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 rpoN Gene of Enterococcus faecalis Directs Sensitivity to Subclass IIa Bacteriocins

The sigma54 factor has been previously described to be involved in Listeria monocytogenes sensitivity to mesentericin Y105, a subclass IIa bacteriocin. Here, we identified the rpoN gene, encoding sigma54, of Enterococcus faecalis JH2-2 and showed that its interruption leads to E. faecalis resistance to different subclass IIa bacteriocins. Moreover, this rpoN mutant remained sensitive to nisin, a class I bacteriocin, suggesting that sigma54 is especially involved in sensitivity to subclass IIa bacteriocins. Received: 5 May 2000 / Accepted 28 June 2000     

Cell-mediated killing of Listeria monocytogenes by leucocin C producing Escherichia coli

Listeria monocytogenes is a foodborne pathogen causing listeriosis. Listeria in foods can be inhibited with bacteriocins or bacteriocin producing cultures. The aim of this study was to enhance the killing of L. monocytogenes by binding bacteriocin producing Escherichia coli cells to Listeria cells. Antilisterial E. coli was obtained by transferring leucocin C production from Leuconostoc carnosum 4010. For binding of E. coli cells to Listeria cells, the Listeria phage endolysin PlyP35 cell wall binding domain (CBD) was displayed on E. coli cell surface as FliC::CBD chimeric protein in flagella. CBD insertion in flagella was confirmed by Western analysis and enterokinase cleavage. By mixing isolated flagella with L. monocytogenes WSLC 1019 cells, the FliC::CBD flagella was shown to bind to Listeria cells. However, the wild type flagella also attached to Listeria cells masking putative additional binding mediated by the CBD. Yet, the cell-mediated leucocin C killing resulted in two-log reduction of Listeria, whereas the corresponding amount of leucocin C in spent culture medium could only inhibit growth without bacteriocidal effect. Cells binding Listeria and secreting antilisterial peptides may have applications in protection against listeriosis as they kill Listeria better than free antilisterial peptides.     

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     

Use of bacteriocinogenic lactic acid bacteria to inhibit spontaneous nisin-resistant mutants of Listeria monocytogenes Scott A

Nisin is a bacteriocin with a broad antibacterial spectrum including strains of Listeria monocytogenes . Populations of L. monocytogenes , however, frequently contain spontaneous nisin-resistant mutants. When a culture of L. monocytogenes Scott A was exposed to nisin concentrations between 10 and 500 IU ml⁻¹, the initial decrease in viable numbers was followed by regrowth of survivors to nisin. Nisin-resistant mutants of L. monocytogenes Scott A were isolated after a single exposure to nisin at 100 IU ml⁻¹ and were shown to be sensitive to the non-nisin bacteriocins, sakacin A and enterocin B, produced by Lactobacillus sake Lb 706 and Enterococcus faecium BFE 900, respectively. The regrowth of L. monocytogenes Scott A following the initial decrease due to exposure to nisin was prevented by nisin-resistant Lact. sake Lb 706–1a and to a somewhat lesser extent, by Ent. faecium BFE 900–6a. Listerial cells surviving nisin action were thus inhibited by the bacteriocin-producing strains that might be used as starter or protective cultures in foods. Growth of a nisin-resistant mutant of L. monocytogenes Scott A (Li3) was also suppressed by the bacteriocinogenic cultures. Use of nisin in combination with a starter culture producing a non-nisin antilisterial bacteriocin may therefore prevent the emergence of nisin-resistant mutants of L. monocytogenes .     

PrfA activation in Listeria monocytogenes increases the sensitivity to class IIa bacteriocins despite impaired expression of the bacteriocin receptor

BackgroundThe scope of the present work was to characterize the activity of class IIa bacteriocins in Listeria (L.) monocytogenes cells that constitutively express an activated form of PrfA, the virulence master regulator, since bacteriocin sensitivity was only characterized in saprophytic cells so far. The mannose phosphotransferase system (Man-PTS) has been shown to be the class IIa bacteriocin receptor in Listeria; hence, special attention was paid to its expression in virulent bacteria.MethodsL. monocytogenes FBprfA* cells were obtained by transconjugation. Bacterial growth was studied in TSB and glucose containing-minimal medium. Sensitivity to antimicrobial peptides was assessed by killing curves. Membranes of L. monocytogenes FBprfA* cells were characterized using proteomic and lipidomic approaches.ResultsThe mannose phosphotransferase system (Man-PTS) was downregulated upon expression of PrfA*, and these cells turned out to be more sensitive to enterocin CRL35 and pediocin PA-1, while not to nisin. Proteomic and lipidomic analysis showed differences between wild type (WT) and PrfA* strains. For instance, phosphatidic acid was only detected in PrfA* cells, whereas, there was a significant decline of plasmalogen-phosphatidylglycerol in the same strain.ConclusionsOur results support a model in which Man-PTS acts just as a docking molecule that brings class IIa bacteriocins to the plasma membrane. Furthermore, our results suggest that lipids play a crucial role in the mechanism of action of bacteriocins.General significanceThis is the first demonstration of the link between L. monocytogenes virulence and the bacterial sensitivity toward pediocin-like peptides.     

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.     

Impairment of the class IIa bacteriocin receptor function and membrane structural changes are associated to enterocin CRL35 high resistance in Listeria monocytogenes

BackgroundEnterocin CRL35 is a class IIa bacteriocin with anti-Listeria activity. Resistance to these peptides has been associated with either the downregulation of the receptor expression or changes in the membrane and cell walls. The scope of the present work was to characterize enterocin CRL35 resistant Listeria strains with MICs more than 10,000 times higher than the MIC of the WT sensitive strain.MethodsListeria monocytogenes INS7 resistant isolates R2 and R3 were characterized by 16S RNA gene sequencing and rep-PCR. Bacterial growth kinetic was studied in different culture media. Plasma membranes of sensitive and resistant bacteria were characterized by FTIR and Langmuir monolayer techniques.ResultsThe growth kinetic of the resistant isolates was slower as compared to the parental strain in TSB medium. Moreover, the resistant isolates barely grew in a glucose-based synthetic medium, suggesting that these cells had a major alteration in glucose transport. Resistant bacteria also had alterations in their cell wall and, most importantly, membrane lipids. In fact, even though enterocin CRL35 was able to bind to the membrane-water interface of both resistant and parental sensitive strains, this peptide was only able to get inserted into the latter membranes.ConclusionsThese results indicate that bacteriocin receptor is altered in combination with membrane structural modifications in enterocin CRL35-resistant L. monocytogenes strains.General significanceHighly enterocin CRL35-resistant isolates derived from Listeria monocytogenes INS7 have not only an impaired glucose transport but also display structural changes in the hydrophobic core of their plasma membranes.     

Partial characterisation of two bacteriocins produced byLactobacillus paracasei subsp.paracasei ST242BZ and ST284BZ and the effect of medium components on their production

The influence of medium components on production of bacteriocins ST242BZ (10.0 kDa) and ST284BZ (3.5 kDa) byLactobacillus paracasei subsp.paracasei ST242BZ and ST284BZ have been studied. Growth in MRS broth (pH of 6.5) yielded bacteriocin levels of 12800 AU/ml. Modified MRS with tryptone as the only nitrogen source, MRS supplemented with KH₂PO₄ (10–100 g/l), or MRS supplemented with thiamine increased bacteriocin ST242BZ production to 25600 AU/ml. Tryptone, combinations of tryptone, meat extract and yeast extract, or thiamine did not increase bacteriocin ST284BZ production. However, MRS supplemented with K₂HPO₄ (50–100 g/l) increased bacteriocin ST284BZ production up to 25600 AU/ml. Our results suggest that production of bacteriocins ST242BZ and ST284BZ are stimulated by potassium ions.     

Antilisterial Activity on Poultry Meat of Amylolysin, a Bacteriocin from Bacillus amyloliquefaciens GA1

This paper describes the production, the purification and the antilisterial activity of amylolysin, a novel bacteriocin from B. amyloliquefaciens GA1. The strain genome was first analysed using PCR techniques for the presence of gene clusters that direct the synthesis of characterised bacteriocins from B. amyloliquefaciens and the closely related B. subtilis. Our results suggest that amylolysin corresponds to a novel bacteriocin. The effect of amylolysin on the growth of different isolates of Listeria monocytogenes was evaluated in poultry meat during 21 days of storage at 4 °C. A potent antilisterial effect was observed for all the indicator strains tested, demonstrating that amylolysin is a novel bacteriocin that could be used as a food preservative.     

Selection and properties of spontaneous mutants ofListeria monocytogenes ATCC 15313 resistant to different bacteriocins produced by lactic acid bacteria strains

The frequency of spontaneous mutants ofListeria monocytogenes ATTC 15313 resistant to the inhibitory action of three bacteriocins of lactic acid bacteria previously discovered in our laboratory (mesenterocin 52, curvaticin 13, and plantaricin C19) was estimated to be in the range of 10^–3 to 10^–4. The phenotypic character of resistance was stable during several generations in the absence of contact with bacteriocins. The resistance was not due to the inactivation of bacteriocins nor to a modification of their adsorption on the target cells. The selected mutants resistant to one of the bacteriocin cited above showed a cross-resistance to the two other bacteriocins, but not to nisin.     

Characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery

Lactococcus lactis CRL 1584 isolated from a Lithobates catesbeianus hatchery inhibits the growth of Citrobacter freundii (a bullfrog pathogen) and Listeria monocytogenes by a synergistic effect between lactic acid, hydrogen peroxide and a bacteriocin-like molecule. The chemical characterization of the bacteriocin in cell-free supernatants indicates that it has a proteinaceous nature. Hexadecane and ethyl acetate did not modify the bacteriocin activity, while 10 and 20 % (v/v) chloroform decreased the activity by 29 and 43 %, respectively. The antimicrobial peptide was heat stable since 85 % of residual activity was detected when neutralized supernatants were heated at 80 °C for 30 min. Moreover, no bacteriocin inactivation was observed when supernatants were kept at ?20 °C for 3 months. The synthesis of the bacteriocin was associated with bacterial growth, highest production (2,100 AU/ml) being detected at the end of the exponential growth phase. At pH ranges of 5–6.5 and 5.0–5.5 the inhibitory molecule was stable when stored for 2 days at 4 and 25 °C, respectively. Moreover, it had a bactericidal effect on L. monocytogenes and the ultrastructural studies of pathogenic cells revealed clumping of the cytoplasmic material, increased periplasmic space and cell wall modifications. The deduced amino acid sequence of the bacteriocin was identical to nisin Z and the genetic determinants for its production are harbored in the chromosome. These results, described for the first time in L. lactis from a bullfrog hatchery, will increase knowledge of the bacteriocin under study with a view to its potential inclusion in probiotics for raniculture or biopreservatives.     

Characterization of Mundticin L,a Class IIa Anti-Listeria Bacteriocin from Enterococcus mundtii CUGF08

Enterococcus mundtii CUGF08, a lactic acid bacterium isolated from alfalfa sprouts, was found to produce mundticin L, a new class IIa bacteriocin that has a high level of inhibitory activity against the genus Listeria. The plasmid-associated operons containing genes for the mundticin L precursor, the ATP binding cassette (ABC) transporter, and immunity were cloned and sequenced. The fifth residue of the conservative consensus sequence YGNGX in the mature bacteriocin is leucine instead of valine in the sequences of the homologous molecules mundticin KS (ATO6) and enterocin CRL35. The primary structures of the ABC transporter and the immunity protein are homologous but unique.Bacteriocins are ribosomally synthesized proteinaceous compounds that inhibit closely related bacteria (19). Due to consumer concerns with chemical and irradiation preservation methods and due to the rising demand for minimally processed food products, alternative methods for shelf life extension and enhanced safety are needed. Bacteriocins are considered “natural” antimicrobials since many bacteriocins are produced by food grade lactic acid bacteria, which are generally recognized as safe. Bacteriocins can be divided into three main classes: the class I lanthionine-containing lantibiotics, exemplified by nisin; the class II non-lanthionine-containing bacteriocins; and the class III heat-labile, large proteins (6). Class III bacteriocins have limited application due to their thermal instability and cytolytic activity against eukaryotic cells. Class II can be further divided into class IIa containing pediocin-like bacteriocins, class IIb containing two-peptide bacteriocins, and class IIc containing other bacteriocins (8). Class IIa bacteriocins have been extensively studied since pediocin PA-1 was first discovered (12) and characterized (20). Currently, only nisin in class I has been approved by the FDA as a natural food additive. Bacteriocins belonging to class IIa are promising alternative antimicrobials since they are more stable over a broader range of heating regimens and pH conditions. In addition, these bacteriocins exhibit stronger antimicrobial activity against the genus Listeria than nisin (17) but have a narrower antimicrobial spectrum.The potential applications of class IIa bacteriocins in both meat and plant-based foods as a means to provide protection against potential food-borne pathogens and extend shelf life continue to expand. In an attempt to use biological methods for controlling food-borne pathogens on fresh sprouts, a number of food grade lactic acid bacteria were isolated from the indigenous microbiota on alfalfa sprouts. Some of these isolates were found to be bacteriocinogenic. This study describes a new class IIa bacteriocin, mundticin L produced by Enterococcus mundtii CUGF08 isolated from alfalfa sprouts.     

Characterization of Anti-Listeria monocytogenes Bacteriocins from Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis Strains Isolated from Raïb, a Moroccan Traditional Fermented Milk

Seventy-four samples of raïb, a Moroccan traditional fermented milk, were screened for their anti-Listeria monocytogenes activity. Nine lactic acid bacteria with antilisterial activity were isolated and identified as Lactococcus lactis[⁴], Enterococcus faecium[⁴], and E. faecalis[¹]. Antibacterial spectra, determined against 45 target strains, led to the selection of four antibacterial-producing strains, which were further characterized. Their anti-microbial agents, inactivated by one or more proteases, were designed as bacteriocins. Lactococcin R9/2 and R10/1 showed the broadest range of inhibitory action. Anti-bacterial spectra and physico-chemical properties suggest that these bacteriocins were similar to nisin. Enterocin R69 had a specificity of action against Listeria spp., whereas Enterocin R18 had a broad spectrum of activity. Lc. lactis R9/2 and E. faecalis R18 were able to coagulate sterilised UHT milk at 30°C in 24 h and induced a 2 log reduction in L. monocytogenes ATCC 15313 population.     

Identification and nucleotide sequence of genes involved in the synthesis of lactocin 705, a two-peptide bacteriocin from Lactobacillus casei CRL 705

The structural gene determinants of lactocin 705, a bacteriocin produced by Lactobacillus casei CRL 705, have been amplified from a plasmid of approximately 35 kb and sequenced. Lactocin 705 is a class IIb bacteriocin, whose activity depends upon the complementation of two peptides (705alpha and 705beta) of 33 amino acid residues each. These peptides are synthesized as precursors with signal sequences of the double-glycine type, which exhibited high identities with the leader peptides of plantaricin S and J from Lactobacillus plantarum, brochocin C from Brochotrix campestris, sakacin P from Lactobacillus sake, and the competence stimulating peptides from Streptococcus gordonii and Streptococcus mitis. However, the two mature bacteriocins 705alpha and 705beta do not show significant similarity to other sequences in the databases.     

Inhibition ofListeria monocytogenes by plantaricin NA, an antibacterial substance fromLactobacillus plantarum

ALactobacillus plantarum of vegetable origin produced a bacteriocin inhibitory toListeria monocytogenes. The antimicrobial agent was inactivated by proteolytic enzymes, was resistant to heat (100°C for 30 min) and stable over a wide pH range (pH 2–10), and displayed a bactericidal mode of action. Growth inhibition ofL. monocytogenes depend on bacteriocin concentration. The antilisterial efficiency depended on the strain ofL. monocytogenes used but was not influenced by the growth phase of this strain. A decrease in absorbance overtime, indicative of cell lysis, was also observed. The significance of the results is discussed in relation to the potential of the bacteriocin in controllingListeria-associated food-borne hazards in foods.     

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 (DeltapH), and the other consisted of cells that maintained DeltapH. The proportion of cells belonging to each subpopulation was estimated, and the concentrations of bacteriocins required to dissipate DeltapH 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.     

Characterization and Antilisterial Effect of Phosphatidylcholine Nanovesicles Containing the Antimicrobial Peptide Pediocin

Encapsulation may provide increased stability and antimicrobial efficiency to bacteriocins. In this work, the antilisterial peptide pediocin was encapsulated in nanovesicles prepared from partially purified soybean phosphatidylcholine. The maintenance of antimicrobial activity and properties of free and encapsulated pediocin was observed during 13 days at 4 °C, and after this period, the encapsulated pediocin retained 50 % its initial activity. The maintenance of the bioactive properties of free and encapsulated pediocin was observed against different species of Listeria, inhibiting Listeria monocytogenes, Listeria innocua and Listeria ivanovii. The size of vesicles containing pediocin was determined by dynamic light scattering as an average of 190 nm, with little change throughout the observation period. Polydispersity index values were around 0.201 and are considered satisfactory, indicating an adequate size distribution of liposomes. The efficiency of encapsulation was 80 %. Considering these results, the protocol used was appropriate for the encapsulation of this bacteriocin. Results demonstrate the production of stable nanoparticulate material. The maintenance of the properties of pediocin encapsulated in liposomes is fundamental to prospect the stability in different conditions of the food matrix.     

Differential Roles of the Two-Component Peptides of Lactocin 705 in Antimicrobial Activity

Lactobacillus casei CRL705 produces a class IIb bacteriocin, lactocin 705, which relies on the complementary action of two components, Lac705α and Lac705β. These peptides exert a bactericidal effect on the indicator strain Lactobacillus plantarum CRL691, with an optimal Lac705α/Lac705β peptide ratio of 1 to 4. Electron microscopy studies showed that treated CRL691 cells have their cell wall severely damaged, with mesosome-like membranous formations protruding into their cytoplasm. Although less pronounced, a similar effect was also observed with the Lac705β peptide alone. Furthermore, Lac705β increased the inhibitory action of a diluted supernatant of L. casei CRL705, while Lac705α protected CRL691 cells from inhibition. Both peptides were required to dissipate the proton motive force (Δψ and ΔpH) of CRL691 cells. These data suggested that of the two components of lactocin 705, the Lac705α peptide is responsible for receptor recognition, and the Lac705β peptide is the active component on the cell membrane of CRL691 cells. Received: 12 April 2002 / Accepted: 24 May 2002