• Controlled functional expression of the bacteriocins pediocin PA-1 and bactofencin A in Escherichia coli

      Mesa-Pereira, Beatriz; O’Connor, Paula M.; Rea, Mary; Cotter, Paul D.; Hill, Colin; Ross, R Paul; Science Foundation Ireland; SFI/12/RC/2273 (Nature Publishing Group, 2017-06-08)
      The bacteriocins bactofencin A (class IId) and pediocin PA-1 (class IIa) are encoded by operons with a similarly clustered gene organization including a structural peptide, an immunity protein, an ABC transporter and accessory bacteriocin transporter protein. Cloning of these operons in E. coli TunerTM (DE3) on a pETcoco-2 derived vector resulted in successful secretion of both bacteriocins. A corresponding approach, involving the construction of vectors containing different combinations of these genes, revealed that the structural and the transporter genes alone are sufficient to permit heterologous production and secretion in this host. Even though the accessory protein, usually associated with optimal disulfide bond formation, was not required for bacteriocin synthesis, its presence did result in greater pediocin PA-1 production. The simplicity of the system and the fact that the associated bacteriocins could be recovered from the extracellular medium provides an opportunity to facilitate protein engineering and the overproduction of biologically-active bacteriocins at industrial scale. Additionally, this system could enable the characterization of new bacteriocin operons where genetic tools are not available for the native producers.
    • Oligosaccharides Isolated from MGO™ Manuka Honey Inhibit the Adhesion of Pseudomonas aeruginosa, Escherichia Coli O157:H7 and Staphylococcus Aureus to Human HT-29 cells

      Lane, Johnathan A.; Calonne, Julie; Slattery, Helen; Hickey, Rita M. (MDPI AG, 2019-10-01)
      Historically, honey is known for its anti-bacterial and anti-fungal activities and its use for treatment of wound infections. Although this practice has been in place for millennia, little information exists regarding which manuka honey components contribute to the protective nature of this product. Given that sugar accounts for over 80% of honey and up to 25% of this sugar is composed of oligosaccharides, we have investigated the anti-infective activity of manuka honey oligosaccharides against a range of pathogens. Initially, oligosaccharides were extracted from a commercially-available New Zealand manuka honey—MGO™ Manuka Honey (Manuka Health New Zealand Ltd.)—and characterized by High pH anion exchange chromatography coupled with pulsed amperiometric detection. The adhesion of specific pathogens to the human colonic adenocarcinoma cell line, HT-29, was then assessed in the presence and absence of these oligosaccharides. Manuka honey oligosaccharides significantly reduced the adhesion of Escherichia coli O157:H7 (by 40%), Staphylococcus aureus (by 30%), and Pseudomonas aeruginosa (by 52%) to HT-29 cells. This activity was then proven to be concentration dependent and independent of bacterial killing. This study identifies MGO™ Manuka Honey as a source of anti-infective oligosaccharides for applications in functional foods aimed at lowering the incidence of infectious diseases.
    • Reincarnation of Bacteriocins From the Lactobacillus Pangenomic Graveyard

      Collins, Fergus W. J.; Mesa-Pereira, Beatriz; O'Connor, Paula M.; Rea, Mary; Hill, Colin; Ross, R Paul; Science Foundation Ireland; SFI/12/RC/227 (Frontiers, 02/07/2018)
      Bacteria commonly produce narrow spectrum bacteriocins as a means of inhibiting closely related species competing for similar resources in an environment. The increasing availability of genomic data means that it is becoming easier to identify bacteriocins encoded within genomes. Often, however, the presence of bacteriocin genes in a strain does not always translate into biological antimicrobial activity. For example, when analysing the Lactobacillus pangenome we identified strains encoding ten pediocin-like bacteriocin structural genes which failed to display inhibitory activity. Nine of these bacteriocins were novel whilst one was identified as the previously characterized bacteriocin “penocin A.” The composition of these bacteriocin operons varied between strains, often with key components missing which are required for bacteriocin production, such as dedicated bacteriocin transporters and accessory proteins. In an effort to functionally express these bacteriocins, the structural genes for the ten pediocin homologs were cloned alongside the dedicated pediocin PA-1 transporter in both Escherichia coli and Lactobacillus paracasei heterologous hosts. Each bacteriocin was cloned with its native leader sequence and as a fusion protein with the pediocin PA-1 leader sequence. Several of these bacteriocins displayed a broader spectrum of inhibition than the original pediocin PA-1. We show how potentially valuable bacteriocins can easily be “reincarnated” from in silico data and produced in vitro despite often lacking the necessary accompanying machinery. Moreover, the study demonstrates how genomic datasets such as the Lactobacilus pangenome harbor a potential “arsenal” of antimicrobial activity with the possibility of being activated when expressed in more genetically amenable hosts.
    • Reincarnation of Bacteriocins From the Lactobacillus Pangenomic Graveyard

      Collins, Fergus W. J.; Mesa-Pereira, Beatriz; O'Connor, Paula M.; Rea, Mary; Hill, Colin; Ross, R Paul; Science Foundation Ireland; SFI/12/RC/227 (Frontiers, 2018-07-02)
      Bacteria commonly produce narrow spectrum bacteriocins as a means of inhibiting closely related species competing for similar resources in an environment. The increasing availability of genomic data means that it is becoming easier to identify bacteriocins encoded within genomes. Often, however, the presence of bacteriocin genes in a strain does not always translate into biological antimicrobial activity. For example, when analysing the Lactobacillus pangenome we identified strains encoding ten pediocin-like bacteriocin structural genes which failed to display inhibitory activity. Nine of these bacteriocins were novel whilst one was identified as the previously characterized bacteriocin “penocin A.” The composition of these bacteriocin operons varied between strains, often with key components missing which are required for bacteriocin production, such as dedicated bacteriocin transporters and accessory proteins. In an effort to functionally express these bacteriocins, the structural genes for the ten pediocin homologs were cloned alongside the dedicated pediocin PA-1 transporter in both Escherichia coli and Lactobacillus paracasei heterologous hosts. Each bacteriocin was cloned with its native leader sequence and as a fusion protein with the pediocin PA-1 leader sequence. Several of these bacteriocins displayed a broader spectrum of inhibition than the original pediocin PA-1. We show how potentially valuable bacteriocins can easily be “reincarnated” from in silico data and produced in vitro despite often lacking the necessary accompanying machinery. Moreover, the study demonstrates how genomic datasets such as the Lactobacilus pangenome harbor a potential “arsenal” of antimicrobial activity with the possibility of being activated when expressed in more genetically amenable hosts.
    • Risk Assessment of E. coli Survival Up to the Grazing Exclusion Period After Dairy Slurry, Cattle Dung, and Biosolids Application to Grassland

      Ashekuzzaman, S.M.; Richards, Karl G.; Ellis, Stephanie; Tyrrel, Sean; O'Leary, Emma; Griffiths, Bryan; Ritz, Karl; Fenton, Owen; European Union; 265269 (Frontiers, 10/07/2018)
      Grassland application of dairy slurry, cattle dung, and biosolids offers an opportunity to recycle valuable nutrients (N, P, and K), which may all introduce pathogens to the soil environment. Herein, a temporal risk assessment of the survival of Escherichia coli (E. coli) up to 40 days in line with the legislated grazing exclusion time points after application was examined across six scenarios: (1) soil and biosolids mixture, (2) biosolids amended soil, (3) dairy slurry application, (4) cattle dung on pasture, (5) comparison of scenario 2, 3, and 4, and (6) maximum legal vs. excess rate of application for scenario 2 and 3. The risk model input parameters were taken or derived from regressions within the literature and an uncertainty analysis (n = 1,000 trials for each scenario) was conducted. Scenario 1 results showed that E. coli survival was higher in the soil/biosolids mixture for higher biosolids portion, resulting in the highest 20 day value of residual E. coli concentration (i.e., C20, log10 CFU g−1 dw) of 1.0 in 100% biosolids or inoculated soil and the lowest C20 of 0.098 in 75/25 soil/biosolids ratio, respectively, in comparison to an average initial value of ~6.4 log10 CFU g−1 dw. The E. coli survival across scenario 2, 3, and 4 showed that the C20 value of biosolids (0.57 log10 CFU g−1 dw) and dairy slurry (0.74 log10 CFU ml−1) was 2.9–3.7 times smaller than that of cattle dung (2.12 log10 CFU g−1 dw). The C20 values of biosolids and dairy slurry associated with legal and excess application rates ranged from 1.14 to 1.71 log10 CFU ha−1, which is a significant reduction from the initial concentration range (12.99 to 14.83 log10 CFU ha−1). The E. coli survival in un-amended soil was linear with a very low decay rate resulting in a higher C20 value than that of biosolids or dairy slurry. The risk assessment and uncertainly analysis showed that the residual concentrations in biosolids/dairy slurry applied soil after 20 days would be 45–57% lower than that of the background soil E. coli concentration. This means the current practice of grazing exclusion times is safe to reduce the risk of E. coli transmission into the soil environment.
    • Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy

      Dalmasso, Marion; Strain, Ronan; Neve, Horst; Franz, C.M.A.P.; Cousin, Fabien J.; Ross, R Paul; Hill, Colin; Science Foundation Ireland; SFI/12/RC/2273 (PLOS, 09/06/2016)
      With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were investigated. All three phages reduced the growth of E. coli strain DPC6051 at multiplicity of infection (MOI) between 10−3 and 105. A cocktail of all three phages completely inhibited the growth of E. coli. The phage cocktail also reduced biofilm formation and prevented the emergence of phage-resistant mutants which occurred with single phage. When combined with ciprofloxacin, phage alone or in cocktail inhibited the growth of E. coli and prevented the emergence of resistant mutants. These three new phages are promising biocontrol agents for E. coli infections.