Effect of bacteriocin-induced cell damage on the branched-chain amino acid transamination by Lactococcus lactis

The effect of the bacteriocin lacticin 3147 on the branched-chain amino acid transamination by Lactococcus lactis IFPL359 was investigated. The bacteriocin provokes membrane permeabilisation of the cells, rendering them non-viable but metabolically active. Free diffusion of amino acids into the cell was facilitated. In addition, membrane permeabilisation promotes further cell lysis. Both facts render the enzymes more accessible to their substrates and hence increase branched-chain amino acid transamination. This research broadens the spectrum of technological applications of lacticin 3147 in the development of cheese flavour.     

Evaluation of a spray-dried lacticin 3147 powder for the control of Listeria monocytogenes and Bacillus cereus in a range of food systems

AIMS: The potential of a powdered preparation of the bacteriocin, lacticin 3147, was investigated for the inhibition of Listeria monocytogenes and Bacillus cereus. METHODS AND RESULTS: A 10% solution of reconstituted demineralized whey powder was fermented with Lactococcus lactis DPC3147 for the generation of a lacticin 3147 containing powdered product. A 99.9% reduction in L. monocytogenes numbers occurred in the presence of the lacticin 3147 powder within 2 h in natural yogurt, and an 85% reduction was observed in cottage cheese within the same time frame. Counts of B. cereus were reduced by 80% in soup, in the presence of 1% (w/w) lacticin 3147 powder, within 3 h. CONCLUSIONS: A powdered preparation of lacticin 3147 was effective for the control of Listeria and Bacillus in natural yogurt, cottage cheese and soup. SIGNIFICANCE AND IMPACT OF THE STUDY: The bioactive lacticin 3147 powder may find broad applications for control of Gram-positive pathogens/spoilage bacteria in a range of foods.     

Heterologous expression of lacticin 3147 in Enterococcus faecalis: comparison of biological activity with cytolysin

Lacticin 3147 is a broad-spectrum, two-component, lanthionine-containing bacteriocin produced by Lactococcus lactis DPC3147 which has widespread food and biomedical applications as a natural antimicrobial. Other two-component lantibiotics described to date include cytolysin and staphylococcin C55. Interestingly, cytolysin, produced by Enterococcus faecalis, has an associated haemolytic activity. The objective of this study was to compare the biological activity of lacticin 3147 with cytolysin. The lacticin 3147-encoding determinants were heterologously expressed in Ent. faecalis FA2-2, a plasmid-free strain, to generate Ent. faecalis pOM02, thereby facilitating a direct comparison with Ent. faecalis FA2-2.pAD1, a cytolysin producer. Both heterologously expressed lacticin 3147 and cytolysin exhibited a broad spectrum of activity against bacterial targets. Furthermore, enterococci expressing active lacticin 3147 did not exhibit a haemolytic activity against equine blood cells. The results thus indicate that the lacticin 3147 biosynthetic machinery can be heterologously expressed in an enterococcal background resulting in the production of the bacteriocin with no detectable haemolytic activity.     

Fate of the Two-Component Lantibiotic Lacticin 3147 in the Gastrointestinal Tract

The component peptides of lacticin 3147 were degraded by α-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit.     

Fate of the two-component lantibiotic lacticin 3147 in the gastrointestinal tract

The component peptides of lacticin 3147 were degraded by alpha-chymotrypsin in vitro with a resultant loss of antimicrobial activity. Activity was also lost in ileum digesta. Following oral ingestion, neither of the lacticin 3147 peptides was detected in the gastric, jejunum, or ileum digesta of pigs, and no lacticin 3147 activity was found in the feces. These observations suggest that lacticin 3147 ingestion is unlikely to have adverse effects, since it is probably inactivated during intestinal transit.     

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva

AIMS: To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. METHODS AND RESULTS: Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. CONCLUSION: A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. SIGNIFICANCE AND IMPACT OF THE STUDY: The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.     

Combination of hydrostatic pressure and lacticin 3147 causes increased killing of Staphylococcus and Listeria

The use of hydrostatic pressure and lacticin 3147 treatments were evaluated in milk and whey with a view to combining both treatments for improving the quality of minimally processed dairy foods. The system was evaluated using two foodborne pathogens: Staphylococcus aureus ATCC6538 and Listeria innocua DPC1770. Trials against Staph. aureus ATCC6538 were performed using concentrated lacticin 3147 prepared from culture supernatant. The results demonstrated a more than additive effect when both treatments were used in combination. For example, the combination of 250 MPa (2.2 log reduction) and lacticin 3147 (1 log reduction) resulted in more than 6 logs of kill. Similar results were obtained when a foodgrade powdered form of lacticin 3147 (developed from a spray dried fermentatation of reconstituted demineralized whey powder) was evaluated for the inactivation of L. innocua DPC1770. Furthermore, it was observed that treatment of lacticin 3147 preparations with pressures greater than 400 MPa yielded an increase in bacteriocin activity (equivalent to a doubling of activity). These results indicate that a combination of high pressure and lacticin 3147 may be suitable for improving the quality of minimally processed foods at lower hydrostatic pressure levels.     

Evaluation of Lacticin 3147 and a Teat Seal Containing This Bacteriocin for Inhibition of Mastitis Pathogens

Lacticin 3147 is a broad-spectrum bacteriocin produced by Lactococcus lactis subsp. lactis DPC3147 which is bactericidal against a range of mastitis-causing streptococci and staphylococci. In this study, both lacticin 3147 and the lantibiotic nisin were separately incorporated into an intramammary teat seal product. The seal containing lacticin 3147 exhibited excellent antimicrobial activity and might form the basis of an improved treatment for the prevention of mastitis in dry cows.     

Lacticin 3147 favours isoleucine transamination by Lactococcus lactis IFPL359 in a cheese-model system

The bacteriocin, lacticin 3147, increased isoleucine transamination by Lactococcus lactis IFPL359 in a cheese model system. The formation of -keto--methyl-n-valeric acid and 2-hydroxy-3-methyl-valeric acid increased by three times in cheese slurries at 12 °C and cheese aroma intensity increased as well, which corresponded with a higher 2-methylbutanal formation.     

Comparison of the Potency of the Lipid II Targeting Antimicrobials Nisin, Lacticin 3147 and Vancomycin Against Gram-Positive Bacteria

While nisin (lantibiotic), lacticin 3147 (lantibiotic) and vancomycin (glycopeptides) are among the best studied lipid II-binding antimicrobials, their relative activities have never been compared. Nisin and lacticin 3147 have been employed/investigated primarily as food preservatives, although they do have potential in terms of veterinary and clinical applications. Vancomycin is used exclusively in clinical therapy. We reveal a higher potency for lacticin 3147 (MIC 0.95?C3.8???g/ml) and vancomycin (MIC 0.78?C1.56???g/ml) relative to that of nisin (MIC 6.28?C25.14???g/ml) against the food-borne pathogen Listeria monocytogenes. A comparison of the activity of the three antimicrobials against nisin resistance mutants of L. monocytogenes also reveals that their susceptibility to vancomycin and lacticin 3147 changed only slightly or not at all. A further assessment of relative activity against a selection of Bacillus cereus, Enterococcus and Staphylococcus aureus targets revealed that vancomycin MICs consistently ranged between 0.78 and 1.56???g/ml against all but one strain. Lacticin 3147 was found to be more effective than nisin against B. cereus (lacticin 3147 MIC 1.9?C3.8???g/ml; nisin MIC 4.1?C16.7???g/ml) and E. faecium and E. faecalis targets (lacticin 3147 MIC from 1.9 to 3.8???g/ml; nisin MIC ??8.3???g/ml). The greater effectiveness of lacticin 3147 is even more impressive when expressed as molar values. However, in agreement with the previous reports, nisin was the more effective of the two lantibiotics against S. aureus strains. This study highlights that in many instances the antimicrobial activity of these leading lantibiotics are comparable with that of vancomycin and emphasizes their particular value with respect to use in situations including foods and veterinary medicine, where the use of vancomycin is not permitted.     

Strategy for manipulation of cheese flora using combinations of lacticin 3147-producing and -resistant cultures

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening, with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10(7) CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora in the test cheeses during ripening.     

Strategy for Manipulation of Cheese Flora Using Combinations of Lacticin 3147-Producing and -Resistant Cultures

The aim of the present study was to develop adjunct strains which can grow in the presence of bacteriocin produced by lacticin 3147-producing starters in fermented products such as cheese. A Lactobacillus paracasei subsp. paracasei strain (DPC5336) was isolated from a well-flavored, commercial cheddar cheese and exposed to increasing concentrations (up to 4,100 arbitrary units [AU]/ml) of lantibiotic lacticin 3147. This approach generated a stable, more-resistant variant of the isolate (DPC5337), which was 32 times less sensitive to lacticin 3147 than DPC5336. The performance of DPC5336 was compared to that of DPC5337 as adjunct cultures in two separate trials using either Lactococcus lactis DPC3147 (a natural producer) or L. lactis DPC4275 (a lacticin 3147-producing transconjugant) as the starter. These lacticin 3147-producing starters were previously shown to control adventitious nonstarter lactic acid bacteria in cheddar cheese. Lacticin 3147 was produced and remained stable during ripening, with levels of either 1,280 or 640 AU/g detected after 6 months of ripening. The more-resistant adjunct culture survived and grew in the presence of the bacteriocin in each trial, reaching levels of 10⁷ CFU/g during ripening, in contrast to the sensitive strain, which was present at levels 100- to 1,000-fold lower. Furthermore, randomly amplified polymorphic DNA-PCR was employed to demonstrate that the resistant adjunct strain comprised the dominant microflora in the test cheeses during ripening.     

An application in cheddar cheese manufacture for a strain of Lactococcus lactis producing a novel broad-spectrum bacteriocin, lacticin 3147.

Lactococcus lactis DPC3147, a strain isolated from an Irish kefir grain, produces a bacteriocin with a broad spectrum of inhibition. The bacteriocin produced is heat stable, particularly at a low pH, and inhibits nisin-producing (Nip+) lactococci. On the basis of the observation that the nisin structural gene (nisA) does not hybridize to DPC3147 genomic DNA, the bacteriocin produced was considered novel and designated lacticin 3147. The genetic determinants which encode lacticin 3147 are contained on a 63-kb plasmid, which was conjugally mobilized to a commercial cheese starter, L. lactis subsp. cremoris DPC4268. The resultant transconjugant, DPC4275, both produces and is immune to lacticin 3147. The ability of lacticin 3147-producing lactococci to perform as cheddar cheese starters was subsequently investigated in cheesemaking trials. Bacteriocin-producing starters (which included the transconjugant strain DPC4275) produced acid at rates similar to those of commercial strains. The level of lacticin 3147 produced in cheese remained constant over 6 months of ripening and correlated with a significant reduction in the levels of nonstarter lactic acid bacteria. Such results suggest that these starters provide a means of controlling developing microflora in ripened fermented products.     

Cross-immunity and immune mimicry as mechanisms of resistance to the lantibiotic lacticin 3147

Lantibiotics are antimicrobial peptides that possess great potential as clinical therapeutic agents. These peptides exhibit many beneficial traits and in many cases the emergence of resistance is extremely rare. In contrast, producers of lantibiotics synthesize dedicated immunity proteins to provide self-protection. These proteins have very specific activities and cross-immunity is rare. However, producers of two peptide lantibiotics, such as lacticin 3147, face the unusual challenge of exposure to two active peptides (α and β). Here, in addition to establishing the contribution of LtnI and LtnFE to lacticin 3147 immunity, investigations were carried out to determine if production of a closely related lantibiotic (i.e. staphylococcin C55) or possession of LtnI/LtnFE homologues could provide protection. Here we establish that not only are staphylococcin C55 producers cross-immune to lacticin 3147, and therefore represent a natural repository of Staphylococcus aureus strains that are protected against lacticin 3147, but that functional immunity homologues are also produced by strains of Bacillus licheniformis and Enterococcus faecium . This result raises the spectre of resistance through immune mimicry, i.e. the emergence of lantibiotic-resistant strains from the environment resulting from the possession/acquisition of immunity gene homologues. These phenomena will have to be considered carefully when developing lantibiotics for clinical application.     

Spontaneous resistance in Lactococcus lactis IL1403 to the lantibiotic lacticin 3147

The ability and frequency at which target organisms can develop resistance to bacteriocins is a crucial consideration in designing and implementing bacteriocin-based biocontrol strategies. Lactococcus lactis ssp. lactis IL1403 was used as a target strain in an attempt to determine the frequency at which spontaneously resistant mutants are likely to emerge to the lantibiotic lacticin 3147. Following a single exposure to lacticin 3147, resistant mutants only emerged at a low frequency (10(-8)-10(-9)) and were only able to withstand low levels of the bacteriocin (100 AU mL(-1)). However, exposure to increasing concentrations, in a stepwise manner, resulted in the isolation of eight mutants that were resistant to moderately higher levels of lacticin 3147 (up to 600 AU mL(-1)). Interestingly, in a number of cases cross-resistance to other lantibiotics such as nisin and lacticin 481 was observed, as was cross-resistance to environmental stresses such as salt. Finally, reduced adsorption of the bacteriocin in to the cell was documented for all resistant mutants.     

Saturation mutagenesis of selected residues of the α‐peptide of the lantibiotic lacticin 3147 yields a derivative with enhanced antimicrobial activity

The lantibiotic lacticin 3147 consists of two ribosomally synthesized and post‐translationally modified antimicrobial peptides, Ltnα and Ltnβ, which act synergistically against a wide range of Gram‐positive microorganisms. We performed saturation mutagenesis of specific residues of Ltnα to determine their functional importance. The results establish that Ltnα is more tolerant to change than previously suggested by alanine scanning mutagenesis. One substitution, LtnαH23S, was identified which improved the specific activity of lacticin 3147 against one pathogenic strain, Staphylococcus aureus NCDO1499. This represents the first occasion upon which the activity of a two peptide lantibiotic has been enhanced through bioengineering.     

Manipulation of charged residues within the two‐peptide lantibiotic lacticin 3147

Lantibiotics are antimicrobial peptides which contain a high percentage of post-translationally modified residues. While most attention has been paid to the role of these critical structural features, evidence continues to emerge that charged amino acids also play a key role in these peptides. Here 16 ‘charge’ mutants of the two-peptide lantibiotic lacticin 3147 [composed of Ltnα (2+, 2−) and Ltnβ (2+)] were constructed which, when supplemented with previously generated peptides, results in a total bank of 23 derivatives altered in one or more charged residues. When examined individually, in combination with a wild-type partner or, in some instances, in combination with one another, these mutants reveal the importance of charge at specific locations within Ltnα and Ltnβ, confirm the critical role of the negatively charged glutamate residue in Ltnα and facilitate an investigation of the contribution of positively charged residues to the cationic Ltnβ. From these investigations it is also apparent that the relative importance of the overall charge of lacticin 3147 varies depending on the target bacteria and is most evident when strains with more negatively charged cell envelopes are targeted. These studies also result in, for the first time, the creation of a derivative of a lacticin 3147 peptide (LtnβR27A) which displays enhanced specific activity.     

Developing applications for lactococcal bacteriocins

While much of the applied research carried out to date with bacteriocins has concerned nisin, lactococci produce other bacteriocins with economic potential. An example is the two component bacteriocin lacticin 3147, which is active over a wide pH range and has a broad spectrum of activity against Gram-positive bacteria. Since the genetic determinants for lacticin 3147 are encoded on a large self-transmissible plasmid, the bacteriocin genes may be conveniently transferred to different lactococcal starters. The resulting food-grade strains can then be used to make a significant impact on the safety and quality of a variety of fermented foods, through the inhibition of undesirable microflora. The bacteriocin is heat stable so it can also be used as an ingredient in a powdered form such as a spray-dried fermentate. Given the observation that lacticin 3147 is effective at physiological pH, there is also considerable potential for biomedical applications. Field trials have demonstrat ed its efficacy in the prevention of mastitis infections in dairy cows. In contrast to lacticin 3147, the lactococcin bacteriocins A, B and M have a narrow spectrum of activity limited to lactococci. Strains which produce these inhibitors can be exploited in the acceleration of cheese ripening by assisting the premature lysis of starter cultures.     

Fate and efficacy of lacticin 3147-producing Lactococcus lactis in the mammalian gastrointestinal tract

Gastrointestinal survival of the bacteriocin-producing strain, Lactococcus lactis DPC6520, was evaluated systematically in vitro and in vivo with a view to using this strain to deliver biologically active lacticin 3147, a broad-spectrum bacteriocin, to the gut. The activity of the lacticin 3147 producer was also evaluated against two clinically relevant pathogens: Clostridium difficile and Listeria monocytogenes. When suspended in an appropriate matrix, the lactococcal strain is capable of surviving simulated gastrointestinal juices similar to the porcine probiotic, Lactobacillus salivarius DPC6005. Upon administration of L. lactis DPC6520 to pigs (n=4), excretion rates of ～10(2) -10(5) CFU g(-1) faeces were observed by day 5. Although passage through the gastrointestinal tract (GIT) did not affect lacticin 3147 production by L. lactis DPC6520 isolates, activity was undetectable in faecal samples by an agar well diffusion assay. Furthermore, L. lactis DPC6520 had no inhibitory effect on C. difficile or other bacterial populations in a human distal colon model, while lactococcal counts declined 10,000-fold over 24 h. The lacticin 3147 producer failed to prevent L. monocytogenes infection in a mouse model, even though a mean L. lactis DPC6520 count of 4.7 × 10(4) CFU g(-1) faeces was obtained over the 5-day administration period. These data demonstrate that L. lactis DPC6520 is capable of surviving transit through the GIT, and yet lacks antimicrobial efficacy in the models of infection used.