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Antimicrobial Agents and Chemotherapy, July 2005, p. 2606-2611, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2606-2611.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Sequential Actions of the Two Component Peptides of the Lantibiotic Lacticin 3147 Explain Its Antimicrobial Activity at Nanomolar Concentrations

Sheila M. Morgan,^1,2 Paula M. O'Connor,^1,2 Paul D. Cotter,³ R. Paul Ross,^1,2* and Colin Hill^2,3

Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland,¹ Microbiology Department, University College, Cork, Co. Cork, Ireland,³ Alimentary Pharmabiotic Centre, Cork, Ireland²

Received 25 August 2004/ Returned for modification 24 November 2004/ Accepted 28 February 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lacticin 3147 is a two-peptide (LtnA1 and LtnA2) lantibiotic produced by Lactococcus lactis subsp. lactis DPC3147 and has inhibitory activity against all gram-positive microorganisms tested. In this study the specific activities of the component peptides (alone or in combination) were determined by using L. lactis subsp. cremoris HP as the target strain. Lacticin 3147 exhibited an MIC₅₀ of 7 nM for each component peptide (in combination), suggesting a peptide stoichiometry of 1:1. Interestingly, the LtnA1 peptide demonstrated independent inhibitory activity, with an MIC₅₀ of 200 nM against L. lactis HP. In parallel studies, the single peptide bacteriocin nisin exhibited an MIC₅₀ of 50 nM against the same target strain. Sequential peptide addition (with an intermediate washing step) demonstrated that LtnA1 must be added before LtnA2 rather than vice versa to observe inhibitory activity. The nanomolar activity of the lacticin peptides suggests the involvement of a docking molecule, speculated to be lipid II. Taken together with the recently determined structure of lacticin 3147 (N. I. Martin, T. Sprules, M. R. Carpenter, P. D. Cotter, C. Hill, R. P. Ross, and J. C. Vederas, Biochemistry, 43:3049-3056, 2004), these data support the hypothesis that the mode of action for lacticin 3147 involves a lipid II binding step (by the mersacidin-like LtnA1 peptide, which would explain its independent inhibitory activity), followed by insertion of the more linear LtnA2 peptide into the target membrane, resulting in pore formation and ultimate cell death.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antimicrobial peptides (AMPs) are produced by genera from all domains and play a major role in host defense mechanisms. Bacteria-derived AMPs produced by both gram-positive and gram-negative species are under investigation as potential alternatives to conventional antibiotics for the treatment of a range of infections caused by human pathogens (9). Of these, the class I bacteriocins (lantibiotics) demonstrate the most potential, since some, such as nisin, display both high specificity and a broad spectrum of inhibition. Lacticin 3147 is an AMP produced by Lactococcus lactis DPC3147 which exhibits inhibitory activity against methicillin-resistant Staphylococcus aureus; vancomycin-resistant Enterococcus faecalis; penicillin-resistant Pneumococcus; Propionibacterium acnes; Streptococcus mutans; and the food-borne pathogens Listeria monocytogenes, Staphylococcus aureus, and Bacillus cereus (10, 27). Lacticin 3147 has been classified as a lantibiotic, a term used to describe a heterogeneous group of lanthionine-containing bacterial AMPs that undergo extensive posttranslational modifications. The lantibiotics are traditionally divided into two groups based on structural characteristics (15). Type A lantibiotics (e.g., Pep5 and nisin) have an elongated linear structure and exert their inhibitory effects via pore formation in the membranes of target species, while type B lantibiotics (e.g., mersacidin and actagardine) are globular in structure and usually inhibit essential steps in peptidoglycan synthesis. The most well characterized lantibiotic is nisin, which has a broad target range and which kills a wide spectrum of gram-positive bacteria, including food-borne pathogens such as Listeria monocytogenes and spoilage bacteria such as Clostridium species. In 1988, nisin was approved by the U.S. Food and Drug Administration for use in food (30), is used in veterinary products for the control of bovine mastitis, and is reported to have successfully undergone phase I clinical trials as an oral treatment for Helicobacter pylori infection (12).

Although nisin has been structurally classified as an elongated type A lantibiotic, it is capable of both pore formation and the inhibition of cell wall biosynthesis, previously associated only with type B lantibiotics, such as mersacidin and actagardine (4, 5, 22). The combination of these dual killing activities in one molecule and the involvement of the docking molecule lipid II (3) results in activity at nanomolar concentrations (31). In contrast, eukaryotic pore-forming AMPs, such as megainin, need to be present at micromolar concentrations to achieve similar levels of killing (3).

In contrast to nisin, lacticin 3147 belongs to an emerging group of two-component lantibiotics which include staphylococcin C55 and cytolysin. Lacticin 3147 is produced by a Lactococcus lactis strain and is similar to nisin in terms of its biological activity, in that it has a broad spectrum of activity against gram-positive organisms and potential widespread applications in both food and biomedicine (10, 17, 21, 23, 24, 28). Indeed, lacticin 3147 has been demonstrated to be inhibitory against clinically significant human pathogens (10). In addition, lacticin 3147 is effective at killing all mastitic staphylococcal and streptococcal isolates tested (26, 29). However, unlike the single peptide nisin, lacticin 3147 is composed of two lanthionine-containing peptides, LtnA1 and LtnA2, with masses of 3,306 Da and 2,847 Da, respectively. These masses represent the true peptide masses, which were reported to be greater in a previous publication (25), due most likely to peptide oxidation. In this study we report on the specific activity of the two-component lantibiotic lacticin 3147 and compare it to that of nisin. In addition, we confirm that the peptides act sequentially at a 1:1 ratio and provide evidence to support a model for the activity of lacticin 3147 which, although similar to nisin, differs from that of nisin, in that each of the two associated activities (cell wall disruption and pore-forming ability) is assigned to a separate peptide.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, culture conditions, and bacteriocin assays. The strain L. lactis subsp. cremoris HP was subcultured in M17 (Difco, Detroit, Mich.) supplemented with 0.5% lactose (LM17) or 0.5% glucose (GM17). Lactobacillus plantarum LMG6907 and Listeria monocytogenes LO28H were subcultured in MRS broth (Oxoid, Basingstoke, England) and GM17, respectively. All strains were stocked at –20°C in 40% glycerol. Bacteriocin activity was assessed in agar plates containing L. lactis subsp. cremoris HP, as outlined by Ryan et al. (26). L. lactis DPC3147 was grown in modified tryptone-yeast (TY) broth (25) for the purpose of peptide purification. All strains are stocked in the Dairy Products Research Centre Culture Collection, Moorepark, Fermoy, County Cork, Ireland.

Purification of the LtnA1 and LtnA2 peptides. The lacticin 3147 peptides were purified as outlined by Ryan et al. (25), with some modifications. An overnight culture of the peptide-producing strain, L. lactis DPC3147, was inoculated into 8 liters of modified TY broth and incubated overnight at 30°C. The cells were removed by centrifugation at 8,000 x g for 15 min, and the supernatant was applied to a column (3 by 23 cm) containing a 60-g bed of XAD-16 resin (Sigma-Aldrich) at a flow rate of 900 ml/h. The column was then washed with 2 liters of 30% ethanol. Inhibitory activity was subsequently eluted with 1 liter of 70% propan-2-ol. The propan-2-ol was removed by rotary evaporation, and the resultant preparation was applied to a 10-g (60-ml volume) Varian C₁₈ Bond Elut column (Varian, Harbor City, Calif.) preequilibrated with methanol and water. The column was washed with 120 ml of 30% ethanol, followed by 60 ml of 40% propan-2-ol, and the component with inhibitory activity was eluted with 100 ml of 70% propan-2-ol (pH 2, by the addition of 1 M HCl). The 100 ml of bacteriocin-containing eluate was concentrated to a volume of 4 to 5 ml through the removal of propan-2-ol by rotary evaporation. Aliquots of 1 ml were then applied to a GROM (GROM, Herrenberg-Kayh, Germany) C₁₈ reverse-phase (RP) high-pressure liquid chromatography (HPLC) column (Nucleosil 100; 250 by 8.0 mm; 5 µm) previously equilibrated with 0.1% aqueous trifluoroacetic acid (TFA). The column was developed in a gradient of 30% propan-2-ol containing 0.1% TFA to 60% propan-2-ol containing 0.1% TFA at a flow rate of 1 ml/min. Fractions were collected and assayed for activity against L. lactis subsp. cremoris HP by the agar well diffusion assay, as described by Ryan et al. (25). Molten agar seeded with the indicator strain, L. lactis subsp. cremoris HP, was dispensed into petri dishes. Wells of 4.6 mm in diameter were bored in the agar, and 10-µl volumes of the putative LtnA1 peptide together with LtnA2 were dispensed into each well, and vice versa. The plates were incubated at 30°C overnight, and the presence of a zone of clearance indicated the presence of active peptides. The two fractions representing the lacticin 3147 peptides LtnA1 and LtnA2, fractions A1 and A2, respectively, were concentrated separately by rotary evaporation, and each fraction was then reapplied to the C₁₈ RP HPLC column. A gradient of 40 to 46% propan-2-ol (0.1% TFA) over 30 min was applied to further fractionate fraction A1, while fraction A2 was developed in a gradient of 44 to 50% propan-2-ol (0.1% TFA) over 30 min. Approximately 2.0 to 4.0 mg of each peptide was recovered from 8 liters of culture. Mass spectrometry and determination of the peptide concentration (by amino acid analysis) were performed in the Department of Molecular and Cell Biology, University of Aberdeen, Aberdeen, Scotland. Mass spectrometry was performed on a matrix-assisted laser desorption ionization-time of flight mass spectrometer (Applied Biosystems DE STR). A 0.5-µl aliquot of sample for analysis was spotted onto a stainless steel matrix-assisted laser desorption ionization target, followed by the addition of a 0.5-µl aliquot of matrix solution (-cyano 4-hydroxy cinnamic acid, 10 mg/ml in acetonitrile-0.1% [vol/vol] trifluoroacetic acid) prior to analysis in positive ion reflectron mode. A pure preparation of nisin was gratefully received from Michiel Kleerebezem of the Wageningen Centre for Food Science, NiZO Food Research, Ede, The Netherlands. This was solubilized in 0.05% acetic acid at a concentration of 1 mg/ml, aliquoted, and stored as stock solutions at –20°C.

Specific activity determination. Ninety-six well microtiter plates were used to determine the MIC₅₀ (the concentration at which 50% growth inhibition can be observed) of L. lactis subsp. cremoris HP by using a combination of purified preparations of LtnA1 and LtnA2. Triplicate wells containing three separate L. lactis subsp. cremoris HP cultures were included in each microtiter plate. The total volume in each well was 200 µl; and the experimental wells comprised broth (media), purified LtnA1, purified LtnA2, and 150 µl of a 1-in-10 dilution (approximately 1 x 10⁸/ml) of overnight L. lactis subsp. cremoris HP cultures (diluted in growth medium, LM17). The plates contained, in addition, a number of blank wells (medium only) and a number of control wells (untreated L. lactis subsp. cremoris HP or L. lactis subsp. cremoris HP treated with LtnA1 alone or LtnA2 alone). The optical density at 620 nm (OD₆₂₀) was recorded (Ceres UV900 Hdi; Biotek Instruments Inc.) at 0 h and 5 h. The plates were incubated at 30°C. Triplicate readings were averaged, and blanks (medium only) were subtracted from these readings. A 50% growth inhibition was determined as half the final OD₆₂₀ ± 0.05. The concentrations of LtnA1 in combination with LtnA2 which caused 50% growth inhibition of L. lactis subsp. cremoris HP were plotted to generate an isobologram. The point of x- and y-axis intersection on the isobologram can be used to determine both the optimal peptide ratio and the specific activities of the peptides under investigation.

Comparison of the specific activities of nisin and lacticin 3147. Ninety-six well microtiter plates were used to assess the effectiveness of lacticin 3147 and nisin for the inhibition of L. plantarum LMG6907 and L. monocytogenes LO28H. Plates were prepared as outlined above, with the total volume in each well being 200 µl. The L. plantarum plates were prepared with MRS broth, and the L. monocytogenes plates were prepared with GM17. Both sets of plates were incubated at 37°C, and OD₆₂₀ readings were taken every hour for up to 6 h.

Potassium ion release. Potassium ion release was determined as outlined by McAuliffe et al. (18). L. lactis subsp. cremoris HP was grown overnight in GM17. The cells were washed and resuspended in 2.5 mM sodium HEPES buffer supplemented with 10 mM glucose. A 27.5 nM quantity of LtnA1 and/or LtnA2 peptide was added to the cell suspension. A cell suspension to which no peptide was added was used as a control. At various intervals 1.5-ml volumes were removed from the suspensions and centrifuged at 10,000 x g for 7 min. The supernatant was removed and stored for the determination of the extracellular K⁺ concentration. The cell pellet was resuspended in 300 µl of 5% trichloroacetic acid and frozen overnight at –20^°C. Samples were thawed and incubated at 95^°C for 10 min. Demineralized water (1,200 µl) was added to each sample, which was then centrifuged at 10,000 x g for 15 min. The supernatant was retained for intracellular K⁺ concentration determination. The K⁺ concentration in the samples was determined by flame photometry (Jenway PFP 7).

Sequential LtnA1 and LtnA2 treatment of L. lactis subsp. cremoris HP and vice versa. The L. lactis subsp. cremoris HP cultures (in triplicate) were diluted 1 in 10, and 150 µl of each culture was added to 500-µl Eppendorf tubes which contained LtnA1 or LtnA2 alone (at concentrations of 0 nM, 5 nM, 9 nM, 15 nM, and 19 nM). The tubes were left at room temperature for approximately 20 min (to enable binding of the peptide molecules to cell surfaces) prior to centrifugation at 13,000 x g for 30 s. The supernatants were removed from each tube, and L. lactis subsp. cremoris HP cell pellets were washed twice with LM17 broth. The cell pellets were resuspended in 150 µl of fresh LM17 broth. Cells that had been exposed to LtnA1 alone were added to microtiter wells which contained LtnA2, and cells that had been exposed to LtnA2 alone were added to microtiter wells which contained LtnA1 (at concentrations of 0 nM, 5 nM, 9 nM, 15 nM, and 19 nM). The microtiter plates were incubated at 30°C and read at hourly intervals for 5 h, with the first reading representing time zero. Control procedures included exposure of the cells to LtnA1 and LtnA2 in combination, in addition to exposure to nisin.

Top Abstract Introduction Materials and Methods Results Discussion References

 
One of the most striking differences between nisin and lacticin 3147 is that the latter requires two peptides for optimal activity. As a consequence, prior to comparison of the specific activity of both lantibiotics, it was necessary to determine the optimum ratio of the individual lacticin 3147 peptides.

Lacticin 3147 is active at nanomolar concentrations in a 1:1 ratio. The isobologram (Fig. 1) indicates that the LtnA1 to the LtnA2 peptides are active at a 1:1 ratio (since the lowest concentration that causes 50% growth inhibition is equal for each peptide). The isobologram also indicates that the lacticin peptides are active in the single-nanomolar concentration range (7 nM LtnA1:7 nM LtnA2). It is noteworthy that while we can conclude that lacticin 3147 displays activity in the single-nanomolar range and in equimolar amounts, the precise value of 7 nM is assay dependent, in that the concentration of peptides required to cause 50% growth inhibition reflects the arbitrarily set initial cell numbers present in the growth medium and, of course, the target strain chosen. Interestingly, in the absence of LtnA2, the LtnA1 peptide displays inhibitory activity against L. lactis subsp. cremoris HP at concentrations in excess of 200 nM (Fig. 1).

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FIG. 1. Concentrations of LtnA1 and LtnA2 required to inhibit the growth of the indicator strain. L. lactis subsp. cremoris HP, by 50%.

Comparison of lacticin 3147 and nisin activities. Nisin and lacticin 3147 have similar spectra of activity, in that both are effective against a wide range of gram-positive microorganisms. The concentrations of nisin and lacticin 3147 required to inhibit a range of strains, including L. monocytogenes LO28H, L. plantarum MG6907, and L. lactis subsp. cremoris HP, revealed that the relative effectiveness of the peptides is also target strain related (Fig. 2). Concentrations of lacticin 3147 between 400 nM and 600 nM were required for an MIC₅₀ for L. monocytogenes, compared to a requirement for MIC₅₀s in excess of 800 nM of nisin against the same strain. However, in the case of L. plantarum MG6907 the situation was reversed, in that higher concentrations of lacticin 3147 (greater than 500 nM) than nisin (200 nM) were required. The lactococcal strain L. lactis subsp. cremoris HP is sensitive to both lantibiotics; however, it was more sensitive to lacticin 3147 (MIC₅₀, 7 nM) than nisin (MIC₅₀, 50 nM).

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FIG. 2. Effects of lacticin 3147 (a) and nisin (b) against Listeria monocytogenes LO28H at concentrations of 0 nM () 200 nM (), 400 nM (), 600 nM (•), and 800 nM (*); and effects of lacticin 3147 (c) and nisin (d) against Lactobacillus plantarum LMG6907 at concentrations of 0 nM (), 200 nM (), 300 nM (), 400 nM (), and 500 nM (+). Error bars represent standard deviations for triplicate data sets.

Lacticin 3147 peptides cause potassium ion release. We have previously observed an efflux of K⁺ ions from sensitive cells upon exposure to crude preparations of lacticin 3147 (18). Thus, the effect of treating L. lactis subsp. cremoris HP cells with individual LtnA1 and LtnA2 peptides at the cellular level was investigated by monitoring potassium ion release (Fig. 3). In the absence of the LtnA1 and LtnA2 peptides, cells of L. lactis subsp. cremoris HP maintained intracellular potassium concentrations of between 9.2 and 8.4 ppm during the 30-min experiment. Similar potassium ion levels were observed when the cells were exposed to the LtnA1 peptide. In the presence of the LtnA2 peptide, the intracellular potassium ion level declined slightly, to a level of 7.9 ppm during the same time period. However, when cells were treated with LtnA1 and LtnA2 in combination, intracellular potassium ion levels declined to 6.7 ppm. Determination of the extracellular potassium ion concentration indicated that the highest levels were found in the supernatants from cells treated with both LtnA1 and LtnA2 in combination (3 ppm, compared to levels less than 0.5 ppm when the cells were untreated or treated with either LtnA1 or LtnA2). These results demonstrate that both peptides (in combination) at low concentrations are required for the creation of K⁺-specific pores.

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FIG. 3. Intracellular (a) and extracellular (b) potassium ion release from L. lactis subsp. cremoris HP cells in the presence of LtnA1 (), LtnA2 (), or LtnA1 plus LtnA2 (•) and in the absence of these peptides (). Error bars represent standard deviations for triplicate data sets.

Lacticin 3147 peptides act sequentially to kill target cells. When LtnA1 and LtnA2 were added in succession to L. lactis subsp. cremoris HP cells, it became apparent that the addition of LtnA1 prior to the addition of LtnA2 was necessary to observe growth inhibition (Fig. 4). The OD₆₂₀ of the L. lactis subsp. cremoris HP cultures initially exposed to LtnA1 and then to LtnA2 demonstrated growth inhibition (Fig. 4a). In the situation in which LtnA2 was the first peptide added to the L. lactis subsp. cremoris HP cells, growth inhibition was not observed (Fig. 4b). When both LtnA1 and LtnA2 were added in combination, growth inhibition occurred (Fig. 4c).

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FIG. 4. Effects of the sequential addition of the LtnA1 and LtnA2 peptides; (a) LtnA1 followed by LtnA2 addition; (b) LtnA2 followed by LtnA1 addition, and (c) LtnA1 and LtnA2 simultaneously. Concentrations of 0 nM (), 5 nM (), 9 nM (•), 15 nM (), and 19 nM (*) were used. Error bars represent standard deviations on triplicate data sets.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this report, we demonstrate that the lacticin 3147 peptides work at a 1:1 ratio and are effective at nanomolar concentrations. The extent of inhibition appears to be target strain dependent and would suggest that lacticin 3147 and nisin do not share an identical mode of action, despite their similar inhibitory spectra.

The 1:1 ratio reported here is similar to the results obtained with a number of other lantibiotic and nonlantibiotic two-component peptide systems (1, 11, 13, 14, 19, 20). We were able to confirm the findings from a previous report (25) that suggested that LtnA1 exhibits independent inhibitory activity with an MIC₅₀ of 200 nM. However when LtnA1 was combined with its companion peptide, LtnA2, there was almost a 30-fold decrease in the amount of LtnA1 required (7 nM) to achieve similar levels of inhibition. Activity at nanomolar concentrations suggests the involvement of a docking molecule, as is the case with nisin, where lipid II was identified as the nisin docking molecule (3). Lipid II has been shown to be not only a docking molecule for nisin but also an intrinsic component of the pore formed by nisin (2), and interaction with lipid II also results in the inhibition of peptidoglycan biosynthesis. In a recent report by Garneau et al. (11), lipid II was mooted as a potential docking molecule for LtnA1. The involvement of a docking molecule in microbial systems enables microbial AMPs to exert their effects at nanomolar concentrations, in comparison to the micromolar concentrations necessary for inhibition with eukaryotic AMPs.

Lipid II has been identified as the target molecule for a number of antimicrobial peptides, including the globular peptide mersacidin. Sequence analysis revealed significant similarities between LtnA1, mersacidin, actagardine, and Plw, one of the components from the two-component lantibiotic plantaricin W (13, 28). The recent structural determination of the LtnA1 peptide (16) has demonstrated that LtnA1 has a specific lanthionine-bridging pattern that resembles that of the globular, lipid II binding lantibiotic mersacidin. The similarities are particularly striking in the three C-terminus rings. Both peptides share residues proposed to be involved in the inhibition of cell wall biosynthesis at the level of transglycosylation by binding to lipid II (4, 5, 6, 7, 8). However, mersacidin is a shorter peptide and lacks a number of residues (which, in the longer peptide LtnA1, may have involvement in LtnA1-LtnA2 interactions). Given the similarities between peptide structures, it may be expected that the activities of LtnA1 alone and mersacidin might be similar. Furthermore, it is interesting to speculate that the conserved CTLT-EC motif of the LtnA1 peptide, mersacidin, and actagardine could represent the site responsible for their modes of action, as suggested for the actagardine and mersacidin peptides by Zimmerman and Jung (32).

At low nanomolar concentrations, LtnA1 alone does not elicit K⁺ efflux, suggesting that pore formation is not a primary mode of action. The independent inhibitory activity of LtnA1 may be explained through lipid II-mediated inhibition of peptidoglycan biosynthesis. The results presented in this report demonstrate that LtnA1 alone has activity (at micromolar concentrations), which is enhanced greatly when it is combined with the cognate peptide LtnA2 (with activity being detected in the single-nanomolar range when both peptides are present). Indeed, the globular structure of LtnA1 indicates that this peptide would not be capable of spanning a cell membrane for pore formation purposes. The LtnA2 peptide, however, has a more elongated, linear structure (16) potentially capable of pore formation. Potassium ion release studies demonstrated that LtnA1 and LtnA2 alone are not capable of inducing pore formation in Lactococcus lactis subsp. cremoris HP target cells. However, when both peptides are combined, there is an efflux of potassium ions from the intracellular to the extracellular environment. The studies performed demonstrated that it is necessary for the LtnA1 peptide to be present before to the LtnA2 molecule is present in order to induce cell death. When both peptides are added together, growth inhibition occurred at rates similar to those observed when LtnA1 addition was followed by LtnA2 addition. Thus, the peptides appear to operate in a sequential fashion, requiring that LtnA1 be cell bound before pore-forming LtnA2 is present to induce cell death.

The results presented here support a dual mechanism for lacticin peptides, where LtnA1 binds to lipid II (similarly to mersacidin lipid II interaction), thereby inhibiting cell wall biosynthesis through the prevention of transglycosylation. This is the initial step in the process which ultimately leads to pore formation and K⁺ release when the allied LtnA2 peptide is present. This mechanism can be contrasted with that of nisin, where both inhibition of cell wall synthesis and pore formation are linked to the one peptide, whereas in the case of lacticin, both functions are assigned to separate peptides. The ability of peptides to develop bifunctionality may be significant in relation to the enhancement of antimicrobial activity and may lead to the generation of alternative, highly active AMP molecules for the treatment and prevention of infections caused by clinically significant gram-positive pathogens.

 
We thank Máire Ryan for initiation of this research and Elaine Lawton, Dan Walsh, and John O'Callaghan for technical assistance during the flame photometry studies.

This research was funded by the Irish Government under the FIRM Programme as part of the National Development Plan, 2000-2006.

 
* Corresponding author. Mailing address: Teagasc, Dairy Products Research Centre, Moorepark, Fermoy, Co. Cork, Ireland. Phone: 353 25 42229. Fax: 353 25 42340. E-mail: pross{at}moorepark.teagasc.ie.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, July 2005, p. 2606-2611, Vol. 49, No. 7
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.7.2606-2611.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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