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Methicillin-resistant Staphylococcus aureus (MRSA) is a major bacterial pathogen that causes hospital- and community-acquired infections. Owing to its multidrug resistance, it is imperative to develop new antimicrobial agents to treat MRSA infections. In this study, using genome mining analysis and a culture-based screening method to detect bacteriocin activity, we screened a strain, Bacillus sp. TL12, which harbored a putative leaderless bacteriocin gene cluster ( bac gene cluster) and exhibited potent anti-MRSA activity. The antimicrobial agents, products of the bac gene cluster, were purified and identified as four novel leaderless bacteriocins: bacin A1, A2, A3, and A4. Bacin A2 was evaluated as a representative antimicrobial agent and showed remarkable antimicrobial activity against S. aureus, MRSA, and the foodborne pathogens Listeria monocytogenes and Bacillus cereus. Mechanistic experiments revealed that bacin A2 damaged cell membranes and exhibited bactericidal activity against MRSA. Bacin A2 effectively inhibited the formation of S. aureus and MRSA biofilms (>0.5× MIC) and killed the cells in their established biofilms (>4× MIC). The hemolytic and NIH/3T3 cytotoxicity assay results for bacin A2 confirmed its biosafety. Thus, bacins have potential as alternative antimicrobial agents for treating MRSA infections. IMPORTANCE Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen that is difficult to treat because of its resistance to several widely used antibiotics. The present study aimed to identify novel anti-MRSA bacteriocins in a prominent producer of bacteriocins, Bacillus cereus group. Four novel leaderless bacteriocins, bacin A1, A2, A3, and A4, which show potent bactericidal effect against S. aureus and MRSA, were identified in Bacillus sp. TL12. Moreover, bacins inhibited biofilm formation and killed cells in the established biofilms of S. aureus and MRSA. These findings suggest that bacins are promising alternatives to treat MRSA infections.

Bacteriocins are bacterial-derived peptides that exhibit antimicrobial activity and can be used as food preservatives. Here, using the indicator strain Bacillus cereus CMCC63301, we screened and identified a Bacillus thuringiensis LX43 strain that exhibits potent antimicrobial activity and harbors a putative leaderless bacteriocin gene cluster ( thn gene cluster). Five novel leaderless bacteriocins, thuricin A1, A2, A3, A4, and A5, encoded by the thn gene cluster, were purified and identified. Thuricin A5 was regarded as a representative and showed remarkable antimicrobial activity against foodborne pathogens B. cereus , Clostridium perfringens , Listeria monocytogenes , and Staphylococcus aureus , likely by damaging their cell envelope. Moreover, thuricin A5 displayed good thermal and pH stability, with no hemolytic activity and cytotoxicity, indicating its wide applicability and biosafety. Furthermore, thuricin A5 effectively inhibited or eradicated foodborne pathogens in skim milk at 25 °C in a dose-dependent manner, affirming its potential for use as a novel biopreservative in foods.

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.

Lantibiotics are antimicrobial peptides with potential applications as the next generation of antimicrobials in the food industry and/or the pharmaceutical industry. Nisin has successfully been used as a food preservative for over 40 years, but its major drawback is its limited stability under neutral and alkaline pH conditions. To identify alternatives with better biochemical properties, we screened more than 100 strains of the Bacillus cereus group. Three novel lantibiotics, ticins A1 (4,062.98 Da), A3 (4,048.96 Da), and A4 (4,063.02 Da), which were highly thermostable (121°C for 30 min) and extremely pH tolerant (pH 2.0 to 9.0), were identified in Bacillus thuringiensis BMB3201. They all showed potent antimicrobial activities against all tested Gram-positive bacteria and greater activities than those of nisin A against Bacillus cereus and Listeria monocytogenes, two important foodborne pathogens. These three novel lantibiotics, with their extremely stable properties and potent antimicrobial activities, have the potential for use as biopreservatives., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Lantibiotics are ribosomally synthesized peptides that contain multiple posttranslational modifications. Research on lantibiotics has increased recently, mainly due to their broad-spectrum antimicrobial activity, especially against some clinical Gram-positive pathogens. Many reports about various bacteriocins in the Bacillus cereus group have been published, but few were about lantibiotics. In this study, we identified 101 putative lanthipeptide gene clusters from 77 out of 223 strains of this group, and these gene clusters were further classified into 20 types according to their gene organization and the homologies of their functional genes. Among them, 18 types were novel and have not yet been experimentally verified. Two novel lantibiotics (thuricin 4A-4 and its derivative, thuricin 4A-4D) were identified in the type I-1 lanthipeptide gene cluster and showed activity against all tested Gram-positive bacteria. The mode of action of thuricin 4A-4 was studied, and we found that it acted as a bactericidal compound. The transcriptional analysis of four structural genes (thiA1, thiA2, thiA3, and thiA4) in the thuricin 4A gene cluster showed that only one structural gene, thiA4, showed efficient transcription in the exponential growth phase; the other three structural genes did not. In addition, the putative transmembrane protein ThiI was responsible for thuricin 4A-4 immunity. Genome analysis and functional verification illustrated that B. cereus group strains were a prolific source of novel lantibiotics., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)