Sensitivity variations of Listeria strains to the bacteriocins, lactocin 705, enterocin CRL35 and nisin

Five strains of Listeria monocytogenes, four strains of Listeria innocua and a strain of Listeria seeligeri showed different sensitivities to lactocin 705 (17 000 AU ml^–1), enterocin CRL35 (8500 AU ml^–1) and nisin (2500 IU ml^–1) at different pHs (5, 6 and 7). The susceptibility of Listeria strains to bacteriocins at each pH was strain dependent, and it was enhanced at the low pH. L. monocytogenes had enhanced nisin tolerance while the non-nisin bacteriocins were more inhibitory with viability losses of 3–3.4 in contrast with 1.5–1.8 log cycles, respectively. Lower viability loss values were obtained with L. innocua strains with all three bacteriocins while L. seeligeri was more sensitive to nisin than to lactocin 705 or enterocin CRL35.     

The Bactericidal Activity of Pediocin PA-1 Is Specifically Inhibited by a 15-mer Fragment That Spans the Bacteriocin from the Center toward the C Terminus

A 15-mer peptide fragment derived from pediocin PA-1 (from residue 20 to residue 34) specifically inhibited the bactericidal activity of pediocin PA-1. The fragment did not inhibit the pediocin-like bacteriocins sakacin P, leucocin A, and curvacin A to nearly the same extent as it inhibited pediocin PA-1. Enterocin A, however, was also significantly inhibited by this fragment, although not as greatly as pediocin PA-1. This is consistent with the fact that enterocin A contains the longest continuous sequence identical to that of pediocin PA-1 in the region spanned by the fragment. The fragment inhibited pediocin PA-1 to a much greater extent than did the other 29 possible 15-mer fragments that span pediocin PA-1. The results suggest that the fragment—by interacting with the target cells and/or pediocin PA-1—interferes specifically with pediocin-target cell interaction.     

Novel method to extract large amounts of bacteriocins from lactic acid bacteria.

Antimicrobial peptides, bacteriocins, produced by lactic acid bacteria were adsorbed on the cells of producing strains and other gram-positive bacteria. pH was a crucial factor in determining the degree of adsorption of these peptides onto cell surfaces. In general, between 93 and 100% of the bacteriocin molecules were adsorbed at pHs near 6.0, and the lowest (< or = 5%) adsorption took place at pH 1.5 to 2.0. On the basis of this property, a novel isolation method was developed for bacteriocins from four genera of lactic acid bacteria. By using this method we made preparations of pediocin AcH, nisin, sakacin A, and leuconocin Lcm1 that were potent and concentrated. This method produced a higher yield than isolation procedures, which rely on precipitation of the bacteriocins from the cell-free culture liquor. It is simple and can be used to produce large quantities of bacteriocins from lactic acid bacteria to be used as food biopreservatives.     

Mechanism of Nisin, Pediocin 34, and Enterocin FH99 Resistance in Listeria monocytogenes

Nisin-, pediocin 34-, and enterocin FH99-resistant variants of Listeria monocytogenes ATCC 53135 were developed. In an attempt to clarify the possible mechanisms underlying bacteriocin resistance in L. monocytogenes ATCC 53135, sensitivity of the resistant strains of L. monocytogenes ATCC 53135 to nisin, pediocin 34, and enterocin FH99 in the absence and presence of different divalent cations was assessed, and the results showed that the addition of divalent cations significantly reduced the inhibitory activity of nisin, pediocin 34, and enterocin FH99 against resistant variants of L. monocytogenes ATCC 53135. The addition of EDTA, however, restored this activity suggesting that the divalent cations seem to affect the initial electrostatic interaction between the positively charged bacteriocin and the negatively charged phospholipids of the membrane. Nisin-, pediocin 34-, and enterocin-resistant variants of L. monocytogenes ATCC 53135 were more resistant to lysozyme as compared to the wild-type strain both in the presence as well as absence of nisin, pediocin 34, and enterocin FH99. Ultra structural profiles of bacteriocin-sensitive L. monocytogenes and its bacteriocin-resistant counterparts revealed that the cells of wild-type strain of L. monocytogenes were maximally in pairs or short chains, whereas, its nisin-, pediocin 34-, and enterocin FH99-resistant variants tend to form aggregates. Results indicated that without a cell wall, the acquired nisin, pediocin 34, and enterocin FH99 resistance of the variants was lost. Although the bacteriocin-resistant variants appeared to lose their acquired resistance toward nisin, pediocin 34, and enterocin FH99, the protoplasts of the resistant variants appeared to be more resistant to bacteriocins than the protoplasts of their wild-type counterparts.     

A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate.

Bacteriocins, including nisin, pediocin PO2, brevicin 286, and piscicolin 126, were extracted from fermentation media by adsorption onto Micro-Cel (a food-grade diatomite calcium silicate anticaking agent) and subsequent desorption. The optimal conditions for desorption of piscicolin 126 were determined and applied to other bacteriocins, and the relative purities of the desorbed preparations were compared. Piscicolin was not successfully desorbed from Micro-Cel at pH 1.0 to 12.0, with organic solvents, or by increase of ionic strength up to 1 M NaCl. However, 25 and 75% of the bacteriocin activity was desorbed by using 1% sodium deoxycholate and 1% sodium dodecyl sulfate (SDS), respectively. Higher levels (up to 100%) of desorption were achieved by repeated elution or by an increase in surfactant concentration. Desorption of piscicolin with 1/10 volume of SDS solution resulted in a preparation with 10 times concentration in activity, equivalent to that of ammonium sulfate preparations (409,600 to 819,200 activity units/ml). Determination of organic nitrogen (N) content revealed that the desorbed piscicolin preparations were substantially free of proteinaceous substances (approximately 92 to 99%) compared with original culture supernatants and ammonium sulfate preparations. Nisin, pediocin, and brevicin were also desorbed with 1% SDS with a similar level of purification.     

Improved Antimicrobial Activities of Synthetic-Hybrid Bacteriocins Designed from Enterocin E50-52 and Pediocin PA-1

Two hybrid bacteriocins, enterocin E50-52/pediocin PA-1 (EP) and pediocin PA-1/enterocin E50-52 (PE), were designed by combining the N terminus of enterocin E50-52 and the C terminus of pediocin PA-1 and by combining the C terminus of pediocin PA-1 and the N terminus of enterocin E50-52, respectively. Both hybrid bacteriocins showed reduced MICs compared to those of their natural counterparts. The MICs of hybrid PE and EP were 64- and 32-fold lower, respectively, than the MIC of pediocin PA-1 and 8- and 4-fold lower, respectively, than the MIC of enterocin E50-52. In this study, the effect of hybrid as well as wild-type (WT) bacteriocins on the transmembrane electrical potential (ΔΨ) and their ability to induce the efflux of intracellular ATP were investigated. Enterocin E50-52, pediocin PA-1, and hybrid bacteriocin PE were able to dissipate ΔΨ, but EP was unable to deplete this component. Both hybrid bacteriocins caused a loss of the intracellular concentration of ATP. EP, however, caused a faster efflux than PE and enterocin E50-52. Enterocin E50-52 and hybrids PE and EP were active against the Gram-positive and Gram-negative bacteria tested, such as Micrococcus luteus, Salmonella enterica serovar Enteritidis 20E1090, and Escherichia coli O157:H7. The hybrid bacteriocins designed and described herein are antimicrobial peptides with MICs lower those of their natural counterparts. Both hybrid peptides induce the loss of intracellular ATP and are capable of inhibiting Gram-negative bacteria, and PE dissipates the electrical potential. In this study, the MIC of hybrid bacteriocin PE decreased 64-fold compared to the MIC of its natural peptide counterpart, pediocin PA-1. Inhibition of Gram-negative pathogens confers an additional advantage for the application of these peptides in therapeutics.     

Novel method to extract large amounts of bacteriocins from lactic acid bacteria.

Antimicrobial peptides, bacteriocins, produced by lactic acid bacteria were adsorbed on the cells of producing strains and other gram-positive bacteria. pH was a crucial factor in determining the degree of adsorption of these peptides onto cell surfaces. In general, between 93 and 100% of the bacteriocin molecules were adsorbed at pHs near 6.0, and the lowest (< or = 5%) adsorption took place at pH 1.5 to 2.0. On the basis of this property, a novel isolation method was developed for bacteriocins from four genera of lactic acid bacteria. By using this method we made preparations of pediocin AcH, nisin, sakacin A, and leuconocin Lcm1 that were potent and concentrated. This method produced a higher yield than isolation procedures, which rely on precipitation of the bacteriocins from the cell-free culture liquor. It is simple and can be used to produce large quantities of bacteriocins from lactic acid bacteria to be used as food biopreservatives.     

Two Subpopulations of Listeria monocytogenes Occur at Subinhibitory Concentrations of Leucocin 4010 and Nisin

In situ analyses of single Listeria monocytogenes cells at subinhibitory concentrations of leucocin 4010 and nisin revealed two subpopulations when measured by fluorescence ratio imaging microscopy (FRIM) after staining with 5(6)-carboxyfluorescein diacetate succinimidyl ester. One subpopulation consisted of cells with a dissipated pH gradient (ΔpH), and the other consisted of cells that maintained ΔpH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate ΔpH for 90% of the cell population (ED₉₀) was predicted. ED₉₀ increased after the addition of sodium chloride (1 to 3% [wt/vol]) to the bacteriocin solutions, while ED₉₀ decreased by the addition of sodium nitrite (60 and 100 ppm). Other meat additives, including sodium phosphate, sodium lactate, sodium citrate, and sodium acetate slightly increased ED₉₀. The inhibitory effect of sodium chloride on the antilisterial activity of leucocin 4010 and nisin was confirmed on the surfaces of meat sausages. This study highlights the important practical implications of applying subinhibitory concentrations of bacteriocins, which results in unaffected target cells. In situ analyses by FRIM in combination with modeling of single-cell data can be applied to ensure that sufficient concentrations of bacteriocins are used in food preservation.     

Bacteriocins from lactic acid bacteria and their potential in the preservation of fruit products

Bacteriocins produced by lactic acid bacteria (LAB) are well-recognized for their potential as natural food preservatives. These antimicrobial peptides usually do not change the sensorial properties of food products and can be used in combination with traditional preservation methods to ensure microbial stability. In recent years, fruit products are increasingly being associated with food-borne pathogens and spoilage microorganisms, and bacteriocins are important candidates to preserve these products. Bacteriocins have been extensively studied to preserve foods of animal origin. However, little information is available for their use in vegetable products, especially in minimally processed ready-to-eat fruits. Although, many bacteriocins possess useful characteristics that can be used to preserve fruit products, to date, only nisin, enterocin AS-48, bovicin HC5, enterocin 416K1, pediocin and bificin C6165 have been tested for their activity against spoilage and pathogenic microorganisms in these products. Among these, only nisin and pediocin are approved to be commercially used as food additives, and their use in fruit products is still limited to certain countries. Considering the increasing demand for fresh-tasting fruit products and concern for public safety, the study of other bacteriocins with biochemical characteristics that make them candidates for the preservation of these products are of great interest. Efforts for their approval as food additives are also important. In this review, we discuss why the study of bacteriocins as an alternative method to preserve fruit products is important; we detail the biotechnological approaches for the use of bacteriocins in fruit products; and describe some bacteriocins that have been tested and have potential to be tested for the preservation of fruit products.     

Natural Variation in Susceptibility of Listeria Strains to Class IIa Bacteriocins

Thirty-one Listeria strains were tested for sensitivity to four class IIa bacteriocins, namely, enterocin A, mesentericin Y105, divercin V41, and pediocin AcH, and to nisin A. Class IIa bacteriocins displayed surprisingly similar antimicrobial patterns ranging from highly susceptible to fully resistant strains, whereas nisin A showed a different pattern in which all Listeria strains were inhibited. Particularly, it was observed that the strain Listeria monocytogenes V7 could not be inhibited by any of the class IIa bacteriocins tested. These observations suggest that Listeria strains resistant to the whole range of class IIa bacteriocins may occur in natural environments, which could be of great concern with regard to the use of these peptides as food preservatives. Received: 22 October 1999 / Accepted: 15 December 1999     

Heterologous coproduction of enterocin A and pediocin PA-1 by Lactococcus lactis: detection by specific peptide-directed antibodies

Antibodies against enterocin A were obtained by immunization of rabbits with synthetic peptides PH4 and PH5 designed, respectively, on the N- and C-terminal amino acid sequences of enterocin A and conjugated to the carrier protein KLH. Anti-PH4-KLH antibodies not only recognized enterocin A but also pediocin PA-1, enterocin P, and sakacin A, three bacteriocins which share the N-terminal class IIa consensus motif (YGNGVXC) that is contained in the sequence of the peptide PH4. In contrast, anti-PH5-KLH antibodies only reacted with enterocin A because the amino acid sequences of the C-terminal parts of class IIa bacteriocins are highly variable. Enterocin A and/or pediocin PA-1 structural and immunity genes were introduced in Lactococcus lactis IL1403 to achieve (co)production of the bacteriocins. The level of production of the two bacteriocins was significantly lower than that obtained by the wild-type producers, a fact that suggests a low efficiency of transport and/or maturation of these bacteriocins by the chromosomally encoded bacteriocin translocation machinery of IL1403. Despite the low production levels, both bacteriocins could be specifically detected and quantified with the anti-PH5-KLH (anti-enterocin A) antibodies isolated in this study and the anti-PH2-KLH (anti-pediocin PA-1) antibodies previously generated (J. M. Martínez, M. I. Martínez, A. M. Suárez, C. Herranz, P. Casaus, L. M. Cintas, J. M. Rodríguez, and P. E. Hernández, Appl. Environ. Microbiol. 64:4536-4545, 1998). In this work, the availability of antibodies for the specific detection and quantification of enterocin A and pediocin PA-1 was crucial to demonstrate coproduction of both bacteriocins by L. lactis IL1403(pJM04), because indicator strains that are selectively inhibited by each bacteriocin are not available.     

Frequency of Bacteriocin Resistance Development and Associated Fitness Costs in Listeria monocytogenes

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in Listeria monocytogenes to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10⁻⁶. However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10⁻⁷ to 10⁻². Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10°C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 × 10⁻⁸). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5°C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5°C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.     

Combined Effect of Bacteriocins on the Survival of Various Listeria Species in Broth and Meat System

The antilisterial efficiency of three bacteriocins from lactic acid bacteria, lactocin 705 (produced by L. casei CRL705, 17000 AU/ml), enterocin CRL35 (produced by E. faecium CRL35, 17000 AU/ml), and nisin (2000 IU/ml), was tested in broth, individually and in combination against Listeria monocytogenes and Listeria innocua. Both Listeria species showed an initial decrease in viable counts followed by the regrowth of the survivors after 1 h in the presence of each bacteriocin. A greater antilisterial effect was observed when the bacteriocins were combined in pairs, maximal inhibition being reached when nisin was involved. When a mix of the three bacteriocins was used, no survivors were observed after 24 h of incubation. Similar results were obtained when the bacteriocin combinations were tested in a meat system, indicating that the use of more than one LAB bacteriocin in combination may be effective in preventing the spontaneous emergence of a bacteriocin-resistant Listeria population. Received: 17 March 2000 / Accepted: 26 June 2000     

Mechanistic action of pediocin and nisin: recent progress and unresolved questions

Nisin and pediocin PA-1 are examples of bacteriocins from lactic acid bacteria (LAB) that have found practical applications as food preservatives. Like other natural antimicrobial peptides, LAB bacteriocins act primarily at the cytoplasmic membranes of susceptible microorganisms. Studies with in vivo as well as in␣vitro membrane systems are directed toward understanding how bacteriocins interact with membranes so as to provide a mechanistic basis for their rational applications. The dissipation of proton motive force was identified early on as the common mechanism for the lethal activity of LAB bacteriocin. Models for nisin/membrane interactions propose that the peptide forms poration complexes in the membrane through a multi-step process of binding, insertion, and pore formation. This review focuses on the current knowledge of: (1) the mechanistic action of nisin and pediocin-like bacteriocins, (2) the requirement for a cell factor such as a membrane protein, (3) the influence of membrane potential, pH, and lipid composition on the of specificity and efficacy of bacteriocins, and (4) the roles of specific amino acids and structural domains of the bacteriocins in their action. Received: 3 April 1998 / Received last revision: 27 July 1998 / Accepted: 29 July 1998     

Frequency of bacteriocin resistance development and associated fitness costs in Listeria monocytogenes

Bacteriocin-producing starter cultures have been suggested as natural food preservatives; however, development of resistance in the target organism is a major concern. We investigated the development of resistance in Listeria monocytogenes to the two major bacteriocins pediocin PA-1 and nisin A, with a focus on the variations between strains and the influence of environmental conditions. While considerable strain-specific variations in the frequency of resistance development and associated fitness costs were observed, the influence of environmental stress seemed to be bacteriocin specific. Pediocin resistance frequencies were determined for 20 strains and were in most cases ca. 10(-6). However, two strains with intermediate pediocin sensitivity had 100-fold-higher pediocin resistance frequencies. Nisin resistance frequencies (14 strains) were in the range of 10(-7) to 10(-2). Strains with intermediate nisin sensitivity were among those with the highest frequencies. Environmental stress in the form of low temperature (10 degrees C), reduced pH (5.5), or the presence of NaCl (6.5%) did not influence the frequency of pediocin resistance development; in contrast, the nisin resistance frequency was considerably reduced (<5 x 10(-8)). Pediocin resistance in all spontaneous mutants was very stable, but the stability of nisin resistance varied. Pediocin-resistant mutants had fitness costs in the form of reduction down to 44% of the maximum specific growth rate of the wild-type strain. Nisin-resistant mutants had fewer and less-pronounced growth rate reductions. The fitness costs were not increased upon applying environmental stress (5 degrees C, 6.5% NaCl, or pH 5.5), indicating that the bacteriocin-resistant mutants were not more stress sensitive than the wild-type strains. In a saveloy-type meat model at 5 degrees C, however, the growth differences seemed to be negligible. The applicational perspectives of the results are discussed.     

Adsorption of nisin and enterocin 4 to polypropylene and glass surfaces and its prevention by Tween 80

H.M.L.J. JOOSTEN AND M. NUÑEZ. 1995. The bacteriocins nisin and enterocin 4 are rapidly adsorbed on polypropylene disposables (pipette tips, microfuge tubes) and on glassware. Losses were estimated to exceed 50% if small volumes were transferred to microfuge tubes. This effect may lead to considerable errors in determination of bacteriocin activity. Addition of Tween 80 prevents adsorption.     

Enhanced nisin and pediocin production on whey supplemented with different nitrogen sources

Production of nisin and pediocin were followed, respectively, in Lactococcus lactis subsp. lactis CECT 539 and Pediococcus acidilactici NRRL B-5627 grown with lactose and four different nitrogen sources. Neither NH₄Cl nor glycine improved production of the bacteriocins. Both yeast extract and Casitone increased pediocin production from 55 BU ml^–1 to 195 BU ml^–1 and 185 BU ml^–1, respectively. Nisin increased from 21 BU ml^–1 to 74 BU ml^–1 and 59 BU ml^–1 with these nitrogen sources.     

Dynamic relationships among type IIa bacteriocins: temperature effects on antimicrobial activity and on structure of the C-terminal amphipathic alpha helix as a receptor-binding region

Dynamic aspects of structural relationships among class IIa bacteriocins, which are antimicrobial peptides from lactic acid bacteria (LAB), have been examined by use of circular dichroism (CD), molecular dynamics (MD) simulations, and activity testing. Pediocin PA-1 is a potent class IIa bacteriocin, which contains a second C-terminal disulfide bond in addition to the highly conserved N-terminal disulfide bond. A mutant of pediocin PA-1, ped[M31Nle], wherein the replacement of methionine by norleucine (Nle) gives enhanced stability toward aerobic oxidation, was synthesized by solid-phase peptide synthesis to study the activity of the peptide in relation to its structure. The secondary structural analysis from CD spectra of ped[M31Nle], carnobacteriocin B2 (cbn B2), and leucocin A (leuA) at different temperatures suggests that the alpha-helical region of these peptides is important for target recognition and activity. Using molecular modeling and dynamic simulations, complete models of pediocin PA-1, enterocin P, sakacin P, and curvacin A in 2,2,2-trifluoroethanol (TFE) were generated to compare structural relationships among this class of bacteriocins. Their high sequence similarity allows for the use of homology modeling techniques. Starting from homology models based on solution structures of leuA (PDB code 1CW6) and cbnB2 (PDB code 1CW5), results of 2-4 ns MD simulations in TFE and water at 298 and 313 K are reported. The results indicate that these peptides have a common helical C-terminal domain in TFE but a more variable beta sheet or coiled N terminus. At elevated temperatures, pediocin PA-1 maintains its overall structure, whereas peptides without the second C-terminal disulfide bond, such as enterocin P, sakacin P, curvacin A, leuA, and cbnB2 experience partial disruption of the helical section. Pediocin PA-1 and ped[M31Nle] were found to be equally active at different temperatures, whereas the other peptides that lack the second C-terminal disulfide bond are 30-50 times less antimicrobially potent at 310 K (37 degrees C) than at 298 K (25 degrees C). These results indicate that the structural changes in the helical region observed at elevated temperatures account for the loss of activity of these peptides. The presence of C-terminal hydrophobic residues on one side of the amphipathic helix in class IIa bacteriocins is an important feature for receptor recognition and specificity toward particular organisms. This study assists in the understanding of structure-activity relationships in type IIa bacteriocins and demonstrates the importance of the conserved C-terminal amphipathic alpha helix for activity.     

Monoclonal antibody-based enzyme immunoassay for pediocins of Pediococcus acidilactici.

Monoclonal antibody (MAb) R2-AR against pediocin RS2 was developed. Mice were immunized for 12 weeks with pediocin RS2 conjugated to a polyacrylamide gel. Two hybridoma fusions yielded an MAb that in Western blots (immunoblots) reacted only with pediocins RS2 and AcH (3 kDa) from Pediococcus acidilactici RS2 and H, respectively, and did not react with any other bacteriocin, including sakacin A from Lactobacillus sake Lb 706, leuconocin LCM1 from Leuconostoc carnosum LM1, nisin from Lactococcus lactis ATCC 11454, and pediocin A from Pediococcus pentosaceus FBB61. Each of the bacteriocin bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels was confirmed to be biologically active by a gel overlay test performed with sensitive indicator organisms. In dot immunoblot assays, the MAb could detect a minimum of 32,000 arbitrary units of pediocin RS2 or AcH per ml. In colony immunoblot assays, the MAb was used successfully to differentiate bac+ and bac- variants of P. acidilactici RS2 strains.