Bacteriocin-producing Strains Research Articles

Elaboration of PCR Primers for Bacteriocin Genetic Determinants Detection

ABSTRACTThe sequences of different genetic determinants of bacteriocins belonging to the classes of lantibiotics and pediocin-like bacteriocins were compared, and in a few cases it was possible to obtain consensus sequences for genes with identical functions. On the basis of these consensus sequences, fifteen primer sets were designed, and a screening of four bacteriocin-producing Enterococcus strains was performed, as well as of a collection of other Enterococci which have not been screened before for antibacterial activity. In the case of the four bacteriocinogenic strains, several amplifications with the expected sizes were obtained with some of the primers.A particular case represented two strains, one of them being bacteriocin producer, and the second one resistant to the bacteriocin produced by the first one. In this case, in both strains amplifications were obtained with primers specific to one of the immunity protein genes, a fact probably suggesting horizontal gene transfer of the immunity determinants.

Coculture with specific bacteria enhances survival of Lactobacillus plantarum NC8, an autoinducer-regulated bacteriocin producer, in olive fermentations

Bacteriocin production in Lactobacillus plantarum NC8 is activated by coculture with specific bacteriocin production-inducing bacterial strains. The system is further regulated by a three-component regulatory system involving a specific autoinducer peptide (PLNC8IF). We have used L. plantarum NC8 as a starter culture in Spanish-style green olive fermentations and examined the influence of coculturing in its survival. We found that L. plantarum NC8 greatly enhanced its growth and survival in the olive fermentations when coinoculated with two specific bacteriocin-production inducing strains, i.e. Enterococcus faecium 6T1a-20 and Pediococcus pentosaceus FBB63, when compared to singly-inoculated fermentations. In addition, a constitutive bacteriocin-producer NC8-derivative strain was used as a control in the olive fermentations and showed also better viability than the parental NC8 strain. Our results suggest the involvement of bacteriocin production in the viability enhancement found in both cases. We postulate that the presence of specific bacteria is recognized by L. plantarum NC8 as an environmental stimulus to switch a specific adaptive response on, most probably involving bacteriocin production. The design of novel bacteriocin-producing starter cultures for food fermentations should consider their constitutive versus regulated character.

Antimicrobial Activity, Inhibition of Urogenital Pathogens, and Synergistic Interactions Between Lactobacillus Strains

Lactobacillus fermentum strain L23 and L. rhamnosus strain L60 were selected as an alternative treatment to prevent or treat urogenital infections based on their probiotic properties and production of bacteriocins. The objectives of the present work were to study the inhibitory activities of these two bacteriocin-producing strains, and to analyze the interactions between pairs of bacteriocins that inhibit urogenital pathogens. Antimicrobial activity tests of L23 and L60 were performed by a diffusion method with 207 bacterial strains, isolated from female patients presenting a urogenital infection. Inhibitory substances interaction tests were carried out by using a streak-diffusion method on agar plates. One hundred percent of the clinical isolates showed sensitivity to the antimicrobial substances produced by L23 and L60. The selected lactobacilli produced larger inhibition halos when compared to several antibiotics commonly used for treating these infections. Synergistic interactions and indifferent interactions were recorded in 68.6% and 31.4% of the cases, respectively. No antagonistic interactions were observed. In conclusion, the bacteriocin-producing strains L23 and L60 are potential candidates for probiotic prophylaxis and treatment of urogenital disorders in women.

Enterococcus faecium CCM7420, bacteriocin PPB CCM7420 and their effect in the digestive tract of rabbits

The effect of <I>Enterococcus faecium</I> CCM7420, bacteriocin-producing strain with probiotic properties and its partially purified bacteriocin PPB CCM7420 on growth performance, microflora, <I>Eimeria</I> sp. oocysts, biochemical blood parameters and glutathione-peroxidase activity in rabbits was determined. An increase in the body weight of rabbits (<I>P</I> < 0.01) was achieved after <I>E. faecium</I> CCM7420 application. A non-significant reduction of faecal <I>E. coli</I> (including haemolytic <I>E. coli</I>), coagulase-positive staphylococci (CPS) and <I>Staphylococcus aureus</I> was found in rabbits administered the <I>E. faecium</I> CCM7420 strain and lower (non-significant) counts of <I>S. aureus</I> and <I>Clostridium</I>-like sp. were detected in PPB CCM7420 group, compared to the control. In the caecum, a significant reduction of CPS was noted in both experimental groups (<I>P</I> < 0.001 and <I>P</I> < 0.05 for EG1 and EG2, respectively). Biochemical blood parameters increased in both experimental groups (<I>P</I> < 0.05 and <I>P</I> < 0.001 for EG2 and EG1, respectively). In the CCM7420 group, the lowest activity of glutathione-peroxidase was measured (<I>P</I> < 0.001). After the application of PPB CCM7420 (<I>P</I> < 0.05; day 21), a reduction of <I>Eimeria </I>sp. oocysts was recorded.

Control of late blowing in cheese by adding lacticin 3147-producing Lactococcus lactis IFPL 3593 to the starter

Bacteriocins produced by lactic acid bacteria offer potential as tools for ensuring food safety and quality. Lacticin 3147 is a two-peptide lantibiotic previously shown to inhibit a broad range of food spoilage and pathogenic bacteria. Lacticin 3147-producing Lactococcus lactis IFPL 3593 was investigated for its ability to inhibit the growth of clostridia as a means to prevent late blowing in cheese. L. lactis IFPL 3593 was shown to inhibit germination of clostridia spores and prevent late blowing in semi-hard cheeses, with a 5 log g −1 reduction in the numbers of spores when compared to control cheeses. Furthermore, this bacteriocin-producing strain demonstrated considerable potential as a biopreservative agent against heterofermentative lactobacilli and their associated blowing defects. The use of this strain to prevent late blowing in cheese thereby represents a promising alternative to the addition of lysozyme particularly given the increasing concerns regarding the potential allergenicity of this additive.

Isolation of lactobacilli from sow milk and evaluation of their probiotic potential

Sow milk protects the piglet against infectious diseases through a variety of mechanisms. In this study, the presence of potentially probiotic lactic acid bacteria in this biological fluid was investigated. Milk samples were obtained from 8 sows and a total of 19 rod-shaped isolates were selected for identification and assessment of their probiotic potential. RAPD profiling revealed the existence of 8 different genetic profiles among them. One representative of each profile was selected for further characterization and they were identified as Lactobacillus reuteri, Lb. salivarius, Lb. plantarum, Lb. paraplantarum, Lb. brevis and Weissella paramesenteroides. Then, their probiotic potential was evaluated through different assays, including survival in conditions simulating those existing in the gastrointestinal tract, production of antimicrobial compounds, adherence to intestinal mucin, production of biogenic amines, degradation of mucin, and pattern of antibiotic sensitivity. Three strains, Lb. reuteri CR20 (a reuterin-producing strain), Lb. salivarius CELA2 (a bacteriocin-producing strain) and Lb. paraplantarum CLB7 displayed the highest probiotic potential.

Effect of inoculated corn silage on rumen fermentation and lipid metabolism in an artificial rumen (RUSITEC)

Effects of providing substrates containing corn silage (12 g of DM/day) and lucerne hay (3 g DM/day) to an artificial rumen (RUSITEC) in which the whole plant corn was not inoculated (CS, control), or inoculated with Lactobacillus plantarum CCM 4000 (CS + LP), Lactobacillus fermentum LF2 (CS + LF) or Enterococcus faecium CCM 4231 (CS + EF) on rumen metabolism was examined. The inoculants were promising probiotic- and bacteriocin-producing strains. The substrates were fermented in fermentation vessels for 11 days, which included a stabilization period of 5 days. The counts of inoculants decreased during ensiling of corn, and at the end of ensiling (105 days), their counts were less than 1.0 log 10 cfu/g; probably for lower pH (3.44–3.54). Organic matter and neutral detergent fibre degradabilities were increased in inoculated CS compared to control. Ammonia N production was decreased in all inoculated substrates. From other fermentation parameters, only total VFA production, production of acetate, n-butyrate (mmol/day) significantly increased during fermentation of CS + LP and CS + LF substrates compared to control. The inoculated CS substrates influenced the efficiency of microbial protein synthesis ( P<0.001). Biohydrogenation of C 18:1, C18:2 and total C18 FA increased in all inoculated CS substrates in fermentation fluid in RUSITEC. The concentration of trans 11 C18:1 (TVA) was unchanged in inoculated CS substrates and the concentration of cis 9, trans 11 C18:2 (CLA) were decreased ( P<0.05) compared in CS. All inoculants of whole plant corn were not effective for increasing CLA and TVA concentration in fermentation fluid and for decreasing of rumen biohydrogenation of C18 fatty acids in RUSITEC.

Effects of curing sodium nitrite additive and natural meat fat on growth control of Listeria monocytogenes by the bacteriocin-producing Lactobacillus curvatus strain CWBI-B28

In realistic model meat systems, the separate and combined effects of fat content and sodium nitrite on the antilisterial activity of the bacteriocin of Lactobacillus curvatus CWBI-B28 were studied. In laboratory fermentations where Listeria monocytogenes was co-cultured at 4 °C with bacteriocin-producing CWBI-B28 in lean pork meat (fat content: 13%) without added nitrite, a strong antilisterial effect was observed after one week. The effect was maintained for an additional week, after which a slight and very gradual rebound was observed. Both added nitrite (20 ppm) and a high-fat content (43%) were found to antagonise this antilisterial effect, the Listeria cfu count reached after six weeks being 200 times as high in high-fat meat with added nitrite than in lean meat without nitrite. This antagonism could not be attributed to slower growth of the bacteriocin-producing strain, since CWBI-B28 grew optimally in fat-rich meat with 20 ppm sodium nitrite. Bacteriocin activity was also measured in the samples. The observed activity levels are discussed in relation to the degree of antilisterial protection conferred.

Molecular and probiotic characterization of bacteriocin-producing Enterococcus faecium strains isolated from nonfermented animal foods

The characterization of four novel bacteriocin-producing enterococcal strains, isolated from nonfermented animal foods, was carried out with a view to evaluate their potential application as probiotics in raw and processed foodstuffs. 16S rRNA sequencing and random amplification of polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis allowed the identification and intra-specific grouping of Enterococcus faecium strains, which inhibited the growth of four relevant food-borne pathogenic and spoilage species. Enterococcus faecium strains exhibited remarkable probiotic profiles, being able to survive to pH 3.0 and to the presence of bile salts, pancreatin and pepsin. Enterococcus faecium strains evaluated did not exhibit bile salt hydrolase or haemolytic activity, but showed good adhesion properties, also exhibiting sensitivity to clinically relevant antimicrobial agents. In our study, DNA sequencing of the 16S rRNA gene and RAPD-PCR analysis were equally discriminatory for typing E. faecium strains. This study also confirmed the potential tolerance and survival of E. faecium strains isolated from nonfermented animal foods to the gastrointestinal tract. This study represents the first report on potential probiotic E. faecium strains isolated from nonfermented meat and fish. Their moderate heat resistance opens the way to their potential use as probiotics in minimally processed foods subjected to moderate heat processing.

Changes in the microbial composition of raw milk induced by thermization treatments applied prior to traditional Greek hard cheese processing.

The microbiological quality, safety, and composition of mixtures of ewe's and goat's milk (90:10) used for cheesemaking were evaluated before and after thermization at 60 and 67 degrees C for 30 s. Such mild thermal treatments are commonly applied to reduce natural contaminants of raw milk before processing for traditional hard Greek cheeses. Raw milk samples had an average total bacterial count of 7.3 log CFU/ml; most of these bacteria were lactic acid bacteria (LAB) and pseudomonads. The LAB flora of raw milk was dominated by enterococci (40.8%), followed by lactococci (20.4%), leuconostocs (18.4%), and mesophilic lactobacilli (10.2%). Enterococcus faecalis (30.1%) and Enterococcus faecium (13.7%) were the most common LAB isolates, followed by Enterococcus durans, Lactococcus lactis subsp. lactis, Lactobacillus plantarum, and Leuconostoc lactis. Thermization at 60 degrees C for 30 s was effective for reducing raw milk contamination by enterobacteria (5.1 log CFU/ml), coagulase-positive staphylococci (3.3 log CFU/ml), and Listeria (present in 25-ml samples) to safe levels, but it also reduced mesophilic lactococci, leuconostocs, lactobacilli, and selected enterococci (72.0%) in thermized milk. Thermization at 67 degrees C for 30 s had a major inactivation effect on all bacterial groups. Two nisin-producing L. lactis subsp. lactis strains (M78 and M104) were isolated from raw milk, but neither nisin-producing nor other bacteriocin-producing LAB strains were isolated from thermized milk. Thus, thermization treatments control harmful bacteria but also may have a negative impact on milk quality by reducing desirable LAB and the biodiversity of raw milk bacteria overall, inactivating potentially protective LAB strains and enhancing the ability of potentially pathogenic enterococci to grow in fresh cheese curds.

Suppression of the bacterial spot pathogen Xanthomonas euvesicatoria on tomato leaves by an attenuated mutant of Xanthomonas perforans.

A bacteriocin-producing strain of the bacterial spot of tomato plant pathogen, Xanthomonas perforans, with attenuated pathogenicity was deployed for biocontrol of a bacteriocin-sensitive strain of the genetically closely related bacterial spot of tomato plant pathogen, X. euvesicatoria. The attenuated mutant (91-118DeltaopgHDeltabcnB) of X. perforans was tested in leaf tissue and shown to significantly inhibit internal populations of the wild-type X. euvesicatoria strain although significantly less than the wild-type 91-118 strain, whereas in a phyllosphere inhibition assay, the mutant strain reduced epiphytic populations comparably to 91-118. Thus, the attenuated mutant limited the sensitive bacterium more efficiently on the leaf surface than inside the leaf. In field experiments, weekly application of 91-118DeltaopgHDeltabcnB significantly reduced X. euvesicatoria populations compared to the growers' standard control (copper hydroxide and mancozeb applied weekly and acibenzolar-S-methyl applied every 2 weeks). The biological control agent, 91-118DeltaopgHDeltabcnB, applied every 2 weeks also significantly reduced X. euvesicatoria populations in one season but was not significantly different from the growers' standard control. Potentially, attenuated pathogenic strains could be deployed as biological control agents in order to improve disease control of foliar plant pathogens.

Characterization of a bacteriocin produced by Enterococcus faecalis N1-33 and its application as a food preservative.

A bacteriocin-producing strain, N1-33, isolated from fermented bamboo shoot was identified as Enterococcus faecalis. The pH-adjusted culture supernatant of this strain consisted of several peptides with bacteriocin activity, and the supernatant inhibited the growth of pathogenic bacteria such as Listeria monocytogenes. The major peptide with bacteriocin activity was purified, and the first 39 amino acid residues of the bacteriocin were found to be identical to enterocin MR10A produced by E. faecalis MRR10-3. Addition of the pH-adjusted and concentrated culture supernatant of strain N1-33 caused a marked reduction in the growth of Bacillus cereus in custard cream and L. monocytogenes in pickled cucumber. These results suggest the potential use of the bacteriocin produced by strain N1-33 in food biopreservation.

Co-cultivation of a bacteriocin-producing mixed culture ofBifidobacterium thermophilumRBL67 andPediococcus acidilacticiUVA1 isolated from baby faeces

We investigated the kinetics of growth, metabolism and antimicrobial activity of Bifidobacterium thermophilum RBL67 and Pediococcus acidilactici UVA1 previously isolated as a consortium from human baby faeces and producing antimicrobial proteinaceous compounds. Cell growth, antimicrobial activity, glucose consumption, organic acid production and pediocin-gene expression were monitored during pure and mixed strain batch cultures with controlled pH (6.0) at 37 degrees C. The balance of the two strains in mixed cultures was stable, yielding high cell count of 10(9) CFU ml(-1) after a very short incubation time of 4 h for UVA1 and 7 h for RBL67, and RBL67 was not affected by the high production of pediocin by UVA1 during co-culture (up to 12.8 microg ml(-1)). Furthermore, a real-time PCR assay was developed and allowed gene-expression analysis of the pediocin-gene pedA. The co-culture of RBL67 and UVA1 showed high stability, cell yields and bacteriocin production during batch cultures and has potential for use as a probiotic mixture with antibacterial properties. In addition, a gene-expression real-time PCR assay was successfully developed, used for the relative quantification of the pediocin transcript pedA and demonstrated to be a valuable complementary tool to activity assay. This is the first study reporting of a stable mixed culture of two bacteriocin-producing strains of human origin.

Purification and Characterization of Phocaecin PI80: An Anti-Listerial Bacteriocin Produced by Streptococcus phocae PI80 Isolated from the Gut of Peneaus indicus (Indian White Shrimp)

A bacteriocin-producing strain PI80 was isolated from the gut of Penaeus indicus (Indian white shrimp) and identified as Streptococcus phocae PI80. The bacteriocin was purified from a culture supernatant to homogeneity as confirmed by Tricine SDS-PAGE. Reverse-phase HPLC analysis revealed a single active fraction eluted at 12.94 min, and MALDI-TOF mass spectrometry analysis showed the molecular mass to be 9.244 kDa. This molecular mass does not correspond to previously described streptococcal bacteriocins. The purified bacteriocin was named phocaecin PI80 from its producer strain, as this is the first report of bacteriocin production by Streptococcus phocae. The bacteriocin exhibited a broad spectrum of activity and inhibited important pathogens: Listeria monocytogenes, Vibrio parahaemolyticus, and V. fischeri. The antibacterial substance was also sensitive to proteolytic enzymes: trypsin, protease, pepsin, and chymotrypsin, yet insensitive to catalase, peroxidase, and diastase, confirming that the inhibition was due to a proteinaceous molecule (i.e., the bacteriocin), and not due to hydrogen peroxide or diacetyl. Phocaecin PI80 moderately tolerated heat treatment (up to 70 degrees for 10 min) and resisted certain solvents (acetone, ethanol, and butanol). A massive leakage of K+ ions from E. coli DH5alpha, L. monocytogenes, and V. parahaemolyticus was induced by phocaecin PI80, as measured by Inductively Coupled Plasma Optical Emission Spectrometry (ICPOES). Therefore, the results of this study show that phocaecin PI80 may be a useful tool for inhibiting L. monocytogenes in seafood products that do not usually undergo adequate heat treatment, whereas the cells of Streptococcus phocae PI80 could be used to control vibriosis in shrimp farming.

Antilisterial Activity of Plantaricin UG1 during Manufacture of Zabady and Kareesh Cheese: Two Arabian Dairy Products

Here, plantaricin UG1 was shown to inhibit Listeria monocytogenes LMG10470 (L. monocytogenes) cells. The inhibition was not caused by lactic acid produced by the bacteriocin producing strain Lactobacillus plantarum UG1 (Lact. plantarum). Partially purified plantaricin UG1 had a higher antilisterial activity than Lact. plantarum cells in both Zabady and Kareesh cheese samples during their maturation. Compared to the antilisterial activity in Kareesh cheese, plantaricin UG1 showed a faster effect during Zabady manufacture.

The dual role of bacteriocins as anti- and probiotics.

Bacteria employed in probiotic applications help to maintain or restore a host's natural microbial floral. The ability of probiotic bacteria to successfully outcompete undesired species is often due to, or enhanced by, the production of potent antimicrobial toxins. The most commonly encountered of these are bacteriocins, a large and functionally diverse family of antimicrobials found in all major lineages of Bacteria. Recent studies reveal that these proteinaceous toxins play a critical role in mediating competitive dynamics between bacterial strains and closely related species. The potential use of bacteriocin-producing strains as probiotic and bioprotective agents has recently received increased attention. This review will report on recent efforts involving the use of such strains, with a particular focus on emerging probiotic therapies for humans, livestock, and aquaculture.

Evaluation of a bacteriocin-producing strain of Pediococcus acidilactici as a biopreservative for “Alheira”, a fermented meat sausage

This study was conducted to evaluate the ability of Pediococcus acidilactici HA-6111-2, a PA-1 bacteriocin-producing lactic acid bacterium (LAB), isolated from “Alheira” to inhibit a cocktail of Listeria innocua strains during production and shelf-life of these products. The bacteriocinogenic culture reduced the Listeria population below the detection limit (1.5log CFU/g) and had no effect on the growth of the natural LAB flora or on the pH. Pathogenic organisms were not detected in any sample. The presence of some virulence factors and antibiotic resistances of the strain to be used as a bioprotective culture were investigated. P. acidilactici HA-6111-2 did not produce any of the biogenic amines tested; no formation of biofilms was observed; more l(+)lactic acid was produced than its isomer d(−); no gelatinase, DNase or lipase activity was recorded; no structural genes for the haemolysin, enterococcal surface protein, hydrolytic compounds, aggregation protein and cell-wall adhesins were detected, no significant antibiotic resistances were found. P. acidilactici HA-6111-2 appears to have potential as a bioprotective culture during “Alheira” fermentation. Moreover, a trained panel considered the protected product to be sensorially acceptable.

Characterization of bacteriocin produced byStreptococcus bovisJ2 40-2 isolated from traditional fermented milk ‘Dahi’

A bacteriocin-producing strain Streptococcus bovis J2 40-2 was isolated from traditional fermented milk 'Dahi' in Bangladesh. Despite its narrow antimicrobial spectrum, it showed strong antimicrobial activity against extremely challenging and problematic organisms in foods, such as Listeria monocytogenes. Bacteriocin was sensitive to several proteolytic enzymes and showed antimicrobial activity over a wide pH range of 2.0-10.0. It was stable when heated to 110 degrees C for 20 min, but lost 25% of its activity when heated to 121 degrees C for 15 or 20 min. Optimum bacteriocin production (5600 AU/mL) was achieved when the strain was cultured at 37 degrees C for 24 h in MRS medium rather than in TYLG, GM17, or skim milk medium. Bacteriocin was partially purified by ammonium sulfate precipitation (80% saturation), dialysis (cut-off MW: 3500) and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that bacteriocin had a molecular weight of approximately 4.5 kDa.

Molecular analysis of the replication region of the pCIZ2 plasmid from the multiple bacteriocin producer strain Enterococcus faecium L50

The sequence analysis of the 7383 bp plasmid pCIZ2 from Enterococcus faecium L50 enabled the identification of a DNA region involved in its replication. The structural organization of the pCIZ2 replication region is highly similar to those of well-known theta-replicating plasmids. It contains an untranslated region, the putative replication origin ( ori), constituted by two sets of direct repeats of 12 and 22 bp (iterons), and followed by three open-reading frames ( orf8 to orf10). orf8 encodes the replication initiation protein (RepE). The transcriptional start site of the replication locus was identified 13 nucleotides upstream of the repE start codon. A two-dimensional agarose gel electrophoresis analysis revealed pCIZ2 intermediates profile typical of the theta-type replication mechanism. Subcloning of different DNA fragments of the pCIZ2 replication region in Escherichia coli and, subsequently, in the plasmidless E. faecium L50/14-2 allowed the determination of the minimal replicon on a 1.2 kb DNA fragment containing only the overall ori and repE which also act in trans. The involvement of orf9 in the plasmid copy number and in the plasmid stability was investigated. The pCIZ2 recombinant plasmids constitute narrow-host range shuttle cloning vectors ( E. coli– E. faecium) that could be very useful for enterococcal genes studies, allowing an easy identification due to their histochemical recognition.

Lactobacillus hordei sp. nov., a bacteriocinogenic strain isolated from malted barley

A high-throughput screening effort, designed to isolate bacteriocin-producing lactic acid bacteria (LAB) from malted cereals, resulted in the isolation of four bacteriocin-producing strains that could not be assigned conclusively to recognized species. The four isolates (UCC128T, UCC125, UCC126 and UCC127) were found to share identical (100%) 16S rRNA gene sequences and were therefore deemed to belong to the same species. The strains were Gram-positive, catalase-negative, non-motile homofermentative LAB. The closest recognized relative to strain UCC128T identified based on comparative 16S rRNA gene sequence analysis was Lactobacillus mali DSM 20444T (97% similarity). The strains were characterized phenotypically to identify specific growth requirements. DNA-DNA hybridization between strain UCC128T and L. mali DSM 20444T revealed a level of relatedness of only 39.4%. This indicates that strain UCC128T does not belong to the species L. mali. The four bacteriocin-producing strains are therefore considered to represent a novel species of the genus Lactobacillus, for which the name Lactobacillus hordei sp. nov. is proposed. The type strain is UCC128T (=DSM 19519T=LMG 24241T).