Pharmaceutical design of a delivery system for the bacteriocin lacticin 3147

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

Nisin Z and lacticin 3147 improve efficacy of antibiotics against clinically significant bacteria

Aim: To determine if bacteriocins improve antibiotic efficacy. Materials & methods: Deferred antagonism assays identified bacteriocins with activity. Growth curves and time kill assays demonstrated bactericidal activity of antimicrobial combinations, and checkerboard assays confirmed synergy. Methicillin-resistant Staphylococcus aureus-infected porcine skin model determined ex vivo efficacy. Results: Subinhibitory concentrations of lacticin with penicillin or vancomycin resulted in complete growth inhibition of strains and the improved inhibitory effect was apparent after 1 h. Nisin with methicillin proved more effective against methicillin-resistant Staphylococcus aureus than either antimicrobial alone, revealing partial synergy and significantly reduced pathogen numbers on porcine skin after 3 h compared with minimal inhibition for either antimicrobial alone. Conclusion: Nisin Z and lacticin 3147 may support the use of certain antibiotics and revive ineffective antibiotics.

Learning the Science Behind Bacteriocins Through Lacticin 3147; a Promising Lantibiotic

As microorganisms continue to develop resistance and survive against many different forms of antimicrobial solutions, such as antibiotics, the threat that antimicrobial resistance poses grows considerably. One solution to this persistent issue could be bacteriocins: ribosomally synthesized antimicrobial peptides that are synthesized by bacteria. In this study, the specific lantibiotic—a subclass of bacteriocin—used was lacticin 3147, which is comprised of two components: A1 and A2. Lacticin 3147 was first purified and isolated in order to properly analyze its antimicrobial effects, which show potential use in antibiotics or food preservation. The procedure started with growing the producer bacteria strain, Lactococcus lactis DPC 3147 in a broth which was later used to inoculate a large volume of media. This media was then separated through centrifugation into two components: the supernatant and cell pellet, both of which were each individually concentrated and purified through a series of columns. Approximately one milliliter of each component was run through a High Performance Liquid Chromatography (HPLC) machine, and the resulting chromatograms interpreted to evaluate and compare the concentrations of lacticin 3147 produced in the liquid media portion (supernatant) and the cell components (cell pellet). Subsequently, fractions were collected from all runs from the HPLC and further subjected to Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI TOF) mass spectrometry. This allows to test the molecular weights of the compounds in the samples to check if they aligned with the known molecular weights of both the A1 and A2 components of lacticin 3147. The final step was to prepare a spot on lawn assay using the indicator species: Lactococcus lactis subsp. cremoris HP. The spot on lawn assay prepared for lacticin 3147 was a visual indicator of the strong antimicrobial effects of the bacteriocin. Ultimately, this highly effective bacteriocin, lacticin 3147, could be utilized in smaller concentrations than current antibiotics, and thus shows great promise in the field of antibiotics. Further studies are being conducted to understand the interactions between the A1 and A2 components of lacticin 3147, including their synergistic effects.

A Multibacteriocin Cheese Starter System, Comprising Nisin and Lacticin 3147 in Lactococcus lactis, in Combination with Plantaricin from Lactobacillus plantarum

ABSTRACT Functional starter cultures demonstrating superior technological and food safety properties are advantageous to the food fermentation industry. We evaluated the efficacies of single- and double-bacteriocin-producing starters of Lactococcus lactis capable of producing the class I bacteriocins nisin A and/or lacticin 3147 in terms of starter performance. Single producers were generated by mobilizing the conjugative bacteriophage resistance plasmid pMRC01, carrying lacticin genetic determinants, or the conjugative transposon Tn5276, carrying nisin genetic determinants, to the commercial starter L. lactis CSK2775. The effect of bacteriocin coproduction was examined by superimposing pMRC01 into the newly constructed nisin transconjugant. Transconjugants were improved with regard to antimicrobial activity and bacteriophage insensitivity compared to the recipient strain, and the double producer was immune to both bacteriocins. Bacteriocin production in the starter was stable, although the recipient strain proved to be a more efficient acidifier than transconjugant derivatives. Overall, combinations of class I bacteriocins (the double producer or a combination of single producers) proved to be as effective as individual bacteriocins for controlling Listeria innocua growth in laboratory-scale cheeses. However, using the double producer in combination with the class II bacteriocin producer Lactobacillus plantarum or using the lacticin producer with the class II producer proved to be most effective for reducing bacterial load. As emergence of bacteriocin tolerance was reduced 10-fold in the presence of nisin and lacticin, we suggest that the double producer in conjunction with the class II producer could serve as a protective culture providing a food-grade, multihurdle approach to control pathogenic growth in a variety of industrial applications. IMPORTANCE We generated a suite of single- and double-bacteriocin-producing starter cultures capable of generating the class I bacteriocin lacticin 3147 or nisin or both bacteriocins simultaneously via conjugation. The transconjugants exhibited improved bacteriophage resistance and antimicrobial activity. The single producers proved to be as effective as the double-bacteriocin producer at reducing Listeria numbers in laboratory-scale cheese. However, combining the double producer or the lacticin-producing starter with a class II bacteriocin producer, Lactobacillus plantarum LMG P-26358, proved to be most effective at reducing Listeria numbers and was significantly better than a combination of the three bacteriocin-producing strains, as the double producer is not inhibited by either of the class I bacteriocins. Since the simultaneous use of lacticin and nisin should reduce the emergence of bacteriocin-tolerant derivatives, this study suggests that a protective starter system produced by bacteriocin stacking is a worthwhile multihurdle approach for food safety applications.

Lantibiotic Reductase LtnJ Substrate Selectivity Assessed with a Collection of Nisin Derivatives as Substrates

ABSTRACTLantibiotics are potent antimicrobial peptides characterized by the presence of dehydrated amino acids, dehydroalanine and dehydrobutyrine, and (methyl)lanthionine rings. In addition to these posttranslational modifications, some lantibiotics exhibit additional modifications that usually confer increased biological activity or stability on the peptide. LtnJ is a reductase responsible for the introduction ofd-alanine in the lantibiotic lacticin 3147. The conversion ofl-serine intod-alanine requires dehydroalanine as the substrate, which is producedin vivoby the dehydration of serine by a lantibiotic dehydratase, i.e., LanB or LanM. In this work, we probe the substrate specificity of LtnJ using a system that combines the nisin modification machinery (dehydratase, cyclase, and transporter) and the stereospecific reductase LtnJ inLactococcus lactis. We also describe an improvement in the production yield of this system by inserting a putative attenuator from the nisin biosynthesis gene cluster in front of theltnJgene. In order to clarify the sequence selectivity of LtnJ, peptides composed of truncated nisin and different mutated C-terminal tails were designed and coexpressed with LtnJ and the nisin biosynthetic machinery. In these tails, serine was flanked by diverse amino acids to determine the influence of the surrounding residues in the reaction. LtnJ successfully hydrogenated peptides when hydrophobic residues (Leu, Ile, Phe, and Ala) were flanking the intermediate dehydroalanine, while those in which dehydroalanine was flanked by one or two polar residues (Ser, Thr, Glu, Lys, and Asn) or Gly were either less prone to be modified by LtnJ or not modified at all. Moreover, our results showed that dehydrobutyrine cannot serve as a substrate for LtnJ.