Antimicrobial antagonists against food pathogens; a bacteriocin perspective

peer-reviewedEfforts 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.This work was funded by the Alimentary Pharmabiotic Centre, a research centre funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan. The authors and their work were supported by SFI (grant no. 12/RC/2273

Saturation Mutagenesis of Lysine 12 Leads to the Identification of Derivatives of Nisin A with Enhanced Antimicrobial Activity

peer-reviewedIt is becoming increasingly apparent that innovations from the “golden age” of antibiotics are becoming ineffective, resulting in a pressing need for novel therapeutics. The bacteriocin family of antimicrobial peptides has attracted much attention in recent years as a source of potential alternatives. The most intensively studied bacteriocin is nisin, a broad spectrum lantibiotic that inhibits Gram-positive bacteria including important food pathogens and clinically relevant antibiotic resistant bacteria. Nisin is gene-encoded and, as such, is amenable to peptide bioengineering, facilitating the generation of novel derivatives that can be screened for desirable properties. It was to this end that we used a site-saturation mutagenesis approach to create a bank of producers of nisin A derivatives that differ with respect to the identity of residue 12 (normally lysine; K12). A number of these producers exhibited enhanced bioactivity and the nisin A K12A producer was deemed of greatest interest. Subsequent investigations with the purified antimicrobial highlighted the enhanced specific activity of this modified nisin against representative target strains from the genera Streptococcus, Bacillus, Lactococcus, Enterococcus and Staphylococcus.This work was supported by the Irish Government under the National Development Plan; by the Irish Research Council for Science Engineering and Technology (IRCSET); by Enterprise Ireland; and by Science Foundation Ireland (SFI), through the Alimentary Pharmabiotic Centre (APC) at University College Cork, Ireland, which is supported by the SFI-funded Centre for Science, Engineering and Technology (SFI-CSET) and provided P.D.C., C.H. and R.P.R. with SFI Principal Investigator funding

Efficacy of nisin A and nisin V semi-purified preparations alone and in combination with plant essential oils to control Listeria monocytogenes

peer-reviewedThe foodborne pathogenic bacterium Listeria is known for relatively low morbidity and high mortality rates reaching up to 25-30%. Listeria is a hardy organism and its control in foods represents a significant challenge. Many naturally occurring compounds, including the bacteriocin nisin and a number of plant essential oils, have been widely studied and are reported to be effective as antimicrobial agents against spoilage and pathogenic microorganisms. The aim of this study was to investigate the ability of semi-purified preparations (spp) containing either nisin A or an enhanced bioengineered derivative nisin V, alone and in combination with low concentrations of the essential oils thymol, carvacrol and trans-cinnamaldehyde, to control L. monocytogenes in both laboratory media and model food systems. Combinations of nisin V-containing spp (25 μg/ml) with thymol (0.02%), carvacrol (0.02%) or cinnamaldehyde (0.02%) produced a significantly longer lag phase than any of the essential oil/nisin A combinations. In addition, the log reduction in cell counts achieved by the nisin V + carvacrol or nisin V + cinnamaldehyde combinations was twice that of the equivalent nisin A + essential oil treatment. Significantly, this enhanced activity was validated in model food systems against L. monocytogenes strains of food origin. We conclude that the fermentate form of nisin V in combination with carvacrol and cinnamaldehyde offers significant advantages as a novel, natural and effective means to enhance food safety by inhibiting foodborne pathogens such as L. monocytogenes.This work was supported by the Irish Government under the National Development Plan, through Science Foundation Ireland Investigator awards to C.H. and R.P.R. (10/IN.1/B3027), and C.H., R.P.R. and P.D.C. (06/IN.1/B98)

Lactococcus lactis subsp. lactis as a natural anti-listerial agent in the mushroom industry

peer-reviewedMushroom 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

Intensive Mutagenesis of the Nisin Hinge Leads to the Rational Design of Enhanced Derivatives

peer-reviewedNisin A is the most extensively studied lantibiotic and has been used as a preservative by the food industry since 1953. This 34 amino acid peptide contains three dehydrated amino acids and five thioether rings. These rings, resulting from one lanthionine and four methyllanthionine bridges, confer the peptide with its unique structure. Nisin A has two mechanisms of action, with the N-terminal domain of the peptide inhibiting cell wall synthesis through lipid II binding and the C-terminal domain responsible for pore-formation. The focus of this study is the three amino acid ‘hinge’ region (N 20, M 21 and K 22) which separates these two domains and allows for conformational flexibility. As all lantibiotics are gene encoded, novel variants can be generated through manipulation of the corresponding gene. A number of derivatives in which the hinge region was altered have previously been shown to possess enhanced antimicrobial activity. Here we take this approach further by employing simultaneous, indiscriminate site-saturation mutagenesis of all three hinge residues to create a novel bank of nisin derivative producers. Screening of this bank revealed that producers of peptides with hinge regions consisting of AAK, NAI and SLS displayed enhanced bioactivity against a variety of targets. These and other results suggested a preference for small, chiral amino acids within the hinge region, leading to the design and creation of producers of peptides with hinges consisting of AAA and SAA. These producers, and the corresponding peptides, exhibited enhanced bioactivity against Lactococcus lactis HP, Streptococcus agalactiae ATCC 13813, Mycobacterium smegmatis MC2155 and Staphylococcus aureus RF122 and thus represent the first example of nisin derivatives that possess enhanced activity as a consequence of rational design.This work was financed by a grant from the Irish Department of Agriculture, Food and the Marine through the Food Institutional Research Measure (08/RD/C/691) and with Science Foundation Investigator award (10/IN.1/B3027)

Discovery and molecular characterisation of novel bacteriocins produced by Gram positive bacteria

Bacteriocins are ribosomally synthesised antimicrobial peptides, produced by many bacterial genera that display potent activity against closely (narrow spectrum) or distantly related (broad-spectrum) bacteria. Bacteriocins, produced by Lactic Acid Bacteria (LAB) that are natural constituents of fermented foods, are ideal natural preservatives to control food spoilage/pathogenic bacteria in minimally processed foods. In addition to their role as food preservatives, bacteriocins have potent activity against medically significant pathogens and are considered attractive alternatives or adjuncts to antibiotics, due to their inherent heat stability, potency at nanomolar scale, resistance to proteases and low levels of acquired resistance in commercial applications. Overall, bacteriocins are versatile antimicrobials with huge potential for use as biopreservatives, antibiotic alternatives, health promoting gut modulators and animal growth promotors. The aim of this thesis was to identify, purify and characterise novel bacteriocins from microorganisms isolated from a wide range of niches, with a view to expanding the number of bacteriocins currently available and exploring novel structures and activities. In this respect, Chapter 2.1 describes the discovery a novel nisin A variant, nisin H, produced by a porcine gut isolate Streptococcus hyointestinalis DPC6484. Nisin H differs from nisin A at five amino acid positions and is an intermediate between naturally occurring nisins of lactococcal and streptococcal origin. The operon encoding nisin H is noteworthy by virtue of the absence of an equivalent of nisI that encodes an immunity protein that protects the cell from its own bacteriocin. This is the first report of natural nisin variant production by an intestinal isolate of streptococcal origin and may confer an advantage to the strain by allowing it to dominate its environment, fight infection or signal the immune system of the host. In a subsequent chapter another natural variant is characterised in the form of nisin J, produced by a human skin isolate Staphylococcus capitis APC2923. Nisin J is more dissimilar to nisin A than nisin H with nine amino acid changes, six of which are unique, and an extra amino acid making it the first nisin variant to contain 35 amino acids. Interestingly, the operon lacks both nisI (immunity) and nisRK (regulatory) equivalents. Nisin J, like nisin A and H, displays activity against a wide number of genera and represents the first natural nisin variant from staphylococci and the first nisin producer from human skin, suggesting a role in competitive colonization for producing organisms. The natural nisin variants described above (nisin H and J), in addition to nisin P produced by Streptococcus agalactiae DPC7040, are all produced by non GRAS strains and are therefore limited in their potential industrial applications. The recent increase in the prevalence of antibiotic resistant pathogens makes it important that all bacteriocins regardless of the producing organism are explored as antibiotic alternatives. As these lantibiotics are gene encoded, bioengineering (Chapter 3.1) was used to enable recombinant expression of peptides naturally expressed by non-GRAS organisms in a host derived from safe origins. Specifically, the Nisin A promotor and nisin A leader sequence were fused to nisin H, J or P structural genes and successfully expressed in the GRAS strain L. lactis NZ9700, demonstrating that the L. lactis production, transport and modification machinery can produce fully functional nisin variants from significantly different genetic backgrounds. In Chapter 4, Bactofencin A produced by Streptococcus salivarius DPC6502 was discovered following a porcine gut mining study. It is a 22 amino acid, class IId bacteriocin that displays activity against Staphylococcus aureus and Listeria monocytogenes. Structurally, it consists of a positively charged N terminus that we propose could bind to the negatively charged cell surface. The small bacteriocin cluster also encodes a DltB homologue that may well be responsible for immunity through D-alanylation of teichoic acids. In order to probe structure/function relationships in bactofencin A, a library of synthetic bactofencin A peptide variants were synthesized. Substituting cysteine residues significantly reduced activity confirming the importance of the disulphide while sequential removal of the positively charged N terminal resulted in a decreasingly active peptide. Substituting each amino acid for alanine revealed that residues 9-17 within the loop were more affected by substitution, suggesting this region contributes significantly to the potency of the bacteriocin. In Chapter 5, bactofencin A was shown to enhance nisin bactericidal activity and reduce the overall frequency of resistance. Interestingly, these studies highlighted the relatively slow or delayed mode of action of bactofencin A. The last two chapters (Chapters 6.1 and 6.2) again focus on the discovery of two novel bacteriocins, namely formicin and actifensin. The first of these, formicin, is a novel bacteriocin that extends the class of two peptide lantibiotics. It was purified from Bacillus paralicheniformis APC1576, a mackerel intestine isolate. Compared with other two component lantibiotics, formicin is most similar to haloduracin and consists of a very hydrophilic Alpha peptide with a charge of +2 whereas the Beta peptide is negatively charged. Formicin displays activity against a broad range of Gram-positive bacteria including clinically relevant pathogens. The second bacteriocin is actifensin a 4091 Da, broad spectrum, Class IId bacteriocin containing three disulphide bridges with more than 50% similarity to eukaryotic defensins that we propose represents a new subclass of bacteriocins. It is produced by Actinomyces ruminicola, isolated from sheep feces. A pangenomic screen of available Actinomyces spp. revealed the presence of very diverse actifensin homologues in 29% of genomes examined, suggesting that production of actifensin like bacteriocins is a common trait. This new class of bacteriocins may provide a template to design new broad-spectrum antimicrobials for treatment of human and animal infections. The developments described in this thesis can be used to contribute to increased commercialisation of bacteriocins in both food systems and human and animal medical treatments

Impact of Environmental Factors on Bacteriocin Promoter Activity in Gut-Derived Lactobacillus salivarius

peer-reviewedBacteriocin production is regarded as a desirable probiotic trait that aids in colonization and persistence in the gastrointestinal tract (GIT). Strains of Lactobacillus salivarius, a species associated with the GIT, are regarded as promising probiotic candidates and have a number of associated bacteriocins documented to date. These include multiple class IIb bacteriocins (salivaricin T, salivaricin P, and ABP-118) and the class IId bacteriocin bactofencin A, which show activity against medically important pathogens. However, the production of a bacteriocin in laboratory media does not ensure production under stressful environmental conditions, such as those encountered within the GIT. To allow this issue to be addressed, the promoter regions located upstream of the structural genes encoding the L. salivarius bacteriocins mentioned above were fused to a number of reporter proteins (green fluorescent protein [GFP], red fluorescent protein [RFP], and luciferase [Lux]). Of these, only transcriptional fusions to GFP generated signals of sufficient strength to enable the study of promoter activity in L. salivarius. While analysis of the class IIb bacteriocin promoter regions indicated relatively weak GFP expression, assessment of the promoter of the antistaphylococcal bacteriocin bactofencin A revealed a strong promoter that is most active in the absence of the antimicrobial peptide and is positively induced in the presence of mild environmental stresses, including simulated gastric fluid. Taken together, these data provide information on factors that influence bacteriocin production, which will assist in the development of strategies to optimize in vivo and in vitro production of these antimicrobials.This work was funded by a SFI PI award “Obesibiotics” (11/PI/1137) to PD

Bioengineered Nisin A Derivatives with Enhanced Activity against Both Gram Positive and Gram Negative Pathogens

peer-reviewedNisin is a bacteriocin widely utilized in more than 50 countries as a safe and natural antibacterial food preservative. It is the most extensively studied bacteriocin, having undergone decades of bioengineering with a view to improving function and physicochemical properties. The discovery of novel nisin variants with enhanced activity against clinical and foodborne pathogens has recently been described. We screened a randomized bank of nisin A producers and identified a variant with a serine to glycine change at position 29 (S29G), with enhanced efficacy against S. aureus SA113. Using a site-saturation mutagenesis approach we generated three more derivatives (S29A, S29D and S29E) with enhanced activity against a range of Gram positive drug resistant clinical, veterinary and food pathogens. In addition, a number of the nisin S29 derivatives displayed superior antimicrobial activity to nisin A when assessed against a range of Gram negative food-associated pathogens, including E. coli, Salmonella enterica serovar Typhimurium and Cronobacter sakazakii. This is the first report of derivatives of nisin, or indeed any lantibiotic, with enhanced antimicrobial activity against both Gram positive and Gram negative bacteria.This work was supported by the Irish Government under the National Development Plan, through Science Foundation Ireland Investigator awards (10/IN.1/B3027) and (06/IN.1/B98) (http://www.sfi.ie)

A Bioengineered Nisin Derivative to Control Biofilms of Staphylococcus pseudintermedius

peer-reviewedAntibiotic resistance and the shortage of novel antimicrobials are among the biggest challenges facing society. One of the major factors contributing to resistance is the use of frontline clinical antibiotics in veterinary practice. In order to properly manage dwindling antibiotic resources, we must identify antimicrobials that are specifically targeted to veterinary applications. Nisin is a member of the lantibiotic family of antimicrobial peptides that exhibit potent antibacterial activity against many gram-positive bacteria, including human and animal pathogens such as Staphylococcus, Bacillus, Listeria, and Clostridium. Although not currently used in human medicine, nisin is already employed commercially as an anti-mastitis product in the veterinary field. Recently we have used bioengineering strategies to enhance the activity of nisin against several high profile targets, including multi-drug resistant clinical pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) and also against staphylococci and streptococci associated with bovine mastitis. However, newly emerging pathogens such as methicillin resistant Staphylococcus pseudintermedius (MRSP) pose a significant threat in terms of veterinary health and as a reservoir for antibiotic resistance determinants. In this study we created a nisin derivative with enhanced antimicrobial activity against S. pseudintermedius. In addition, the novel nisin derivative exhibits an enhanced ability to impair biofilm formation and to reduce the density of established biofilms. The activities of this peptide represent a significant improvement over that of the wild-type nisin peptide and merit further investigation with a view to their use to treat S. pseudintermedius infections.This work was supported by the Irish Government under the National Development Plan, through Science Foundation Ireland Investigator awards (10/IN.1/B3027 (http://www.sfi.ie). DF would like to acknowledge receipt of a Society for Applied Microbiology (http://www.sfam.org.uk) Students into Work Grant for FL

Development of Technologies for Separation and Functional Improvement of Individual Milk Protein Fractions

End of Project ReportMilk proteins can be hydrolysed (i.e. fragmented) using proteolytic enzymes to give enhanced functional and nutritional properties. There is an increasing demand for hydrolysed protein ingredients with specific properties for nutrition of individuals with specialised dietary requirements including infants, the critically ill, the immuno-compromised and athletes. Such hydrolysed proteins can be specifically designed to provide distinctive tailor-made solutions to meet customer needs in these areas. This project explored the technologies for the production of two types of hydrolysates i.e. acid-soluble and glutamine-rich. Acid-soluble protein hydrolysates have potential in the fortification of acidic beverages, including soft drinks. Glutamine-rich hydrolysates are suggested as an optimal glutamine source for administration during periods of stress, such as recovery from strenuous exercise, or from surgery. Casein was selected as the protein for development of acid-soluble product and cereal protein for the glutamine-rich product. The main conclusions were as follows: A number of protein hydrolysate products with value added properties and the processes required for their manufacture have been developed and are available for uptake by the food industry. Laboratory investigations identified conditions for the generation of two casein hydrolysates with desirable functional properties. Scale-up conditions for the manufacture of these hydrolysates in the pilot plant were successfully developed. Both hydrolystates were 100% soluble at pH 4.6, exhibited clarity in solution at low pH in clear soft drinks and in caramelised beverages and were stable in solution over a wide temperature range (from 4 to 30ºC) for extended periods. Solutions containing these hydrolysates exhibited no foaming properties and had acceptable sensory properties, being considered as weakly bitter compared to unsupplemented solutions. These performance characteristics make the acid-soluble hydrolysates useful supplements for caramelised beverages, such as colas, and clear soft drinks. Six glutamine-enriched peptide products were produced at laboratory scale using two commercially available enzyme preparations. These products had desirable characteristics such as increased levels of peptide bound glutamine, low free amino acid and free pyroglutamate levels. Pilot plant processes were developed for manufacture of the two glutamine-rich hydrolysates with most suitable compositional properties and these were fully characterised chemically. The manufacturing process was modified to enable industrial scale batches (5,000 litres) to be produced.Department of Agriculture, Food and the Marin