Isolation and Characterization of a Bacteriocin-Like Substance Produced by <Emphasis Type=Italic>Geobacillus toebii</Emphasis> Strain HBB-247

A total of 201 thermophilic bacteria isolated from various thermal spring, mud and soil were tested for their antibacterial activity. Among the mostly active isolates, Geobacillus toebii HBB-247 was further examined. Bacteriocin-like inhibitory substance (BLIS) produced by strain HBB-247 was found to be stable up to 60°C, sensitive to proteolytic enzymes and effective against Enterococcus faecalis, Listeria sp., E. avium, Clostridium pasteurianum, Cellulomonas fimi and some thermophilic strains isolated and identified in this study. As a result of Tricine-SDS-PAGE molecular weight of BLIS was estimated about 38 kDa. Production studies showed that G. toebii HBB-247 starts to produce antibacterial substance at early logarithmic phase of growth and maximum production was detected at the end of the logarithmic phase.     

筛选产类细菌素乳酸菌及类细菌素特性研究

从健康鸡肠道内容物及新鲜粪便中分离到21株乳酸菌,通过单层琼脂平板扩散实验,筛选出1株对藤黄微球菌(Micrococcus luteus)、金黄色葡萄球菌(Staphylococcus aureus)和枯草杆菌(Bacillus subtilis)等革兰氏阳性菌和大肠杆菌(Escherichia coli)等革兰氏阴性菌有明显抑菌活性代谢产物的乳酸菌,经细菌鉴定为乳杆菌属。排除酸和过氧化氢的干扰后,发酵液上清对指示菌仍有抑菌活性;用胰蛋白酶和蛋白酶K处理后抑菌活性明显降低,而胃蛋白酶对其活性无影响;用过氧化氢酶作用上清液,抑菌效果不变,说明过氧化氢未起作用;pH在2.0~7.0时,发酵上清液有抑菌活性;培养液粗提物经120℃处理20 min仍有部分活性,表明培养上清中有蛋白质类细菌素。     

Characterization of a bacteriocin-like substance produced by a vaginal Lactobacillus salivarius strain

A novel bacteriocin-like substance produced by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328 with activity against Enterococcus faecalis, Enterococcus faecium, and Neisseria gonorrhoeae was characterized. The highest level of production of this heat-resistant peptide or protein occurred during the late exponential phase. Its mode of action was shown to be bactericidal. L. salivarius subsp. salivarius CRL 1328 could be used for the design of a probiotic to prevent urogenital infections.     

Characterization of d-amino acid aminotransferase from Lactobacillus salivarius

We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K_m values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.     

Characterization of endogenous plasmids from Lactobacillus salivarius UCC118

The genome of Lactobacillus salivarius UCC118 comprises a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and two smaller plasmids of 20 kb (pSF118-20) and 44 kb (pSF118-44). Annotation and bioinformatic analyses suggest that both of the smaller plasmids replicate by a theta replication mechanism. Furthermore, it appears that they are transmissible, although neither possesses a complete set of conjugation genes. Plasmid pSF118-20 encodes a toxin-antitoxin system composed of pemI and pemK homologs, and this plasmid could be cured when PemI was produced in trans. The minimal replicon of pSF118-20 was determined by deletion analysis. Shuttle vector derivatives of pSF118-20 were generated that included the replication region (pLS203) and the replication region plus mobilization genes (pLS208). The plasmid pLS203 was stably maintained without selection in Lactobacillus plantarum, Lactobacillus fermentum, and the pSF118-20-cured derivative strain of L. salivarius UCC118 (strain LS201). Cloning in pLS203 of genes encoding luciferase and green fluorescent protein, and expression from a constitutive L. salivarius promoter, demonstrated the utility of this vector for the expression of heterologous genes in Lactobacillus. This study thus expands the knowledge base and vector repertoire of probiotic lactobacilli.     

Salivaricin P, One of a Family of Two-Component Antilisterial Bacteriocins Produced by Intestinal Isolates of Lactobacillus salivarius

Lactobacillus salivarius DPC6005, a porcine intestinal isolate, produces a two-component bacteriocin, salivaricin P, with homology to ABP-118 produced by a human probiotic L. salivarius strain. Indeed, molecular characterization revealed that while the peptides Sln1 and ABP-118α are identical, their companion peptides (Sln2 and ABP-118β, respectively) differ by two amino acids. This observation suggests that two-component bacteriocins may be a common feature of intestinal L. salivarius strains.     

Inhibition of Vancomycin and High-Level Aminoglycoside-Resistant Enterococci Strains and Listeria monocytogenes by Bacteriocin-Like Substance Produced by Enterococcus faecium E86

Three hundred and thirty nine lactic bacteria strains isolated from food samples were screened for antimicrobial activity. Only one strain isolated from meat pie and identified as Enterococcus faecium produced a bacteriocin-like inhibitory substance (BLIS) showing activity against Enterococcus, Leuconostoc, Lactobacillus, Listeria, Corynebacterium and Staphylococcus aureus. The BLIS produced was resistant to acid and alkali treatment and 121oC for 15 min. The addition of BLIS in BHI contaminated with Listeria monocytogenes decreased the contamination in 4.8 log cycles in 24 h. The inhibition of listeria was also obtained in milk. Forty multiresistant enterococci strains were inhibited in the well-diffusion test. Two vancomycin resistant strains tested in liquid with BLIS were also inhibited. The BLIS producer showed no pathogenicity marker.     

Characterization of Reutericyclin Produced by Lactobacillus reuteri LTH2584

Lactobacillus reuteri LTH2584 exhibits antimicrobial activity that can be attributed neither to bacteriocins nor to the production of reuterin or organic acids. We have purified the active compound, named reutericyclin, to homogeneity and characterized its antimicrobial activity. Reutericyclin exhibited a broad inhibitory spectrum including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, Staphylococcus aureus, and Listeria innocua. It did not affect the growth of gram-negative bacteria; however, the growth of lipopolysaccharide mutant strains of Escherichia coli was inhibited. Reutericyclin exhibited a bactericidal mode of action against Lactobacillus sanfranciscensis, Staphylococcus aureus, and B. subtilis and triggered the lysis of cells of L. sanfranciscensis in a dose-dependent manner. Germination of spores of B. subtilis was inhibited, but the spores remained unaffected under conditions that do not permit germination. The fatty acid supply of the growth media had a strong effect on reutericyclin production and its distribution between producer cells and the culture supernatant. Reutericyclin was purified from cell extracts and culture supernatant of L. reuteri LTH2584 cultures grown in mMRS by solvent extraction, gel filtration, RP-C₈ chromatography, and anion-exchange chromatography, followed by rechromatography by reversed-phase high-pressure liquid chromatography. Reutericyclin was characterized as a negatively charged, highly hydrophobic molecule with a molecular mass of 349 Da. Structural characterization (A. Höltzel, M. G. Gänzle, G. J. Nicholson, W. P. Hammes, and G. Jung, Angew. Chem. Int. Ed. 39:2766–2768, 2000) revealed that reutericyclin is a novel tetramic acid derivative. The inhibitory activity of culture supernatant of L. reuteri LTH2584 corresponded to that of purified as well as synthetic reutericyclin.     

拮抗细菌B9菌株抗菌物质的初步研究

B9菌株是从番茄灰霉病发生严重植株的健康叶片上分离到的拮抗细菌。采用酸沉淀的方法从B9菌株的发酵液中得到抗菌物质的粗提物。该物质在中性和碱性条件下都能溶解于水和有机溶剂,在270~280 nm处有吸收峰,其成分可能是蛋白质。平板抑菌试验表明抗菌物质抑菌谱广,对供试的6种病原菌都有不同程度的抑菌作用。稳定性试验结果表明抗菌物质对温度比较稳定,经80~100℃处理10 min或者121℃(1.4 Pa/cm2)高温抑菌活性才显著降低;对紫外光稳定性差,随着紫外照射时间的延长抑菌活性逐渐减弱。     

野生Bc菌株产黑色素的研究

Bc58是一株野生蜡状芽抱杆菌菌株,经L-酪氨酸诱导后可产生红棕色色素。通过红外光谱及各种化学测定证明该色素与Sigma公司标准黑色素（Melanin）的性质相似。生测结果显示添加Bc58黑色素的Bt制剂经紫外照射5h后的LC50为16．1μg／mL,与未经紫外照射的Bt制剂的LC50 15．2μg／mL基本相同,而比未添加黑色素的Bt制剂经紫外照射后的杀虫毒力高出近1倍。经SDS—PAGE检测表明该黑色素可保护苏云金杆菌晶体蛋白在紫外光下基本不降解,表明Bc58黑色素是一种优良的紫外保护剂。     

Bacteriocin-Like Substances Produced by Rhizobium japonicum and Other Slow-Growing Rhizobia

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80°C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.     

Characterization of a new Lactobacillus salivarius strain engineered to express IBV multi-epitope antigens by chromosomal integration

To obtain adhesive and safe lactic acid bacteria (LAB) strains for expressing heterologous antigens, we screened LAB inhabitants in intestine of Tibetan chickens by analyzing their adhesion and safety properties and the selected LAB was engineered to express heterologous antigen (UTEpi C-A) based on chromosomal integration strategy. We demonstrated that a new Lactobacillu salivarius TCMM17 strain is strongly adhesive to chicken intestinal epithelial cells, contains no endogenous plasmids, is susceptible to tested antimicrobials, and shows no toxicities. In order to examine the potential of TCMM17 strain as heterogenous antigen delivering vehicle, we introduced a UTEpi C-A expression cassette in its chromosome by constructing a non-replicative plasmid (pORI280-UUTEpi C-AD). The recombinant TCMM17 strain (?TCMM17) stably was found to keep the gene cassette through 50 generations, and successfully displayed EpiC encoded by the cassette on its surface. This work provides a universal platform for development of novel oral vaccines and expression of further antigens of avian pathogens.     

Optimization of Low-Cost Culture Media for the Production of Biomass and Bacteriocin by a Urogenital Lactobacillus salivarius Strain

The aim of this work was to formulate a culture medium of lower cost than conventional laboratory media, in order to simultaneously obtain high amounts of both biomass and bacteriocin of vaginal Lactobacillus salivarius CRL 1328. The growth assays under different culture conditions were performed by using a 2^8?2 central composite experimental design, with a central point and sixteen additional points. The factors taken into consideration were glucose, lactose, yeast extract, tryptone, ammonium citrate, sodium acetate, MgSO₄ and MnSO₄. The simultaneous presence of a carbon source (mainly glucose), a nitrogen source (mainly yeast extract) and salts (mainly MnSO₄, MgSO₄ and sodium acetate) allowed the highest cell biomass and bacteriocin levels to be reached in the experimental design. Through the application of the desirability function, several optimal medium compositions to achieve efficient production of biomass and bacteriocin were predicted. The optimized growth media allow a cost reduction of around 25 to 40% compared with conventional broths. The results obtained represent an advance in the search of the most suitable strategies for the production of bioactive compounds for pharmaceutical products to prevent or treat female urogenital infections.     

Streptococcal Bacteriocin-Like Inhibitory Substances: Some Personal Insights into the Bacteriocin-Like Activities Produced by Streptococci Good and Bad

The background to the discovery and commercial development of the first Streptococcus salivarius probiotic is documented. A 40-year search of the genus Streptococcus for a harmless natural antagonist of Streptococcus pyogenes had as its operational basis a simple deferred antagonism “fingerprinting” procedure, the application of which results in each tested strain being accorded an inhibitor production (P)-type and inhibitor sensitivity (S)-type profile. Systematic application of this schema has opened a “Pandora’s Box” of novel streptococcal bacteriocin-like inhibitory substances (BLIS). The numerically prominent commensal S. salivarius is proposed to have a pivotal population-modulating role within the oral microbiota of humans. The probiotic strain S. salivarius K12 produces several megaplasmid-encoded BLIS including the lantibiotics salivaricin A and salivaricin B. Strain K12 and other BLIS-producing S. salivarius are currently in use or under development for application to the control of a variety of common maladies and infections of the human oral cavity.     

Lactocin 160, a Bacteriocin Produced by Vaginal Lactobacillus rhamnosus, Targets Cytoplasmic Membranes of the Vaginal Pathogen, Gardnerella vaginalis

Bacterial vaginosis (BV) is a commonly occurring vaginal infection that is associated with a variety of serious risks related to the reproductive health of women. Conventional antibiotic treatment for this condition is frequently ineffective because the antibiotics tend to inhibit healthy vaginal microflora along with the pathogens. Lactocin 160, a bacteriocin produced by healthy vaginal lactobacilli, is a promising alternative to antibiotics; this compound specifically inhibits the BV-associated vaginal pathogens such as Gardnerella vaginalis and Prevotella bivia without affecting the healthy microflora. This study investigates the molecular mechanism of action for lactocin 160 and reveals that this compound targets the cytoplasmic membrane of G. vaginalis, causing the efflux of ATP molecules and dissipation of the proton motive force.     

一株布氏乳杆菌所产类细菌素的初步纯化与部分特性

从分离自内蒙古传统乳制品的67株乳酸菌中筛选得到一株产生类细菌素的布氏乳杆菌KLDS1.0364, 对其所产类细菌素进行初步分离纯化, 同时研究其所产类细菌素的生物学特性。KLDS1.0364无细胞发酵上清液经阳离子交换树脂纯化后, 采用tricine-SDS-PAGE测定类细菌素分子量, 并测定了类细菌素的部分特性。KLDS1.0364产生的类细菌素分子量约为21.6kD, 对热和pH值稳定, 可被多种蛋白酶失活, 不能被过氧化氢酶和α-淀粉酶失活。KLDS1.0364产生的类细菌素的作用方式是杀菌, 且抑菌谱广, 可抑制多种革兰氏阳性菌、革兰氏阴性菌和真菌。     

一株布氏乳杆菌所产类细菌素的初步纯化与部分特性

从分离自内蒙古传统乳制品的67株乳酸菌中筛选得到一株产生类细菌素的布氏乳杆菌KLDS1.0364,对其所产类细菌素进行初步分离纯化,同时研究其所产类细菌素的生物学特性.KLDS1.0364无细胞发酵上清液经阳离子交换树脂纯化后,采用tricine-SDS-PAGE测定类细菌素分子量,并测定了类细菌素的部分特性.KLDS1.0364产生的类细菌素分子量约为21.6kD,对热和pH值稳定,可被多种蛋白酶失活,不能被过氧化氢酶和α-淀粉酶失活.KLDS1.0364产生的类细菌素的作用方式是杀菌,且抑菌谱广,可抑制多种革兰氏阳性菌、革兰氏阴性菌和真菌.     

A Novel Bacteriocin-Like Substance Produced by <Emphasis Type="Italic">Enterococcus faecium</Emphasis> 3587

Secretion of a novel bacteriocin-like inhibitory substance from Enterococcus faecium 3587 is described herein for the first time. Whereas some bacteriocins receive their denomination based on the species or genus name of the producer microorganism, the newly discovered bacteriocin-like substance was named “enterocin 3587.” The growth characteristics of the producer strain, as well as the type of production and the primary characteristic of the peptide, were investigated. It was found by sodium dodecyl sulfate–polyacrylamide gel electrophoresis that the molecule possesses a molecular weight <6.5 kDa; its secretion is growth-phase dependent; and it shows activities only against other closely related enterococci but not against other Gram-positive bacteria, such as L. innocua, S. aureus 209, and Lactobacillus delbrueckii B2, nor against some Gram-negative species, such as Escherichia coli HB101.     

Characteristics and Effects of Temperature and Surfactants on Bacteriocin-Like Inhibitory Substance Production of Soil-Isolated Lactobacillus animalis C060203

A soil isolate of Lactobacillus animalis was found to produce a bacteriocin-like inhibitory substance (BLIS) with a wide inhibitory spectrum against Gram-positive bacteria. The isolate exhibited high BLIS production in broth containing surfactants, such as Tween 20 and 80, but low BLIS production in the absence of surfactants. Culture temperature also had an effect on BLIS production. This is the first report to study the production and characteristics of L. animalis BLIS.     

Genomic diversity of Lactobacillus salivarius

Strains of Lactobacillus salivarius are increasingly employed as probiotic agents for humans or animals. Despite the diversity of environmental sources from which they have been isolated, the genomic diversity of L. salivarius has been poorly characterized, and the implications of this diversity for strain selection have not been examined. To tackle this, we applied comparative genomic hybridization (CGH) and multilocus sequence typing (MLST) to 33 strains derived from humans, animals, or food. The CGH, based on total genome content, including small plasmids, identified 18 major regions of genomic variation, or hot spots for variation. Three major divisions were thus identified, with only a subset of the human isolates constituting an ecologically discernible group. Omission of the small plasmids from the CGH or analysis by MLST provided broadly concordant fine divisions and separated human-derived and animal-derived strains more clearly. The two gene clusters for exopolysaccharide (EPS) biosynthesis corresponded to regions of significant genomic diversity. The CGH-based groupings of these regions did not correlate with levels of production of bound or released EPS. Furthermore, EPS production was significantly modulated by available carbohydrate. In addition to proving difficult to predict from the gene content, EPS production levels correlated inversely with production of biofilms, a trait considered desirable in probiotic commensals. L. salivarius displays a high level of genomic diversity, and while selection of L. salivarius strains for probiotic use can be informed by CGH or MLST, it also requires pragmatic experimental validation of desired phenotypic traits.