• Antimicrobial antagonists against food pathogens; a bacteriocin perspective

      O'Connor, Paula M.; Ross, R Paul; Hill, Colin; Cotter, Paul D.; Science Foundation Ireland; 12/RC/2273 (Elsevier, 03/02/2015)
      Efforts are continuing to find novel bacteriocins with enhanced specificity and potency. Traditional plating techniques are still being used for bacteriocin screening studies, however, the availability of ever more bacterial genome sequences and the use of in silico gene mining tools have revealed novel bacteriocin gene clusters that would otherwise have been overlooked. Furthermore, synthetic biology and bioengineering-based approaches are allowing scientists to harness existing and novel bacteriocin gene clusters through expression in different hosts and by enhancing functionalities. The same principles apply to bacteriocin producing probiotic cultures and their application to control pathogens in the gut. We can expect that the recent developments on bacteriocins from Lactic Acid Bacteria (LAB) described here will contribute greatly to increased commercialisation of bacteriocins in food systems.
    • Bactofencin A, a New Type of Cationic Bacteriocin with Unusual Immunity

      O'Shea, Eileen F.; O'Connor, Paula M.; O'Sullivan, Orla; Cotter, Paul D.; Ross, R Paul; Hill, Colin; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; 04R; 07/CE/B1368 (American Society for Microbiology, 29/10/2013)
      Bacteriocin production is an important probiotic trait of intestinal bacteria. In this study, we identify a new type of bacteriocin, bactofencin A, produced by a porcine intestinal isolate Lactobacillus salivarius DPC6502, and assess its potency against pathogenic species including Staphylococcus aureus and Listeria monocytogenes. Genome sequencing of the bacteriocin producer revealed bfnA, which encodes the mature and highly basic (pI 10.59), 22-amino-acid defensin-like peptide. Matrixassisted laser desorption ionization–time of flight (MALDI-TOF) mass spectral analysis determined that bactofencin A has a molecular mass of 2,782 Da and contains two cysteine residues that form an intramolecular disulfide bond. Although an ABC transporter and transport accessory protein were also present within the bacteriocin gene cluster, a classical bacteriocin immunity gene was not detected. Interestingly, a dltB homologue was identified downstream of bfnA. DltB is usually encoded within the dlt operon of many Gram-positive bacteria. It is responsible for D-alanylation of teichoic acids in the cell wall and has previously been associated with bacterial resistance to cationic antimicrobial peptides. Heterologous expression of this gene conferred bactofencin A-specific immunity on sensitive strains of L. salivarius and S. aureus (although not L. monocytogenes), establishing its role in bacteriocin immunity. An analysis of the distribution of bfnA revealed that it was present in four additional isolates derived from porcine origin and absent from five human isolates, suggesting that its distribution is host specific. Given its novelty, we anticipate that bactofencin A represents the prototype of a new class of bacteriocins characterized as being cationic, with a DltB homologue providing a cognate immunity function.
    • Lactococcus lactis subsp. lactis as a natural anti-listerial agent in the mushroom industry

      Dygico, Lionel K.; O'Connor, Paula M.; Hayes, Maria; Gahan, Cormac G M; Grogan, Helen; Burgess, Catherine; Department of Agriculture, Food & the Marine; 14F881 (Elsevier, 2019-01-28)
      Mushroom growth substrates from different commercial producers of mushrooms (Agaricus bisporus) were screened for the presence of bacteria with potential for use as biocontrol agents for controlling Listeria monocytogenes in the mushroom production environment. Eight anti-listerial strains were isolated from different sources and all were identified using 16s rRNA gene sequencing as Lactococcus lactis subsp. lactis. Whole-genome sequencing of the Lc. lactis isolates indicated that strains from different sites and substrate types were highly similar. Colony MALDI-TOF mass spectrometry found that these strains were Nisin Z producers but inhibitory activity was highly influenced by the incubation conditions and was strain dependant. The biofilm forming ability of these strains was tested using a crystal violet assay and all were found to be strong biofilm formers. Growth of Lc. lactis subsp. lactis using mixed-biofilm conditions with L. monocytogenes on stainless steel resulted in a 4-log reduction of L. monocytogenes cell numbers. Additional sampling of mushroom producers showed that these anti-listerial Lc. lactis strains are commonly present in the mushroom production environment. Lc. lactis has a generally regarded as safe (GRAS) status and therefore has potential for use as an environmentally benign solution to control L. monocytogenes in order to prevent product contamination and to enhance consumer confidence in the mushroom industry.
    • Production of multiple bacteriocins from a single locus by gastrointestinal strains of Lactobacillus salivarius

      O'Shea, Eileen F.; O'Connor, Paula M.; Raftis, Emma J.; O'Toole, Paul W.; STANTON, CATHERINE; Cotter, Paul D.; Ross, R Paul; Hill, Colin; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; et al. (American Society for Microbiology, 07/10/2011)
      Bacteriocins produced by Lactobacillus salivarius isolates derived from gastrointestinal origin have previously demonstrated efficacy for in vivo protection against Listeria monocytogenes infection. In this study, comparative genomic analysis was employed to investigate the intraspecies diversity of seven L. salivarius isolates of human and porcine intestinal origin, based on the genome of the well characterised bacteriocin-producing strain L. salivarius UCC118. This revealed a highly conserved megaplasmid-encoded gene cluster in these strains involved in the regulation and secretion of two-component class IIb bacteriocins. However, considerable intraspecific variation was observed in the structural genes encoding the bacteriocin peptides. These ranged from close relatives of abp118 such as salivaricin P, which differs by 2 amino acids, to completely novel bacteriocins such as salivaricin T, which is characterized in this study. Salivaricin T inhibits closely related lactobacilli and bears little homology to previously characterized salivaricins. Interestingly, the two peptides responsible for salivaricin T activity, SalTα and SalTβ, share considerable identity with the component peptides of thermophilin 13, a bacteriocin produced by Streptococcus thermophilus. Furthermore, the salivaricin locus of strain DPC6488 also encodes an additional novel one-component class IId anti-listerial bacteriocin, salivaricin L. These findings suggest a high level of redundancy in the bacteriocins that can be produced by intestinal L. salivarius isolates using the same enzymatic production and export machinery. Such diversity may contribute to their ability to dominate and compete within the complex microbiota of the mammalian gut.
    • Subspecies diversity in bacteriocin production by intestinal Lactobacillus salivarius strains

      O'Shea, Eileen F.; O'Connor, Paula M.; Raftis, Emma J.; O'Toole, Paul W.; STANTON, CATHERINE; Cotter, Paul D.; Ross, R Paul; Hill, Colin; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; et al. (Landes Bioscience, 2012-10)
      A recent comparative genomic hybridisation study in our laboratory revealed considerable plasticity within the bacteriocin locus of gastrointestinal strains of Lactobacillus salivarius. Most notably these analyses led to the identification of two novel unmodified bacteriocins salivaricin L and salivaricin T produced by the neonatal isolate L. salivarius DPC6488 with immunity, regulatory and export systems analogous to those of abp118, a two-component bacteriocin produced by the well characterized reference strain L. salivarius UCC118. In this addendum we discuss the intraspecific diversity of our seven bacteriocin-producing L. salivarius isolates on a genome-wide level, and more specifically, with respect to their salivaricin loci.
    • Synthesis of trypsin-resistant variants of the Listeria-active bacteriocin salivaricin P

      O'Shea, Eileen F.; O'Connor, Paula M.; Cotter, Paul D.; Ross, R Paul; Hill, Colin; Department of Agriculture, Food and the Marine, Ireland; Science Foundation Ireland; Teagasc Walsh Fellowship Programme (American Society for Microbiology, 25/06/2010)
      Two-component Salivaricin P-like bacteriocins have demonstrated potential as antimicrobials capable of controlling infections in the gastrointestinal tract (GIT). The anti-Listeria activity of salivaricin P is optimal when the individual peptides, Sln1 and Sln2, are added in succession in a 1:1 ratio. However, as degradation by digestive proteases may compromise the functionality of these peptides within the GIT we investigated the potential to create salivaricin variants with enhanced resistance to the intestinal protease, trypsin. A total of 11 variants of the salivaricin P components were generated in which conservative modifications at the trypsin-specific cleavage sites were explored in order to protect the peptides from trypsin degradation while maintaining their potent antimicrobial activity. Analysis of these variants revealed that eight were resistant to trypsin digestion while retaining antimicrobial activity. Combining the complementary trypsin resistant variants Sln1-5 and Sln2-3 resulted in a MIC50 of 300 nM against Listeria monocytogenes, a 3.75-fold reduction in activity compared to wild-type salivaricin P. This study demonstrates the potential of engineering bacteriocins variants which are resistant to specific protease action but which retain significant antimicrobial activity.