Bacteriocin plasmids ofPediococcus acidilactici

Summary Pediococcus acidilactici strains E, F and H isolated from fermented sausages produced bacteriocins which were protein in nature and inhibitory to a variety of spoilage and pathogenic microorganisms often encountered in foods. These strains harbored two to three plasmids ranging in size from 7.4 to 40.2 megadaltons. Curing experiments and plasmid profile analysis indicated the involvement of plasmid DNA with bacteriocin activity in all three strains. Carbohydrate fermentation and antibiotic resistance phenotypes did not appear to be associated with bacteriocin plasmids. Both bacteriocin activity and resistance determinants were linked in strain H and mediated by a 7.4-megadalton plasmid, whereas in strains E and F these two traits were not linked.     

Purification and mass spectrometry based characterization of a pediocin produced by Pediococcus acidilactici 13

Bacteriocins are antimicrobial peptides produced by several bacterial species. Among the bacteriocins pediocin-like bacteriocins have a significant inhibitory activity on the foodborne pathogens especially on Listeria monocytogenes. This study aims to select a simple and usable purification method to purify/concentrate the antimicrobial peptide and characterization of the bacteriocin produced by Pediococcus acidilactici 13 by using proteomic approaches which is a recent omic technology. For purification dialysis, ultrafiltration method was used, and as a result of this study the bacteriocin activity reached 819,200 AU/mL from 102,400 AU/mL initially. Two dimensional gel electrophoresis and then matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) analysis were carried out to identify the current bacteriocin and related proteins. Obtained data revealed similarity to pediocin PA-1 transport/processing ATP-binding protein PedD (accession number: P36497), pediocin operon PedC (accession number: Q68GC4) and bacteriocin pediocin PA-1 (accession number: P29430) from UniProtKB/Swiss-Prot databank, thus the bacteriocin produced by P. acidilactici 13 is considered similar to pediocin PA-1.     

Multilocus Hybridization Typing in Pediococcus acidilactici Strains

In previous a study we demonstrated the presence of several genomic subpopulations within a collection of Pediococcus acidilactici strains isolated from different environments, through a multilocus typing analysis taking into consideration housekeeping conserved loci and protein coding genes of the primary metabolism. In this study, representative strains of five genomic subpopulations previously described (I, II, III, V, VII) were analyzed by restriction analysis of chromosomal DNA and subsequent hybridization assays using as probes amplified fragments obtained from five housekeeping genes (16S rDNA, rpoC, ldhD, ldhL, and metS). A computer similarity and clustering analysis of hybridization data showed the subdivision of P. acidilactici strains in five distinct genotypes according to the grouping previously obtained confirming that pediocin AcH/PA-1 producer strains represent one genomic lineage within the species P. acidilactici. Received: 30 January 2001 / Accepted: 16 May 2001     

rep-PCR fingerprinting and taxonomy based on the sequencing of the 16S rRNA gene of 54 elite commercial rhizobial strains

In tropical soils, diversity and biotechnological potential of symbiotic diazotrophic bacteria are high. However, the phylogenetic relationships of prominent strains are still poorly understood. In addition, in countries such as Brazil, despite the broad use of rhizobial inoculants, molecular methods are rarely used in the analysis of strains or determination of inoculant performance. In this study, both rep-PCR (BOX) fingerprintings and the DNA sequences of the 16S rRNA gene were obtained for 54 rhizobial strains officially authorized for the production of commercial inoculants in Brazil. BOX-PCR has proven to be a reliable fingerprinting tool, reinforcing the suggestion of its applicability to track rhizobial strains in culture collections and for quality control of commercial inoculants. On the other hand, the method is not adequate for grouping or defining species or even genera. Nine strains differed in more than 1.03% (15) nucleotides of the 16S rRNA gene in relation to the closest type strain, strongly indicative of new species. Those strains were distributed across the genera Burkholderia, Rhizobium, and Bradyrhizobium.     

Isolation and Selection of Potential Probiotic Bacteria from the Pig Gastrointestinal Tract

The present study aimed to isolate bacterial strains from the pig gastrointestinal tract that have antagonistic activity against potential pathogens and are able to produce antimicrobial compounds. That ability would be a first requirement for the strains’ possible use as probiotics. Samples obtained from pig intestinal mucosa and contents were screened for the presence of antagonistic activity against pathogenic indicator strains of Escherichia coli, Salmonella, and Listeria by means of the double-layer technique. Samples displaying the largest inhibitory halos were further studied for the production of inhibitory substances using the agar diffusion and microtitration methods. The three most promising isolates were identified by sequencing of the 16S rRNA gene and showed highest affiliation to Lactobacillus salivarius. Optimal growth conditions and bacteriocin production were recorded in de Man, Rogosa, and Sharpe broth under anaerobic conditions at 37 °C. The antimicrobial substances were found to be sensitive to proteolytic enzymes but showed good stability at pH values below 6. Our findings suggest that these three intestinal strains are able to produce antimicrobial substances capable of inhibiting the growth of potential enteric pathogens and might have potential as probiotic feed additives for the prevention of gastrointestinal diseases.     

Development and application of a multiple typing system for Clostridium difficile

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Development and application of a multiple typing system for Clostridium difficile.

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Isolation and characterization of bacteriocin‐producing bacteria from the gastrointestinal tract of broiler chickens for probiotic use

Aims: To isolate and characterize the bacteriocin‐producing bacteria (BPB) from the gastrointestinal tract of broiler chickens for probiotic use. Methods and Results: In total, 291 bacterial strains were isolated from broilers and screened for bacteriocin‐producing ability. The bacteriocins produced by Enterococcus faecium SH 528, Ent. faecium SH 632 and Pediococcus pentosaceus SH 740 displayed inhibitory activity against pathogens including Clostridium perfringens and Listeria monocytogenes. Activity of the bacteriocins remained unchanged after 30 min of heat treatment at 60°C or exposure to organic solvents, but diminished after treatment with proteolytic enzymes. PCR was used to detect the structural genes enterocin A and B in SH 528, enterocin L50 and P in SH 632, and pediocin PA‐1 in SH 740. Most of them were resistant to 0·5% bile salts and remained viable after 2 h at pH 3·0. Ent. faecium SH 528 exhibited the highest amylase activity among the strains tested. Conclusions: We selected Ent. faecium SH 528 and SH 632 and Ped. pentosaceus SH 740 by probiotic selection criteria including inhibition activity against pathogens. Significance and Impact of the Study: The isolated BPB could potentially be used in the poultry industry as probiotics to control pathogens.     

Isolation and Characterization of Pediocin NV 5 Producing <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> LAB 5 from Vacuum-Packed Fermented Meat Product

A potentially novel antimicrobial compound producing Pediococcus acidilactici LAB 5 was isolated from vacuum-packed fermented meat product. This compound was found active against some species of Enterococcus, Leuconostoc, Staphylococcus and Listeria, many of which are associated with food spoilage and food related health hazards. The strain was found to be a paired cocci which can utilize a broad range of carbohydrates and produce acid identical to the P. acidilactici and P. pentoseus. Since the antimicrobial agent was sensitive to proteolytic enzymes but quite resistant to heat, it was identified as a bacteriocin and was designated as Pediocin NV 5. The molecular weight of the bacteriocin was 10.3 kDa and the bacterium possessed a 5 kbp plasmid responsible for bacteriocin production and also for vancomycin resistance phenotype.     

Purification and characterization of enterocin FH 99 produced by a faecal isolate <Emphasis Type="Italic">Enterococcus faecium</Emphasis> FH 99

Enterococcus faecium FH 99 was isolated from human faeces and selected because of its broad spectrum of inhibitory activity against several Gram-positive foodborne spoilage and pathogenic bacteria. Ent. faecium FH 99 accumulates enterocin in large number in early stationary phase of the growth. The enterocin FH 99 was stable over a wide pH range (2–10) and recovered activity even after treatment at high temperatures (10 min at 100°C). The enterocin was subjected to different purification techniques viz., gel filteration, cation exchange chromatography and reverse-phase high-performance liquid chromatography. The activity was eluted as one individual active fraction. SDSPAGE revealed a molecular weight of less than 6.5 kDa. Studies carried out to identify the genetic determinants for bacteriocin production showed that this trait may be plasmid encoded as loss in both of the plasmids (size>chromosomal DNA) led to loss in bacteriocin production by Ent. faecium FH 99. Ent. faecium strain FH 99 is a newly discovered high bacteriocin producer with Activity Units 1.8 × 10⁵ AU ml⁻¹ and its characteristics indicate that it may have strong potential for application as a protective agent against pathogens and spoilage bacteria in foods.     

Effect of prebiotics on bacteriocin production and cholesterol lowering activity of <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> LAB 5

The in vitro influence of three prebiotics viz. mannitol, maltodextrin and sorbitol was evaluated on probiotic aspects like bile salt tolerance, cholesterol lowering efficiency and bacteriocin production of the strain, Pediococcus acidilactici LAB 5 which was isolated from vacuum packed fermented meat product. Optimum temperature for bacteriocin production in MRS medium was 37°C. The strain deconjugated bile salt (sodium taurocholate) to 607.66 ± 10.894 μg/ml from initial bile salt concentration 3 mg/ml. In vitro cholesterol removal capability of the strain P. acidilactici LAB 5 was 62 ± 2.742 μg/ml. Prebiotic sorbitol had a positive influence on the probiotic parameters like better cell growth, bacteriocin production and cholesterol removal capability. Anaerobic condition had influenced largely on bile salt deconjugation, cholesterol removal and bacteriocin production. Synbiotic treatment of P. acidilactici LAB 5 with sorbitol for 1 month lowered the plasma cholesterol level to 176.34 ± 12.66 μg/ml in comparison to untreated one (208.76 ± 20.27 μg/ml) in Swiss albino mice. Intestinal adherence of P. acidilactici LAB 5 was also more in synbiotic condition than only in probiotic and control treatments.     

Characterization and application of a native lactic acid bacterium isolated from tannery fleshings for fermentative bioconversion of tannery fleshings

Lactic acid bacteria (LAB) species isolated from limed and delimed tannery fleshings (TF) were evaluated for their fermentation efficiency and antibacterial property. The native LAB isolates efficiently fermented TF and resulted in a fermented mass with antioxidant properties, indicating their potential for effective eco-friendly bioconversion of TF. From among the LAB isolated, a proteolytic isolate showing better antimicrobial spectrum and reasonably good fermentation efficiency was identified as Enterococcus faecium HAB01 based on various biochemical and molecular tests. This isolate afforded a better degree of hydrolysis (81.36%) of TF than Pediococcus acidilactici (54.64%) that was previously reported by us. The bacteriocin produced by E. faecium was found to be antagonistic to several human pathogens including Listeria, Aeromonas, Staphylococcus and Salmonella. Further, E. faecium HAB01 bacteriocin was thermostable and had a molecular weight of around 5 kDa, apart from being stable at both acidic and alkaline conditions. The bacteriocin was unstable against proteases.     

Thermophilin 110: A Bacteriocin of Streptococcus thermophilus ST110

A screen of thermophilic lactic acid bacteria identified Streptococcus thermophilus strain ST110 as the putative producer of a bacteriocin with high level of activity against pediococci. Thermophilin 110 was isolated from culture supernatant after 16 h of growth and partially purified by a chloroform extraction procedure. The bacteriocin inhibited the growth of several lactic acid bacteria and in the case of Pediococcus acidilactici, it induced cell lysis with the concomitant release of OD₂₆₀ - absorbing material and intracellular enzymes. SDS-PAGE analysis revealed two components with estimated sizes between 4.0 kDa and 4.5 kDa, respectively, with possible involvement in bacteriocin activity as indicated by agar overlay assays with P. acidilactici as the target organism. Thermophilin 110 was inactivated by several proteolytic enzymes and also by α-amylase, which indicated the putative requirement of a glycosidic component for activity. The bacteriocin produced by S. thermophilus may be especially useful in the food processing industries to control spoilage caused by pediococci.     

Improved antimicrobial activity of Pediococcus acidilactici against Salmonella Gallinarum by UV mutagenesis and genome shuffling

Pediococcus acidilactici is a widely used probiotic, and Salmonella enterica serovar Gallinarum (SG) is a significant pathogen in the poultry industry. In this study, we improved the antimicrobial activity of P. acidilactici against SG using UV mutation and genome shuffling (GS). To improve antimicrobial activity against SG, UV mutagenesis was performed against wild-type P. acidilactici (WT), and five mutants showed improved antimicrobial activity. To further improve antimicrobial activity, GS was performed on five UV mutants. Following GS, four mutants showed improved antimicrobial activity compared with the UV mutants and WT. The antimicrobial activity of GS1 was highest among the mutants; however, the activity was reduced when the culture supernatant was treated with proteinase K, suggesting that the improved antimicrobial activity is due to a proteinous substance such as bacteriocin. To validate the activity of GS1 in vivo, we designed multi-species probiotics and performed broiler feeding experiments. Groups consisted of no treatment (NC), avilamycin-treated (PC), probiotic group 1 containing WT (T1), and probiotic group 2 containing GS1 (T2). In broiler feeding experiments, coliform bacteria were significantly reduced in T2 compared with NC, PC, and T1. The cecal microbiota was modulated and pathogenic bacteria were reduced by GS1 oral administration. In this study, GS1 showed improved antimicrobial activity against SG in vitro and reduced pathogenic bacteria in a broiler feeding experiment. These results suggest that GS1 can serve as an efficient probiotic, as an alternative to antibiotics in the poultry industry.

     

稻瘟病菌拮抗性大豆根瘤内生细菌的筛选及抑制效果

【目的】从河南大豆根瘤的内生细菌资源中筛选对稻瘟病菌有拮抗作用的菌株,初步探讨其抑菌效果,为进一步研究其抑菌机理提供菌种资源。【方法】以稻瘟病菌为供试病原菌,采用对峙法进行拮抗性菌株筛选,显微观察法研究受抑制病原菌菌丝变化,对筛选拮抗性菌株进行细胞形态学及生理生化特性试验、16S rRNA基因测序和系统发育分析及接种防效试验。【结果】经复筛有17株内生菌拮抗效果较明显,最高抑制率为62.16%;受抑制病原菌丝呈现弯曲打结、断裂、原生质浓缩等畸形状态。拮抗性筛选过程中内生菌快速生长形成生物薄膜,包埋菌丝并使其断裂。拮抗菌株分布在7属9种,稻瘟病拮抗性大豆根瘤内生菌呈现种属多样性。防效试验表明内生菌处理组稻苗发病率和病情指数均显著降低,防治效果最高达74.19%。【结论】大豆根瘤内生拮抗性菌株具有种属多样性,拮抗性菌株处理组稻苗发病率和病情指数均显著降低,防治效果显著,为进一步研究其抑菌机理提供菌种资源。     

Rapid discrimination and classification of the <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> group based on a partial <Emphasis Type="Italic">dnaK</Emphasis> sequence and DNA fingerprinting techniques

The Lactobacillus plantarum group comprises five very closely related species. Some species of this group are considered to be probiotic and widely applied in the food industry. In this study, we compared the use of two different molecular markers, the 16S rRNA and dnaK gene, for discriminating phylogenetic relationships amongst L. plantarum strains using sequencing and DNA fingerprinting. The average sequence similarity for the dnaK gene (89.2%) among five type strains was significantly less than that for the 16S rRNA (99.4%). This result demonstrates that the dnaK gene sequence provided higher resolution than the 16S rRNA and suggests that the dnaK could be used as an additional phylogenetic marker for L. plantarum. Species-specific profiles of the Lactobacillus strains were obtained with RAPD and RFLP methods. Our data indicate that phylogenetic relationships between these strains are easily resolved using sequencing of the dnaK gene or DNA fingerprinting assays.     

贵州兴义喀斯特洞穴土可培养细菌多样性及其产蛋白酶、淀粉酶活性筛选

【目的】进一步了解贵州喀斯特洞穴土可培养细菌的物种多样性组成及其产蛋白酶、淀粉酶生物活性能力。【方法】选取11种分离培养基,利用稀释直接涂布平板法对贵州黔西南兴义市喀斯特地区白碗窑镇魔家大溶洞洞内土壤进行可培养细菌分离;利用两种鉴定培养基对相关细菌进行生物活性判定。【结果】根据16S rRNA基因序列的系统进化分析,将分离得到的217株细菌分别归类到24个属的63个不同种类,其中红球菌属(Rhodococcus)和链霉菌属(Streptomyces)为该洞内土壤可培养细菌的优势菌群,分别占24.42%和21.66%。大多数菌株与已知典型菌株的16S rRNA基因序列相似性为97.90%-99.99%,其中至少有4株菌株(D3T01、D911、D961和D502)为潜在的新分类单元。对217株细菌进行蛋白酶和淀粉酶活性筛选,其中具有蛋白酶或淀粉酶活性的99株,占分离菌株的45.62%,分别属于18个属的38个不同种;同时具有蛋白酶和淀粉酶活性的36株,占具有酶活性菌株的36.36%,占分离菌株的16.59%。【结论】贵州兴义喀斯特洞穴土中存在丰富多样的细菌类群,且蕴藏着一定数量的潜在新物种资源;此外功能酶菌株在喀斯特洞穴土壤中大量存在,为工业应用奠定了资源基础,极具进一步发掘和研究的价值。     

Culturable Actinobacteria from the Marine Sponge Hymeniacidon perleve: Isolation and Phylogenetic Diversity by 16S rRNA gene-RFLP Analysis

A total of 106 actinobacteria associated with the marine sponge Hymeniacidon perleve collected from the Yellow Sea, China were isolated using eight different media. The number of species and genera of actinobacteria recovered from the different media varied significantly, underlining the importance of optimizing the isolation conditions. The phylogenetic diversity of the actinobacteria isolates was assessed using 16S rRNA gene amplification–restriction fragment length polymorphism (RFLP) analysis of the 106 strains with different morphologies. The RFLP fingerprinting of selected strains by HhaI-digestion of the 16S rRNA genes resulted in 11 different patterns. The HhaI-RFLP analysis gave good resolution for the identification of the actinobacteria isolates at the genus level. A phylogenetic analysis using 16S rRNA gene sequences revealed that the isolates belonged to seven genera of culturable actinobacteria including Actinoalloteichus, Micromonospora, Nocardia, Nocardiopsis, Pseudonocardia, Rhodococcus, and Streptomyces. The dominant genus was Streptomyces, which represented 74% of the isolates. Three of the strains identified are candidates for new species.     

马尾松毛虫肠道产细菌素细菌的筛选及抑菌特性

[背景]细菌素是一类由细菌产生的抗菌肽活性物质,对多种致病菌有抑制和杀灭作用.昆虫是世界上种类最多、肠道菌群资源最为丰富且多样的动物类群之一.昆虫肠道很可能蕴含着丰富的产细菌素菌种资源.[目的]以马尾松毛虫(Dendrolimuspunctatu)幼虫为研究材料,对其肠道细菌进行分离、培养和鉴定,获得对典型致病菌有明显...     

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0–12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain’s genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.