Capacity of human nisin- and pediocin-producing lactic Acid bacteria to reduce intestinal colonization by vancomycin-resistant enterococci

This study demonstrated the capacity of bacteriocin-producing lactic acid bacteria (LAB) to reduce intestinal colonization by vancomycin-resistant enterococci (VRE) in a mouse model. Lactococcus lactis MM19 and Pediococcus acidilactici MM33 are bacteriocin producers isolated from human feces. The bacteriocin secreted by P. acidilactici is identical to pediocin PA-1/AcH, while PCR analysis demonstrated that L. lactis harbors the nisin Z gene. LAB were acid and bile tolerant when assayed under simulated gastrointestinal conditions. A well diffusion assay using supernatants from LAB demonstrated strong activity against a clinical isolate of VRE. A first in vivo study was done using C57BL/6 mice that received daily intragastric doses of L. lactis MM19, P. acidilactici MM33, P. acidilactici MM33A (a pediocin mutant that had lost its ability to produce pediocin), or phosphate-buffered saline (PBS) for 18 days. This study showed that L. lactis and P. acidilactici MM33A increased the concentrations of total LAB and anaerobes while P. acidilactici MM33 decreased the Enterobacteriaceae populations. A second in vivo study was done using VRE-colonized mice that received the same inocula as those in the previous study for 16 days. In L. lactis-fed mice, fecal VRE levels 1.73 and 2.50 log(10) CFU/g lower than those in the PBS group were observed at 1 and 3 days postinfection. In the P. acidilactici MM33-fed mice, no reduction was observed at 1 day postinfection but a reduction of 1.85 log(10) CFU/g was measured at 3 days postinfection. Levels of VRE in both groups of mice treated with bacteriocin-producing LAB were undetectable at 6 days postinfection. No significant difference in mice fed the pediocin-negative strain compared to the control group was observed. This is the first demonstration that human L. lactis and P. acidilactici nisin- and pediocin-producing strains can reduce VRE intestinal colonization.     

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products

Aims:  To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides.
Method and Results:  Seven LAB strains [ Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121°C for 15 min, stable at pH 2–10, sensitive to protease, insensitive to α-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N -terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins.
Conclusions:  Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics.
Significance and Impact of the Study:  The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori .     

Continuous production of bacteriocins, brevicin, nisin and pediocin, using calcium alginate-immobilized bacteria

Bacteriocins including brevicin 286, nisin and pediocin PO2 have been produced successfully with immobilized cells of Lactobacillus brevis VB286, Lactococcus lactis subsp. lactis and Pediococcus acidilactici PO2 respectively encapsulated in calcium alginate beads. The beads encapsulating the bacteriocin-producing cells were loaded into a column, and continuously supplied with fresh medium at a flow rate of 1 bed volume per 20 min. The concentrations of the three bacteriocins produced in the eluents were at least as high as those obtained from conventional free-cell batch fermentations.     

Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T

AIMS: To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins. METHODS AND RESULTS: Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity. It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE). However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA). The inhibitory activity of Ent. faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase. The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10. The molecular weight of the partially purified bacteriocin(s) was approx. 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent. faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin. CONCLUSION: Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent. faecium JCM 5804T among LAB type and reference strains.     

Lactic acid bacteria isolated from fermented flour of finger millet,its probiotic attributes and bioactive properties

This study aims to isolate and identify lactic acid bacteria from fermented flour of selected finger millet varieties grown in Sri Lanka and to evaluate their probiotic attributes and bioactive properties in vitro. Fifteen lactic acid bacteria were isolated from three varieties of fermented finger millet flour namely ravi, raavana and oshadha. These isolates were screened for phenotypical and biochemical characteristics. The selected isolates were identified by 16 S rRNA sequencing as Bacillus cereus (five strains), Streptococcus lutetiensis, Lactobacillus plantarum, Lactobacillus fermentum (two strains), Brevibacillus borstelensis, Paenibacillus species, Lactococcus lactis subspecies lactis, Enterococcus faecium, Pediococcus acidilactici, and Enterococcus lactis, and their partial sequences were deposited in GenBank. Among them, five isolates including two isolates, L. plantarum MF405176.1 and L. fermentum MF033346.1 isolated from ravi; two isolates, L. lactis MF480428.1 and E. faecium MF480431.1 isolated from raavana; and P. acidilactici MF480434.1 isolated from oshadha varieties respectively, exhibited in vitro safety attributes and could tolerate acid, gastric juice, bile, salt, phenol, and temperature under simulated gastric conditions, and also were susceptible to antibiotics tested. Further, they demonstrated bactericidal activity against both drug-sensitive and multidrug-resistant pathogens. Among the selected isolates, L. plantarum MF405176.1 demonstrated highest hydrophobicity and adhesion to both colon colorectal adenocarcinoma and colon colorectal carcinoma cell lines. L. lactis subspecies lactis MF480428.1 exhibited the highest auto-aggregation and 2, 2, diphenyl-1-pricrylhydrazyl free radical scavenging activity. P. acidilactici MF480434.1 demonstrated the lowest IC50 values against HCT-116 and HT-29 cells. None of the LAB isolates could assimilate > 10% cholesterol in vitro.     

Phylogenetic diversity of lactic acid bacteria associated with paddy rice silage as determined by 16S ribosomal DNA analysis

A total of 161 low-G+C-content gram-positive bacteria isolated from whole-crop paddy rice silage were classified and subjected to phenotypic and genetic analyses. Based on morphological and biochemical characters, these presumptive lactic acid bacterium (LAB) isolates were divided into 10 groups that included members of the genera Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, and WEISSELLA: Analysis of the 16S ribosomal DNA (rDNA) was used to confirm the presence of the predominant groups indicated by phenotypic analysis and to determine the phylogenetic affiliation of representative strains. The virtually complete 16S rRNA gene was PCR amplified and sequenced. The sequences from the various LAB isolates showed high degrees of similarity to those of the GenBank reference strains (between 98.7 and 99.8%). Phylogenetic trees based on the 16S rDNA sequence displayed high consistency, with nodes supported by high bootstrap values. With the exception of one species, the genetic data was in agreement with the phenotypic identification. The prevalent LAB, predominantly homofermentative (66%), consisted of Lactobacillus plantarum (24%), Lactococcus lactis (22%), Leuconostoc pseudomesenteroides (20%), Pediococcus acidilactici (11%), Lactobacillus brevis (11%), Enterococcus faecalis (7%), Weissella kimchii (3%), and Pediococcus pentosaceus (2%). The present study, the first to fully document rice-associated LAB, showed a very diverse community of LAB with a relatively high number of species involved in the fermentation process of paddy rice silage. The comprehensive 16S rDNA-based approach to describing LAB community structure was valuable in revealing the large diversity of bacteria inhabiting paddy rice silage and enabling the future design of appropriate inoculants aimed at improving its fermentation quality.     

Purification and characterization of two bacteriocins produced by lactic acid bacteria isolated from Mongolian airag

AIMS: The aim of this study was to isolate and identify bacteriocin-producing lactic acid bacteria (LAB) issued from Mongolian airag (traditional fermented mare's milk), and to purify and characterize bacteriocins produced by these LAB. METHODS AND RESULTS: Identification of the bacteria (Enterococcus durans) was carried out on the basis of its morphological, biochemical characteristics and carbohydrate fermentation profile and by API50CH kit and 16S rDNA analyses. The pH-neutral cell-free supernatant of this bacterium inhibited the growth of several Lactobacillus spp. and food-borne pathogens including Escherichia coli, Staphylococcus aureus and Listeria innocua. The antimicrobial agent (enterocin A5-11) was heat stable and was not sensitive to acid and alkaline conditions (pH 2-10), but was sensitive to several proteolytic enzymes. Its inhibitory activity was completely eliminated after treatment with proteinase K and alpha-chymotrypsin. The activity was however not completely inactivated by other proteases including trypsin and pepsin. Three-step purification procedure with high recovery yields was developed to separate two bacteriocins. The applied procedure allowed the recovery of 16% and 64% of enterocins A5-11A and A5-11B, respectively, present in the culture supernatant with purity higher than 99%. SDS-PAGE analyses revealed that enterocin A5-11 has a molecular mass of 5000 Da and mass spectrometry analyses demonstrates molecular masses of 5206 and 5218 Da for fractions A and B, respectively. Amino acid analyses of both enterocins indicated significant quantitative difference in their contents in threonine, alanine, isoleucine and leucine. Their N-termini were blocked hampering straightforward Edman degradation. CONCLUSIONS: Bacteriocins A5-11A and B from Ent. durans belong to the class II of bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: Judging from molecular masses, amino acid composition and spectrum of activities, bacteriocins A5-11A and B from Ent. durans show high degree of similarity with enterocins L50A and L50B isolated from Enterococcus faecium (Cintas et al. 1998, 2000) and with enterocin I produced by Ent. faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation (Floriano et al. 1998).     

Widespread distribution of the bacteriocin-producing lactic acid cocci in Miso-paste products

AIMS: A survey was conducted on the ecological distribution of bacteriocin-producing lactic acid bacteria in Miso-pastes, a typical fermented food in Japan. METHODS AND RESULTS Nine Miso-pastes were sampled for isolation of bacteriocin-producers. Almost all isolated enterococcal strains produced bacteriocins but no isolated tetragenococci did so. The bacteriocin-producing isolates were divided into nine groups by phenotypic tests. As the phenotypic characters were highly diverse, these strains could not be identified to species level on the basis of their phenotypes. The nine representative strains from each group were identified by 16S rRNA analysis. These bacteriocin-producers with one exception (Lactococcus sp.) were identified as strains of the Enterococcus faecium 'species group'. The bacteriocins of the nine strains were classified into five types according to their antibacterial spectral patterns and their SDS-PAGE profiles. The bacteriocins inhibited undesirable bacteria in Miso-pastes, such as Bacillus subtilis, but did not inhibit the useful Tetragenococcus halophila. CONCLUSIONS: The bacteriocin-producing lactic acid cocci were widespread at high frequencies in Miso-pastes. They were considered to play an important role in preventing the growth of undesirable bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that bacteriocin-producers act effectively as safe biopreservatives and may contribute to the biopreservation in Miso-pastes.     

Characterization of some bacteriocins produced by lactic acid bacteria isolated from fermented foods

Lactic acid bacteria (LAB) isolated from different sources (dairy products, fruits, fresh and fermented vegetables, fermented cereals) were screened for antimicrobial activity against other bacteria, including potential pathogens and food spoiling bacteria. Six strains have been shown to produce bacteriocins: Lactococcus lactis 19.3, Lactobacillus plantarum 26.1, Enterococcus durans 41.2, isolated from dairy products and Lactobacillus amylolyticus P40 and P50, and Lactobacillus oris P49, isolated from bors. Among the six bacteriocins, there were both heat stable, low molecular mass polypeptides, with a broad inhibitory spectrum, probably belonging to class II bacteriocins, and heat labile, high molecular mass proteins, with a very narrow inhibitory spectrum, most probably belonging to class III bacteriocins. A synergistic effect of some bacteriocins mixtures was observed. We can conclude that fermented foods are still important sources of new functional LAB. Among the six characterized bacteriocins, there might be some novel compounds with interesting features. Moreover, the bacteriocin-producing strains isolated in our study may find applications as protective cultures.     

Redox potential to discriminate among species of lactic acid bacteria

AIMS: To verify to what degree reducing capacity is a characterizing parameter of a species, and of the strains themselves within a given species, of lactic acid bacteria. METHODS AND RESULTS: Eighty-eight strains belonging to 10 species of lactic acid bacteria (LAB) isolated from traditional Italian cheeses were studied for their reduction activity: Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Streptococcus thermophilus, Lactococcus lactis ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus and Pediococcus pentosaceus. It was observed that the lactococci reached minimum redox potential before the lactobacilli. The reduction rate of Enterococcus spp. and L. lactis ssp. lactis was higher than that of the streptococci and Lactobacillus spp. All the P. pentosaceus strains had poor reduction activity compared with the other species. CONCLUSIONS: The evolution of the redox potential in milk over a time span of 24 h has been found to be a parameter that characterizes a species: the different courses corresponding to the species in question are clearly evident, and interesting differences can also be noted within the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: The reduction aptitude of strains might be used to select and adapt appropriate strains for use as starters for dairy products.     

Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain

Aims:  The aim of this study was to purify and identify the bacteriocin produced by Pediococcus acidilactici MM33, a strain previously isolated from human gut.
Methods and Results:  Purification of the bacteriocin was performed by cationic exchange chromatography followed by a reverse phase step. Biochemical and mass spectrometry analysis showed homology with pediocin PA-1. To verify if P. acidilactici MM33 carried the pediocin PA-1 gene, total DNA was used to amplify the pediocin gene. The PCR product obtained was then sequenced and the nucleotide sequence revealed to be identical to that of pediocin PA-1. Treatment of P. acidilactici MM33 with novobiocin resulted in a plasmid-cured strain without bacteriocin-producing capacity. Antimicrobial assay and molecular analysis demonstrated that this strain was ped ⁻ suggesting that the ped cluster is plasmid encoded. Antimicrobial assay revealed that pediocin was bactericidal against Listeria monocytogenes , showing a minimal inhibitory concentration (MIC) of 200 AU ml⁻¹.
Conclusions:  A two-step purification procedure was elaborated in this study. The bacteriocin secreted by the human strain P. acidilactici MM33 is carried on a plasmid and the amino acid sequence is identical to pediocin PA-1.
Significance and Impact of the Study:  Pediococcus acidilactici MM33 is the first human pediocin-producing strain reported and could be used as probiotic to prevent enteric pathogen colonization.     

Antibacterial metabolites of lactic acid bacteria: their diversity and properties

The review is devoted to literature data on antimicrobial metabolites produced by lactic acid bacteria (LAB), which have long been used for the preparation of cultured dairy products. This paper summarizes data on low-molecular-weight antimicrobial substances, which are primary products or by-products of lactic fermentation. Individual sections are devoted to a variety of antifungal agents and bacteriocins produced by LAB; their potential use as food preservatives has been discussed. The characteristics and classification of bacteriocins are presented in a greater detail; their synthesis and mechanism of action are described using the example of nisin A, which belongs to class I lantibiotics synthesized by the bacterium Lactococcus lactis subsp. lactis. The mechanism of action of class II bacteriocins has been demonstrated with lacticin. Prospective directions for using LAB antimicrobial metabolites in industry and medicine are discussed in the Conclusion.     

Growth,acid production and bacteriocin production by probiotic candidates under simulated colonic conditions

Aims

The aim of this study is to evaluate the capacity of three bacteriocin producers, namely Lactococcus lactis subsp. lactis biovar diacetylactis UL719 (nisin Z producer), L. lactis ATCC 11454 (nisin A producer) and Pediococcus acidilactici UL5 (pediocin PA‐1 producer), and to grow and produce their active bacteriocins in Macfarlane broth, which mimics the nutrient composition encountered in the human large intestine.

Methods and Results

The three bacteriocin‐producing strains were grown in Macfarlane broth and in De Man–Rogosa–Sharpe (MRS) broth. For each strain, the bacterial count, pH drop and production of organic acids and bacteriocins were measured for different period of time. The ability of the probiotic candidates to inhibit Listeria ivanovii HPB 28 in co‐culture in Macfarlane broth was also examined. Lactococcus lactis subsp. lactis biovar diacetylactis UL719, L. lactis ATCC 11454 and Ped. acidilactici UL5 were able to grow and produce their bacteriocins in MRS broth and in Macfarlane broth. Each of the three candidates inhibited L. ivanovii HPB 28, and this inhibition activity was correlated with bacteriocin production. The role of bacteriocin production in the inhibition of L. ivanovii in Macfarlane broth was confirmed for Ped. acidilactici UL5 using a pediocin nonproducer mutant.

Conclusions

The data provide some evidence that these bacteria can produce bacteriocins in a complex medium with carbon source similar to those found in the colon.

Significance and Impact of the Study

This study demonstrates the capacity of lactic acid bacteria to produce their bacteriocins in a medium simulating the nutrient composition of the large intestine.     

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

Cloning and heterologous production of Hiracin JM79, a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, in lactic acid bacteria and Pichia pastoris

Hiracin JM79 (HirJM79), a Sec-dependent bacteriocin produced by Enterococcus hirae DCH5, was cloned and produced in Lactococcus lactis, Lactobacillus sakei, Enterococcus faecium, Enterococcus faecalis, and Pichia pastoris. For heterologous production of HirJM79 in lactic acid bacteria (LAB), the HirJM79 structural gene (hirJM79), with or without the HirJM79 immunity gene (hiriJM79), was cloned into the plasmid pMG36c under the control of the constitutive promoter P(32) and into the plasmid pNZ8048 under the control of the inducible P(NisA) promoter. For the production of HirJM79 in P. pastoris, the gene encoding the mature HirJM79 protein was cloned into the pPICZalphaA expression vector. The recombinant plasmids permitted the production of biologically active HirJM79 in the supernatants of L. lactis IL1403, L. lactis NZ9000, L. sakei Lb790, E. faecalis JH2-2, and P. pastoris X-33, the coproduction of HirJM79 and nisin A in L. lactis DPC5598, and the coproduction of HirJM79 and enterocin P in E. faecium L50/14-2. All recombinant LAB produced larger quantities of HirJM79 than E. hirae DCH5, although the antimicrobial activities of most transformants were lower than that predicted from their production of HirJM79. The synthesis, processing, and secretion of HirJM79 proceed efficiently in recombinant LAB strains and P. pastoris.     

Susceptibility of Pediococcus spp. to antimicrobial agents

AIM: To investigate the susceptibility of Pediococcus species to antimicrobial agents. METHODS AND RESULTS: The susceptibility to 14 antimicrobial agents of 31 genotypically distinct strains of six Pediococcus species was assessed by using Etests on ISO-sensitest agar supplemented with horse blood. The species included were Pediococcus acidilactici, Pediococcus damnosus, Pediococcus dextrinicus, Pediococcus inopinatus, Pediococcus parvulus and Pediococcus pentosaceus. For several antimicrobial agents, some species were more susceptible than others. The two industrially important species, P. acidilactici and P. pentosaceus, differed with respect to erythromycin and trovafloxacin susceptibility, and in general both species had higher minimum inhibitory concentrations than the other species. In an erythromycin-resistant P. acidilactici, an erythromycin resistance methylase B [erm(B)] gene was identified by PCR. Using a plasmid preparation from strain P. acidilactici 6990, a previously erythromycin-sensitive Lactococcus lactis strain was made resistant. Transformants harboured a single plasmid, sized at 11.6 kb through sequence analysis. In addition, the erm(B) gene was identified within the plasmid sequence. CONCLUSIONS: The phenotypic test indicated the absence of acquired antimicrobial resistance genes in 30 of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help in selection of the best Pediococcus strains for use as starter cultures.     

Isolation,identification and characterization of <Emphasis Type="Italic">Pediococcus pentosaceus</Emphasis> LB44 and <Emphasis Type="Italic">Weissella confusa</Emphasis> LM85 for the presence of bacteriocin-like inhibitory substances (BLIS)

Lactic acid bacteria (LAB) were isolated from different soil samples such as dairy, garden, rhizospheric soil and sediments of lake. Thirty nine strains of bacteria were isolated using direct spreading method but only one showed the characteristics of LAB. Whereas, using accumulation with incubation method, fifty seven strains were found to be LAB out of sixty eight strains of bacteria isolated. Therefore, accumulation with incubation method was found to be more effective and faster. On the basis of higher growth and antimicrobial activity, two potential isolates LB44 and LM85 were selected for the further characterization. Strain LB44 and LM85 were identified using 16S rDNA amplification and sequencing as Pediococcus pentosaceus LB44 and Weissella confusa LM85, respectively. Cell free supernatant (CFS) of these strains showed antimicrobial activity, stability at higher temperatures, acidic pH and in the presence of organic solvents, detergents and surfactants. Antimicrobial activity was not affected by treatment with catalase and lipase but reduced after treatment with proteinase K reveals the presence of bacteriocin-like inhibitory substance in CFS. CFS inhibited the growth of other Gram-positive and Gram-negative bacteria such as Micrococcus luteus, Lactobacillus delbrueckii, L. curvatus, L. plantarum, L. acidophilus, Enterobacter cloaceae, Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Bacillus subtilis, B. licheniformis, Salmonella typhi and Escherichia coli.     

Phylogenetic analysis of antimicrobial lactic acid bacteria from farmed seabass Dicentrarchus labrax

The use of lactic acid bacteria (LAB) in the prevention or reduction of fish diseases is receiving increasing attention. In the present study, 47 LAB strains were isolated from farmed seabass ( Dicentrarchus labrax ) and were phenotypically and phylogenetically analysed by 16S rDNA and randomly amplified polymorphic DNA - polymerase chain reaction (RAPD-PCR). Their antimicrobial effect was tested in vitro against a wide variety of pathogenic and spoilage bacteria. Most of the strains isolated were enterococci belonging to the following species: Enterococcus faecium (59%), Enterococcus faecalis (21%), Enterococcus sanguinicola (4 strains), Enterococcus mundtii (1 strain), Enterococcus pseudoavium (1 strain), and Lactococcus lactis (1 strain). An Aerococcus viridans strain was also isolated. The survey of their antimicrobial susceptibility showed that all isolates were sensitive to vancomycin and exhibited resistance to between 4 and 10 other antibiotics relevant for therapy in human and animal medicine. Different patterns of resistance were noted for skin and intestines isolates. More than 69% (32 strains) of the isolates inhibited the growth of the majority of pathogenic and spoilage bacteria tested, including Listeria monocytogenes, Staphylococcus aureus, Aeromonas hydrophila, Aeromonas salmonicida, Vibrio anguillarum, and Carnobacterium sp. To our knowledge, this is the first report of bioactive enterococcal species isolated from seabass that could potentially inhibit the undesirable bacteria found in food systems.     

Biological and genetic classification of canine intestinal lactic acid bacteria and bifidobacteria

To investigate the distribution of lactic acid bacteria (LAB) inhabiting canine intestines, a total of 374 gram-positive LAB and bifidobacteria (BF) isolated from large intestinal contents in 36 dogs were classified and identified by phenotypic and genetic analyses. Based on cell morphological sizes, these isolates were divided into seven biotypes containing the genera Lactobacillus, Bifidobacterium, Enterococcus, and Streptococcus. The LAB and BF isolates were classified into 38 chemotypes based on SDS-PAGE protein profile analysis of whole cells. Furthermore, partial 16S rDNA sequencing analysis demonstrated the presence of 24 bacterial species in the 38 chemotypes from 36 dogs. The identified species consisted of ten species belonging to the genus Lactobacillus (78.8%), seven species to the genus Bifidobacterium (6.8%), five species to the genus Enterococcus (11.6%), one species of Streptococcus bovis (2.0%), and one species of Pediococcus acidilactici (0.8%). In particular, the most predominant species in canine intestines were L. reuteri, L. animalis, and L. johnsonii and were found in the high frequency of occurrence of 77.8, 80.6, and 86.1%, respectively. Besides these, Enterococcus faecalis, Bifidobacterium animalis subsp. lactis, Pediococcus acidilactici, and Streptococcus bovis were also isolated in the present study. The sequences of the isolates also showed high levels of similarity to those of the reference strains registered previously in the DDBJ and the similarity was above 97.2%. Their partial 16S rRNA genes were registered in the DDBJ.     

Intra-specific variation of Lactobacillus plantarum and Lactobacillus pentosus in sensitivity towards various bacteriocins

Fifty-two strains belonging to the Lactobacillus plantarum species group were identified and typed. They represented 32 clones of Lactobacillus plantarum and 7 clones of Lactobacillus pentosus. Sensitivity of all strains towards bacteriocins of four different producer strains was investigated using a deferred inhibition test (DIT). Substantial intra-specific variation in sensitivity of clones was observed towards bacteriocinogenic lactic acid bacteria producing nisin ( Lactococcus lactis ATCC 11454) or pediocin PA-1 ( Pediococcus acidilactici PAC-1.0), while none of the strains were sensitive towards the two remaining bacteriocin producers. The minimum inhibitory concentration (MIC) of nisin towards selected strains confirmed the DIT results. No correlation between the susceptibility of fourteen selected strains towards nisin and an array of antibiotics was found. The present study indicates that the variation in bacteriocin-sensitivity within target species might be a potential limitation for the application of bacteriocins as biopreservatives.