution mass spectrometry (HRMS), and tandem mass spectrometry. Both compounds exhibited potent antibacterial activities against Gram-positive strains with minimum inhibitory concentration (MIC) values of 0.05-0.8 μg/mL and did not show cytotoxicity in the MTT assay up to a concentration of 10 μM. This study adds a new promising member, micrococcin P3, to the family of thiopeptide antibiotics, which shows potential for the development of new antibiotics targeting Gram-positive bacteria.

Lactic acid bacteria (LAB) are widely known for their probiotic activities for centuries. These bacteria synthesise some secretory proteinaceous toxins, bacteriocins, which help destroy similar or interrelated bacterial strains. This study was aimed at characterising bacteriocins extracted from Lactobacillus spp. found in yoghurt and assessing their bactericidal effect on foodborne bacteria. Twelve isolated Lactobacillus spp. were examined to produce bacteriocins by the organic solvent extraction method. Bacteriocins produced by two of these strains, Lactobacillus helveticus (BLh) and Lactobacillus plantarum (BLp), showed the most significant antimicrobial activity, especially against Staphylococcus aureus and Acinetobacter baumannii . Analysis of SDS-PAGE showed that L . plantarum and L . helveticus bacteriocins have a molecular weight of ~10 kDa and ~15 kDa, respectively. L . plantarum (BLp) bacteriocin was heat stable while L . helveticus (BLh) bacteriocin was heat labile. Both bacteriocins have shown activity at acidic pH. Exposure to a UV light enhances the activity of the BLh; however, it had negligible effects on the BLp. Different proteolytic enzymes confirmed the proteinaceous nature of both the bacteriocins. From this study, it was concluded that bacteriocin extracts from L . helveticus (BLh) can be considered a preferable candidate against foodborne pathogens as compared to L . plantarum (BLp). These partially purified bacteriocins should be further processed to attain purified product that could be useful for food spoilage and preservation purposes., Competing Interests: The authors report no conflict of interest., (Copyright © 2020 Mahreen Ul Hassan et al.)

Streptomyces spp. are Gram-positive bacteria well-known for their ability to produce antibiotics and other metabolites, but few studies on bacteriocins produced by these bacteria have been reported. We tested eight Streptomyces strains against different pathogenic bacteria, and selected S. griseus, S. nigrescens, S. bottroprensis, and S. violaceoruber for further study based on their inhibitory effects against bacteria, including human pathogens. S. bottropensis reached its highest activity at 312 h and was higher than the activities of S. violaceoruber and S. nigrescens. The best condition for bacteriocin precipitation was using diammonium sulfate at 50% saturation. Bacteriocins were susceptible to proteinase treatments and stable at high temperature (up to 100 °C). The highest inhibitory activities were observed between pH 5 and 6. Cross-activity assays indicated that each Streptomyces strain produced different bacteriocins. When preparations of S. griseus and S. nigrescens were subjected to SDS-PAGE, bands of inhibition were observed in the gel overlay assay at a position corresponding to ~ 2 and 3 kDa, respectively, suggesting that both strains are potential sources for novel bacteriocins.

Lantibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d-amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N-dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram-positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: An increasing rate of antibiotic resistance among Gram-negative bacterial pathogens has created an urgent need to discover novel therapeutic agents to combat infectious diseases. Use of bacteriocins as therapeutic agents has immense potential due to their high potency and mode of action different from that of conventional antibiotics., Results: In this study, a novel bacteriocin E20c of molecular weight 6.5 kDa was purified and characterized from the probiotic strain of Enterococcus hirae. E20c had bactericidal activities against several multidrug resistant (MDR) Gram-negative bacterial pathogens. Flow cytometry and scanning electron microscopy studies showed that it killed the Salmonella enterica cells by forming ion-permeable channels in the cell membrane leading to enhanced cell membrane permeability. Further, checkerboard titrations showed that E20c had synergistic interaction with antibiotics such as ampicillin, penicillin, ceftriaxone, and ciprofloxacin against a ciprofloxacin- and penicillin-resistant strain of S. enterica., Conclusion: Thus, this study shows the broad spectrum antimicrobial activity of novel enterocin E20c against various MDR pathogens. Further, it highlights the importance of bacteriocins in lowering the minimum inhibitory concentrations of conventional antibiotics when used in combination.

The bacteriocinogenic lactic acid bacterium Pediococcus pentosaceus LJR1 isolated from rumen liquor of goat had strong anti-bacterial activity toward Listeria monocytogenes in vitro. This antibacterial activity was lost on treatment with protease indicating that the bacteriocin is proteinaceous in nature. The bacteriocin LJR1 produced by P. pentosaceus was purified following a three step procedure consisting of ammonium sulphate precipitation, gel filtration chromatography and reverse phase-high performance liquid chromatography. The molecular weight of purified bacteriocin was determined to be 4.6 kDa using Tricine SDS-PAGE. Further, we found that the proteinaceous bacteriocin was stable at 100 °C as well as 121 °C for 30 min and 15 min respectively and also at different pH ranging from 4 to 10 when stored for 15 min at 37 °C. Its minimum inhibitory concentration for S. typhi MTCC134 and L. monocytogenes MTCC 1143 was 7.81 µg/ml and 15.63 µg/ml respectively. Scanning electron microscopy analysis of the surface of S. typhi treated with the bacteriocin showed the presence of craters; while in the case of treated L. monocytogenes blebs were observed. The addition of the bacteriocin to shrimp (white leg shrimp) has led to reduction of about 1 log units of L. monocytogenes on day 1 and maintained for 7 days on storage at 4 °C. It is clear that the purified bacteriocin has good potential as a bio preservative for application in food industry.

Thuricin 4AJ1, produced by Bacillus thuringiensis strain 4AJ1, showed inhibition activity against Bacillus cereus 0938 and ATCC 10987. It began to appear in the stationary phase and reached its maximum activity level of 209.958 U at 18 h against B. cereus 0938 and 285.689 U at 24 h against B. cereus ATCC 10987. Tricine-SDS-PAGE results showed that the partly purified thuricin 4AJ1 was about 6.5 kDa. The molecular weights of the known B. thuringiensis bacteriocins and the ones obtained by the two mainstream websites for predicting bacteriocins were inconsistent with the size of the thuricin 4AJ1, indicating that the bacteriocin obtained in this study may have a novel structure. Based on the biochemical properties, the thuricin 4AJ1 activities increased after treatment with proteinase K and lipase II, and were not affected by a-amylase, catalase, α-chymotrypsin VII and α-chymotrypsin II. It was heat tolerant, being active up to 90º C. In the pH 3-10 range, it maintained most of its activity. Finally, the sensitivity of the strain 4AJ1 to commonly used antibiotics was tested. In view of its stability and antibacterial activity, thuricin 4AJ1 may be applied as a food biopreservative.

Streptococcus pneumoniae infections are one of the major causes of morbility and mortality worldwide. Although vaccination and antibiotherapy constitute fundamental and complementary strategies against pneumococcal infections, they present some limitations including the increase in non-vaccine serotypes and the emergence of multidrug-resistances, respectively. Ribosomally-synthesized antimicrobial peptides (i.e. bacteriocins) produced by Lactic Acid Bacteria (LAB) may represent an alternative or complementary strategy to antibiotics for the control of pneumococal infections. We tested the antimicrobial activity of 37 bacteriocinogenic LAB, isolated from food and other sources, against clinical S. pneumoniae strains. Streptococcus infantarius subsp. infantarius LP90, isolated from Venezuelan water-buffalo milk, was selected because of its broad and strong anti-pneumococcal spectrum. The in vitro safety assessment of S. infantarius LP90 revealed that it may be considered avirulent. The analysis of a 19,539-bp cluster showed the presence of 29 putative open reading frames (ORFs), including the genes encoding 8 new class II-bacteriocins, as well as the proteins involved in their secretion, immunity and regulation. Transcriptional analyses evidenced that the induction factor (IF) structural gene, the bacteriocin/IF transporter genes, the bacteriocin structural genes and most of the bacteriocin immunity genes were transcribed. MALDI-TOF analyses of peptides purified using different multichromatographic procedures revealed that the dairy strain S. infantarius LP90 produces at least 6 bacteriocins, including infantaricin A1, a novel anti-pneumococcal two-peptide bacteriocin., Competing Interests: The authors have declared that no competing interests exist.

Aims: We aimed to evaluate some specific conditions for growth of Pediococcus pentosaceus ST65ACC and its bacteriocin expression through ABC transporters; to purify the bacteriocin and determine its sequence; and to evaluate the cytotoxicity potential of the purified bacteriocin(s)., Methods and Results: The results presented for growth behaviour of P. pentosaceus ST65ACC showed that the bacterial growth was slightly influenced when cultured in MRS broth with different amounts of inoculum: 1, 2, 5 and 10%. The bacteriocin activity increased when 5 and 10% inocula were used. The carbon source (glucose) used in different amounts (1, 2, 3 or 4%) had no significant effect on growth and bacteriocin production. The studied strain P. pentosaceus ST65ACC was able to metabolize xylooligosaccharide (XOS) as the sole carbon source, resulting in the production of an antimicrobial peptide. The genes involved in the ABC transport system and sugar metabolism of P. pentosaceus ST65ACC were expressed at different levels. The bacteriocin produced by P. pentosaceus ST65ACC was partially purified by precipitation with ammonium sulphate (40% saturation), followed by reversed-phase liquid chromatography, resulting in the identification of an active bacteriocin. Tandem mass spectrometry was used to identify the partial sequence KYYGNGVTCGKHSCSVDWGK sharing high similarity to coagulin A. The semi-purified bacteriocin had low cytotoxicity based on estimated values for maximal nontoxic concentration (MNC) and cytotoxicity concentration (CC 50 )., Conclusions: The bacteriocin produced by P. pentosaceus ST65ACC is similar to coagulin, with low cytotoxicity, strong antimicrobial activity and possible additional metabolite routes in the producer cell. In addition to MRS broth, bacteriocin was produced also in medium containing XOS (as the single carbon source)., Significance and Impact of the Study: To the best of our knowledge, this is the first report of evaluation of the role of ABC transporters in the expression of bacteriocin by P. pentosaceus, cultured in MRS and XOS., (© 2019 The Society for Applied Microbiology.)

Enterococcus faecium TJUQ1 with high bacteriocin-producing ability was isolated from pickled Chinese celery. In this study, enterocin TJUQ1 was purified by ammonium sulfate precipitation, reversed-phase chromatography (Sep-Pak C 8 ) and cation-exchange chromatography. The activity of the purified bacteriocin was 44,566.41 ± 874.69 AU/mg, which corresponds to a purification fold of 35.89 ± 2.34. The molecular mass was 5520 Da by MALDI-TOF MS and Tris-Tricine SDS-PAGE. The result of LC-MS/MS showed that the bacteriocin shared 59.15% identity with enterocin produced by E. faecium GN (accession no. O34071). PCR amplification revealed that E. faecium TJUQ1 possesses a gene encoding enterocin B with 60% identity to enterocin B. Circular dichroism (CD) spectroscopy showed that the molecular conformation was 32.6% helix, 19.5% beta, 12.9% turn and 35.0% random. The stability of enterocin TJUQ1 was measured. After exposure at 121 °C for 15 min, the residual antimicrobial activity of enterocin TJUQ1 was 85.95 ± 1.32%. The antimicrobial activity of enterocin TJUQ1 was still active over a pH range of 3-11. Enterocin TJUQ1 was inactivated after exposure to proteolytic enzymes but was not inactivated by lipase or amylase. These results showed that enterocin TJUQ1 was a novel class II bacteriocin. Enterocin TJUQ1 showed wide antibacterial activity against food-borne gram-negative and gram-positive pathogens, such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli and Salmonella enterica. The MIC was 5.26 ± 0.24 μg/mL against L. monocytogenes CMCC 1595. SEM and TEM were used to observe the changes in the morphological and intracellular organization of L. monocytogenes CMCC 1595 cells treated with enterocin TJUQ1. The results demonstrated that enterocin TJUQ1 increased extracellular electrical conductivity, facilitated pore formation, triggered the release of UV-absorbing materials, ATP and LDH, and even caused cell lysis in L. monocytogenes CMCC 1595 cells. Based on the characterization, the wide inhibitory spectrum and mode of action determined so far, enterocin TJUQ1 is a potential preservative for the food industry., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lantibiotics are lanthionine ring containing natural products that belong to the class of ribosomally synthesized and posttranslationally modified peptides (RiPPs). Recent expansion in the availability of microbial genome data and in silico analysis tools have accelerated the discovery of these promising alternatives to antibiotics. Following the genome-mining approach, a biosynthetic gene cluster for a putative two-component lantibiotic, roseocin, was identified in the genome of an Actinomycete, Streptomyces roseosporus NRRL 11379. Posttranslationally modified lanthipeptides of this cluster were obtained by heterologous expression of the genes in Escherichia coli, and were in vitro reconstituted to their bioactive form by exploiting commercial proteases like endoproteinase GluC, and proteinase K. The two peptides displayed synergistic antimicrobial activity against Gram-positive bacteria including the WHO high-priority pathogens, MRSA and VRE. Structural characterization confirmed the installation of four (methyl)lanthionine rings with an indispensable disulfide bond in the α-peptide, and six (methyl)lanthionine rings in the β-peptide, by a single promiscuous lanthionine synthetase, RosM. Roseocin is the first two-component lantibiotic from a non-Firmicute, with extensive lanthionine bridging., (© 2019 John Wiley & Sons Ltd.)

A bacterial strain inhibiting the growth of Vibrio anguillarum , the causative agent of vibriosis, was isolated from fish intestines. The isolated strain HS36 was identified as Aerococcus urinaeequi based on the characteristics of the genus according to Bergey's Manual of Systematic Bacteriology and by 16S rRNA sequencing. The growth rate and antibacterial activity of strain HS36 in shaking culture were higher than those in static culture, while the optimal pH and temperature for antibacterial activity were 7.0 and 30°C, respectively. The active antibacterial substance was purified from a culture broth of A. urinaeequi HS36 by Sephadex G-75 gel chromatography, Sephadex G-25 gel chromatography, and reverse-phase high-performance liquid chromatography. Its molecular weight, as estimated by Tricine SDS-polyacrylamide gel electrophoresis, was approximately 1,000 Da. The antibacterial substance produced by strain HS36 was stable after incubation for 1 h at 100°C. Although its antibacterial activity was optimal at pH 6-8, activity was retained at a pH range from 2 to 11. The purified antibacterial substance was inactivated by proteinase K, papain, and β-amylase treatment. The newly purified antibacterial substance, classified as a class II bacteriocin, inhibited the growth of Klebsiella pneumoniae , Salmonella enterica , and Vibrio alginolyticus .

Most clinical antibiotics are derived from actinomycete natural products discovered at least 60 years ago. However, the repeated rediscovery of known compounds led the pharmaceutical industry to largely discard microbial natural products (NPs) as a source of new chemical diversity. Recent advances in genome sequencing have revealed that these organisms have the potential to make many more NPs than previously thought. Approaches to unlock NP biosynthesis by genetic manipulation of strains, by the application of chemical genetics, or by microbial cocultivation have resulted in the identification of new antibacterial compounds. Concomitantly, intensive exploration of coevolved ecological niches, such as insect-microbe defensive symbioses, has revealed these to be a rich source of chemical novelty. Here, we report the new lanthipeptide antibiotic kyamicin, which was generated through the activation of a cryptic biosynthetic gene cluster identified by genome mining Saccharopolyspora species found in the obligate domatium-dwelling ant Tetraponera penzigi of the ant plant Vachellia drepanolobium Transcriptional activation of this silent gene cluster was achieved by ectopic expression of a pathway-specific activator under the control of a constitutive promoter. Subsequently, a heterologous production platform was developed which enabled the purification of kyamicin for structural characterization and bioactivity determination. This strategy was also successful for the production of lantibiotics from other genera, paving the way for a synthetic heterologous expression platform for the discovery of lanthipeptides that are not detected under laboratory conditions or that are new to nature. IMPORTANCE The discovery of novel antibiotics to tackle the growing threat of antimicrobial resistance is impeded by difficulties in accessing the full biosynthetic potential of microorganisms. The development of new tools to unlock the biosynthesis of cryptic bacterial natural products will greatly increase the repertoire of natural product scaffolds. Here, we report a strategy for the ectopic expression of pathway-specific positive regulators that can be rapidly applied to activate the biosynthesis of cryptic lanthipeptide biosynthetic gene clusters. This allowed the discovery of a new lanthipeptide antibiotic directly from the native host and via heterologous expression., (Copyright © 2020 Vikeli et al.)

Background and Objective: The bacteriocin isolated from fermented buffalo milk from West Sumatra-Indonesia, called Dadih, can be considered as a natural and safe antimicrobial compound for food products. The objective of this research was to evaluate the antimicrobial activity of bacteriocin from Dadih and its effectiveness as a preservative in chicken meat., Materials and Methods: This study used experimental method followed by statistical analysis using 3 experiments with duplication including experiment of meat samples (0 and 10% bacteriocin), storage temperatures (7 and 26°C) and storage duration (0, 1, 2, 3, 4, 5, 6 days and 0, 6, 12 hrs). Each experiment consists of a bacteriocin test, antimicrobial activity assay, physicochemical measurement and storability., Results: From 10 LAB isolates successfully obtained from Dadih, two isolates with D7 code and D10 code had the highest antimicrobial activity, reaching 11.75 mm and 12 mm, respectively. The meat treated by 10% of bacteriocin gave the lower total microbial (3rd and 5th day) and total E. coli (5th day) at 7 and 26°C. The pH and water activity (aw) values of chicken meat with 10% of bacteriocin showed lower values at 7 and 26°C. The application of bacteriocin to chicken meat was able to inhibit the microbial growth that was still below standard for 3 days at 7°C and 6 hrs at 26°C., Conclusion: Based on research, lactic acid bacteria isolated from buffalo milk curd produced bacteriocin compound which has antimicrobial properties. This bacteriocin showed potential as a natural preservative for chicken meat by inhibiting the growth of pathogen microorganisms.

Background: Catheter-associated urinary tract infections are one of the most common types of hospital-acquired infections that start with bacterial adhesion and lead to biofilm formation. The antagonistic activity of lactic acid bacteria against pathogenic organisms makes them important for medical applications., Objective: This study evaluated the precise method for purification of bacteriocin from Lactobacillus plantarum subsp. argentoratensis SJ33, and its characterization and effectiveness for biofilm inhibition on urinary catheters coated with bacteriocin., Methods: Purification of bacteriocin was carried out using various methods such as cell adsorption-desorption, gel permeation chromatography, and hydrophobic interaction chromatography. Bacteriocin preparation was analysed using reverse-phase high-performance liquid chromatography (HPLC) and further characterised by Tricine SDS-PAGE and Q-TOF ESI MS. Antibacterial activity of bacteriocin was assessed against 16 different Gram-positive and Gram-negative bacterial strains, and their effect on morphology was observed under scanning electron microscopy (SEM). Biofilm adherence and inhibition were evaluated by crystal violet assay, fluorescence microscopy and SEM., Results and Conclusions: Bacteriocin preparation exhibited broad-spectrum activity against both Gram-positive and Gram-negative bacteria, and SEM analysis revealed membrane pore formation. On treating with various enzymes, bacteriocin was found to be sensitive to proteases, which confirmed its proteinaceous nature. Bacteriocin showed its applicability at acidic pH in the urinary tract. Antibiofilm activity of bacteriocin established its significance in catheter-associated biofilm inhibition against Pseudomonas aeruginosa and Staphylococcus aureus. Molecular weight of bacteriocins, namely Bac F1 and Bac F2 as resolved by RP-HPLC, was estimated to be 4039 Da and 1609 Da, respectively., (Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.)

Bacteriocins are ribosomally synthesized peptides/proteins produced by bacteria. These compounds have antibacterial activity against other bacteria that are usually closely related to the producer strain. Here we describe bacteriocin geobacillin 26 from a thermophilic Gram-positive bacterium Geobacillus stearothermophilus 15. We have purified native bacteriocin, determined its amino acid sequence and heterologously expressed in Gram-negative Escherichia coli. Geobacillin 26 is a heat-labile, high molecular weight antibacterial protein belonging to class III bacteriocins. It has a narrow antibacterial spectrum against other thermophilic bacteria. Our study suggests that this bacteriocin is not a cell wall hydrolyzing enzyme as most of high molecular weight bacteriocins. In addition, geobacillin 26 has no amino acid sequence similarities to other known function proteins. No other class III bacteriocin from a thermophilic bacterium has been reported and well characterized before. Geobacillin 26 as a natural antibacterial agent has a great potential in industry where contamination with other thermophilic bacteria is unwanted. Moreover, this study may prompt to disclose more novel geobacillin 26-like antibacterial proteins, which could find their applications in food industry or medicine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The inhibitory action that a Brevibacillus laterosporus strain isolated from the honeybee body causes against the American Foulbrood (AFB) etiological agent Paenibacillus larvae was studied by in-vitro experiments. A protein fraction isolated from B. laterosporus culture supernatant was involved in the observed inhibition of P. larvae vegetative growth and spore germination. As a result of LC-MS/MS proteomic analyses, the bacteriocin laterosporulin was found to be the major component of this fraction, followed by other antimicrobial proteins and substances including lectins, chaperonins, various enzymes and a number of putative uncharacterized proteins. The results obtained in this study highlight the potential of B. laterosporus as a biological control agent for preserving and improving honeybee health., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Methodology . Biochemical and molecular methods were used to identify 100 lactobacilli isolated from rectal swabs. Among these, L. paracasei ssp. paracasei LP5 and L. brevis LP9 showed significant antibacterial activity against S. agalactiae and L. monocytogenes . Accordingly, characterization of their bacteriocins, BacLP5 and BacLP9, was conducted to obtain information on their kinetic production, sensitivity to chemico-physical parameters and molecular weight. To investigate the possible use of the two Lactobacillus strains as probiotics, their gastrointestinal resistance, cellular adhesiveness and sensitivity to antibiotics were also studied. Results . The obtained data show that BacLP5 and BacLP9 most likely belong to class II bacteriocins and both have a molecular weight of approximately 3 kDa. The production of BacLP5 and BacLP9 started after 4 h (40 and 80 AU ml -1 ), respectively. Both of the Lactobacillus strains survived gastric and intestinal juices well and showed adhesive capability on HEp-2 cells. Conclusion . Due to their peculiar antimicrobial characteristics, L. paracasei ssp. paracasei LP5 and L. brevis LP9 are suitable for use in the treatment of vaginal disorders, through both oral and transvaginal administration.

There is a significant increase in the discovery of new antimicrobial compounds in recent past to combat drug resistant pathogens. Members of the genus Bacillus and related genera have been screened extensively due to their ability to produce wide range of antimicrobial compounds. In this study, we have isolated and characterized a new antimicrobial peptide from a marine bacterium identified as Virgibacillus species. The low molecular mass and stability of the antimicrobial substance pointed towards the bacteriocinogenic nature of the compound. The RAST analysis of genome sequence showed presence of a putative bacteriocin biosynthetic cluster containing genes necessary for synthesis of a lanthipeptide. Translated amino acid sequence of mature C-terminal propeptide showed identity with salivaricin A (52.2%) and lacticin A (33.3%). Accordingly, the mass (2417 Da) obtained by MALDI analysis was in agreement with posttranslational modifications of the leader peptide to yield three methyl lanthionine rings and a disulfide bond between two free cysteine residues. The lanthipeptide was named as virgicin, which selectively inhibited the growth of Gram-positive bacteria and biofilm formation by Enterococcus faecalis. Inhibition of biofilm formation by E. faecalis was also observed in in vitro model experiments using hydroxyapatite discs. Thus, virgicin appears to be a promising new bacteriocin to control oral biofilm formation by selective pathogens.

Little is known about the migration of bacteriocins across human cells. In this study, we report on migration of three bacteriocins nisin, plantaricin 423 and bacST4SA across colonic adenocarcinoma (Caco-2) cells and human umbilical vein endothelial cells (HUVECs). Bacteriocins were fluorescently labelled while still maintaining antimicrobial activity. Migration of fluorescently labelled bacteriocins across monolayers was assessed in vitro using transmigration well inserts. After 3 h, 75% of nisin, 85% of plantaricin 423 and 82% of bacST4SA migrated across the Caco-2 cell monolayer. Over the same time span, 88% nisin, 93% plantaricin 423 and 91% bacST4SA migrated across the HUVEC monolayer. The viability of both cell types remained unchanged when exposed to 50 µM of nisin, plantaricin 423 or bacST4SA. The effect of human plasma on bacteriocin activity was also assessed. Activity loss was dependent on bacteriocin type and concentration, with the class-IIa bacteriocins retaining more activity compared to nisin. This is the first report of bacteriocins migrating across simulated gastrointestinal- and vascular-barriers. This study provides some of the first evidence that bacteriocins are capable of crossing the gut-blood-barrier. However, in vivo studies need to be performed to confirm these findings and expand on the role of bacteriocin migration across cell barriers.

The need for new antibiotic compounds is rising and antimicrobial peptides are excellent candidates to fulfill this object. The bacteriocin subgroup lantibiotics, for example, are active in the nanomolar range and target the membranes of mainly Gram-positive bacteria. They bind to lipid II, inhibit cell growth and in some cases form pores within the bacterial membrane, inducing rapid cell death. Pharmaceutical usage of lantibiotics is however hampered by the presence of gene clusters in human pathogenic strains which, when expressed, confer resistance. The human pathogen Streptococcus agalactiae COH1, expresses several lantibiotic resistance proteins resulting in resistance against for example nisin. This study presents a highly potent, pore forming nisin variant as an alternative lantibiotic which bypasses the SaNSR protein. It is shown that this nisin derivate nisin C28P keeps its nanomolar antibacterial activity against L. lactis NZ9000 cells but is not recognized by the nisin resistance protein SaNSR. Nisin C28P is cleaved by SaNSR in vitro with a highly decreased efficiency, as shown by an cleavage assay. Furthermore, we show that nisin C28P is still able to form pores in the membranes of L. lactis and is three times more efficient against SaNSR-expressing L. lactis cells than wildtype nisin., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Intestinal commensal bacteria can inhibit dense colonization of the gut by vancomycin-resistant Enterococcus faecium (VRE), a leading cause of hospital-acquired infections 1,2 . A four-strained consortium of commensal bacteria that contains Blautia producta BP SCSK can reverse antibiotic-induced susceptibility to VRE infection 3 . Here we show that BP SCSK reduces growth of VRE by secreting a lantibiotic that is similar to the nisin-A produced by Lactococcus lactis. Although the growth of VRE is inhibited by BP SCSK and L. lactis in vitro, only BP SCSK colonizes the colon and reduces VRE density in vivo. In comparison to nisin-A, the BP SCSK lantibiotic has reduced activity against intestinal commensal bacteria. In patients at high risk of VRE infection, high abundance of the lantibiotic gene is associated with reduced density of E. faecium. In germ-free mice transplanted with patient-derived faeces, resistance to VRE colonization correlates with abundance of the lantibiotic gene. Lantibiotic-producing commensal strains of the gastrointestinal tract reduce colonization by VRE and represent potential probiotic agents to re-establish resistance to VRE.

Bacillus velezensis BS2 was isolated from meongge (common sea squirt) jeotgal, a Korean fermented seafood, and produces a bacteriocin, BacBS2, which strongly inhibits Listeria monocytogenes and Bacillus cereus . BacBS2 was partially purified by Q-Sepharose column chromatography after ammonium sulfate precipitation of the culture supernatant, then further purified by Sephadex G-50 column chromatography. Partially purified BacBS2 was estimated to be 6.5 kDa in size by Tricine-SDS PAGE and activity detection by gel-overlay. Enzyme treatment and FT-IR spectrum of partially purified BacBS2 confirmed its proteinaceous nature. BacBS2 was fully stable at pH 4-9, and half of activity was retained at pH 1-3. Full activity was retained after exposure to 80°C for 15 min, but half of the activity was retained upon exposure to 90°C for 15 min or 100°C for 10 min. BacBS2 inhibited L. monocytogenes by bactericidal mode of action. B. velezensis BS2 and its BacBS2 seem useful as biopreservatives for fermented foods such as jeotgal.

The production of a bacteriocin-like substance with antimicrobial activity, named peocin, by the probiotic Paenibacillus ehimensis NPUST1 was previously reported by our laboratory. The present study aimed to identify peocin and increase the peocin yield by heterologous expression in Escherichia coli BL21(DE3). Peocin was identified as a DNA starvation/stationary phase protection protein, also called DNA-binding protein from starved cells (Dps), by gel overlay and LC-MS/MS analysis. For mass production of peocin, fed-batch cultivation of E. coli was performed using a pH-stat control system. Purification by simple nickel affinity chromatography and dialysis yielded 45.3 mg of purified peocin from a 20-mL fed-batch culture (49.3% recovery). The biological activity of the purified peocin was confirmed by determination of the MIC and MBC against diverse pathogens. Purified peocin exhibited antimicrobial activity against aquatic, food spoilage, clinical and antibiotic-resistant pathogens. In an in vivo challenge test, zebrafish treated with purified peocin exhibited significantly increased survival rates after A. hydrophila challenge. The present study is the first to show the antimicrobial activity of Dps and provides an efficient strategy for production of bioactive peocin, which will aid the development of peocin as a novel antimicrobial agent with potential applications in diverse industries.