Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius

Streptococcus salivarius strains commonly produce bacteriocins as putative anticompetitor or signalling molecules. Here we report that bacteriocin production by the oral probiotic strain S. salivarius K12 is encoded by a large (ca. 190 kb) plasmid. Oral cavity transmission of the plasmid from strain K12 to a plasmid-negative variant of this bacterium was demonstrated in two subjects. Tests of additional S. salivarius strains showed large (up to ca. 220 kb) plasmids present in bacteriocin-producing isolates. Various combinations (up to 3 per plasmid) of loci encoding the known streptococcal lantibiotics salivaricin A, salivaricin B, streptin and SA-FF22 were localised to these plasmids. Since all bacteriocin-producing strains of S. salivarius tested to date appear to harbour plasmids, it appears that they may function as mobile repositories for bacteriocin loci, especially those of the lantibiotic class.     

Bacteriocin Production: a Probiotic Trait?

Bacteriocins are an abundant and diverse group of ribosomally synthesized antimicrobial peptides produced by bacteria and archaea. Traditionally, bacteriocin production has been considered an important trait in the selection of probiotic strains, but until recently, few studies have definitively demonstrated the impact of bacteriocin production on the ability of a strain to compete within complex microbial communities and/or positively influence the health of the host. Although research in this area is still in its infancy, there is intriguing evidence to suggest that bacteriocins may function in a number of ways within the gastrointestinal tract. Bacteriocins may facilitate the introduction of a producer into an established niche, directly inhibit the invasion of competing strains or pathogens, or modulate the composition of the microbiota and influence the host immune system. Here we review the role of bacteriocin production in complex microbial communities and their potential to enhance human health.     

Antimicrobial Activity,Inhibition of Urogenital Pathogens,and Synergistic Interactions Between <Emphasis Type="Italic">Lactobacillus</Emphasis> 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.     

An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley

AIMS: The aim of this study was to perform a detailed characterization of bacteriocins produced by lactic acid bacteria (LAB) isolated from malted barley. METHODS AND RESULTS: Bacteriocin activities produced by eight LAB, isolated from various types of malted barley, were purified to homogeneity by ammonium sulphate precipitation, cation exchange, hydrophobic interaction and reverse-phase liquid chromatography. Molecular mass analysis and N-terminal amino acid sequencing of the purified bacteriocins showed that four non-identical Lactobacillus sakei strains produced sakacin P, while four Leuconostoc mesenteroides strains were shown to produce bacteriocins highly similar or identical to leucocin A, leucocin C or mesenterocin Y105. Two of these bacteriocin-producing strains, Lb. sakei 5 and Leuc. mesenteroides 6, were shown to produce more than one bacteriocin. Lactobacillus sakei 5 produced sakacin P as well as two novel bacteriocins, which were termed sakacin 5X and sakacin 5T. The inhibitory spectrum of each purified bacteriocin was analysed and demonstrated that sakacin 5X was capable of inhibiting the widest range of beer spoilage organisms. CONCLUSION: All bacteriocins purified in this study were class II bacteriocins. Two of the bacteriocins have not been described previously in the literature while the remaining purified bacteriocins have been isolated from environments other than malted barley. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a thorough analysis of bacteriocin-producing LAB from malt and demonstrates, for the first time, the variety of previously identified and novel inhibitory peptides produced by isolates from this environment. It also highlights the potential of these LAB cultures to be used as biological controlling agents in the brewing industry.     

Production of multiple bacteriocins from a single locus by gastrointestinal strains of Lactobacillus salivarius

Bacteriocins produced by Lactobacillus salivarius isolates derived from a gastrointestinal origin have previously demonstrated efficacy for in vivo protection against Listeria monocytogenes infection. In this study, comparative genomic analysis was employed to investigate the intraspecies diversity of seven L. salivarius isolates of human and porcine intestinal origin, based on the genome of the well-characterized bacteriocin-producing strain L. salivarius UCC118. This revealed a highly conserved megaplasmid-borne gene cluster in these strains involved in the regulation and secretion of two-component class IIb bacteriocins. However, considerable intraspecific variation was observed in the structural genes encoding the bacteriocin peptides. They ranged from close relatives of abp118, such as salivaricin P, which differs by 2 amino acids, to completely novel bacteriocins, such as salivaricin T, which is characterized in this study. Salivaricin T inhibits closely related lactobacilli and bears little homology to previously characterized salivaricins. Interestingly, the two peptides responsible for salivaricin T activity, SalTα and SalTβ, share considerable identity with the component peptides of thermophilin 13, a bacteriocin produced by Streptococcus thermophilus. Furthermore, the salivaricin locus of strain DPC6488 also encodes an additional novel one-component class IId anti-listerial bacteriocin, salivaricin L. These findings suggest a high level of redundancy in the bacteriocins that can be produced by intestinal L. salivarius isolates using the same enzymatic production and export machinery. Such diversity may contribute to their ability to dominate and compete within the complex microbiota of the mammalian gut.     

The role of ‘soaking’ in spiteful toxin production in Pseudomonas aeruginosa

The ubiquitous production of antibacterial toxins, such as bacteriocins, is an ecologically significant class of interbacterial interactions that have primarily evolved through their indirect fitness benefits to the producer. Bacteria release bacteriocins into the environment at a cost to individual cell, but individual bacteriocin-producing cells are unlikely to gain any direct benefit from their own toxin; indeed, cell lysis is required in many species. There is a growing body of research describing the ecological conditions that can favour the evolution of bacteriocin production. However, an important aspect of many bacteriocins has yet to be investigated: the ability of bacteriocin-producing cells to neutralize toxin (‘soaking’) produced by other clonemates. By competing Pseudomonas aeruginosa bacteriocin-producing wild-type and ‘non-soaking’ strains against a bacteriocin-susceptible strain, we find that soaking markedly reduces the fitness of a bacteriocin-producing strain at both high and low frequencies.     

Selection and fitness in bacteriocin-producing bacteria.

Bacteriocins are proteinaceous anticompetitor molecules produced by bacteria against closely related species. A number of theoretical models have been used to explain experimental data that indicate high polymorphisms among bacteriocins and a frequency-dependent nature of selection for bacteriocin-producing strains. The majority of these experimental data were, however, obtained from investigations into the colicin group of bacteriocins produced by Gram-negative bacteria. The conclusions drawn from these models have been extrapolated to other bacteriocins and allelopathic compounds in general. Examination of more recent experimental investigations into the bacteriocins of Gram-positive bacteria indicate a lower degree of polymorphism and a less frequency dependent mode of selection among these strains them among the colicin-producing strains. Here we examine these contradictions in the light of the assumptions and conclusions of the theoretical models and reported data. We show that fitness costs as indicated by decreased relative maximum growth rate associated with bacteriocin production may be much lower in many cases than is assumed in the present models. A lower fitness cost associated with bacteriocin production adequately explains the newer data from Gram-positive bacteria cited here, and indicates that extrapolation of existing models to all bacteriocins and other allelopathic compounds is not appropriate.     

Group H Streptococcal Bacteriocins Having No Relation to Bacterial Transformation

The culture filtrate of group H streptococcus strain Challis produced a competence factor (CF) for bacterial transformation as well as a bactericidal factor(s) against Wicky cells. Strain 36658, in the same streptococcal group, also produced the bactericidal factor(s) but not CF. The effect of the Challis bacteriocin was limited to strains Wicky and 58, whereas the 36658 bacteriocin affected 67% of 49 strains tested. Strain 58, one of the indicator strains, was affected by the bactericidal activity of these bacteriocins but not by CF activity, and failed to transform. No relationship between the bacteriocin-producing strains and indicator strains was observed. Both Challis and 36658 bacteriocin activities decreased markedly either when the bacteriocins were heated at 50 C for 30 min or with the addition of a protein synthesis inhibitor, but showed different sensitivities to trypsin, papain and lipase. The bacteriocins were of at least protein nature and their molecular weight was roughly estimated as 100,000 daltons by membrane filtration experiments. The 36658 bacteriocin is a new type of streptocin previously not reported. The possible absence of bacteriophage or phage-like particles in the preparations is discussed.     

Diversity of bacteriocins and activity spectrum in Streptococcus pneumoniae

The production of bacteriocins can be favorable for colonization of the host by eliminating other bacterial species that share the same environment. In Streptococcus pneumoniae, the pnc (blp) locus encoding putative bacteriocins, immunity, and export proteins is controlled by a two-component system similar to the comCDE system required for the induction of genetic competence. A detailed comparison of the pnc clusters of four genetically distinct isolates confirmed the great plasticity of this locus and documented several repeat sequences. Members of the multiple-antibiotic-resistant Spain23F-1 clone, one member of the Spain9V-3 clone, sensitive 23F strain 2306, and the TIGR4 strain produced bactericidal substances active against other gram-positive bacteria and in some cases against S. pneumoniae as well. However, other strains did not show activity against the indicator strains despite the presence of a bacteriocin cluster, indicating that other factors are required for bacteriocin activity. Analysis of strain 2306 and mutant derivatives of this strain confirmed that bacteriocin production was dependent on the two-component regulatory system and genes involved in bacteriocin transport and processing. At least one other bacteriocin gene, pncE, is located elsewhere on the chromosome and might contribute to the bacteriocin activity of this strain.     

Antagonistic interactions amongst bacteriocin-producing enteric bacteria in dual species biofilms

AIMS: The objective of this study was to investigate the antagonistic interactions between bacteriocin-producing enteric bacteria in dual species biofilms and the interspecies interactions correlated with sensitivity to biocides. METHODS AND RESULTS: When compared with their single species counterparts, the dual species biofilms formed by bacteriocin-producing strains exhibited a decrease in biofilm size and an increase in sensitivity to the antimicrobial agents hypochlorite, triclosan and benzalkonium chloride. The five dual species biofilms studied all resulted in biofilms containing a mixture of the two strains. This was attributed to the spatial distribution of cells within the biofilm, with each strain forming its own microcolonies. The production of a bacteriocin also gave a strain a competitive advantage when interacting with a bacteriocin-sensitive strain within a biofilm, both in gaining a foothold in a new environment and in preventing the colonization of a potential competitor into a pre-established biofilm. CONCLUSIONS: It was concluded that bacteriocins might be used specifically for interacting with competing strains within a biofilm, as opposed to a planktonic, environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike planktonically grown bacteriocin-producing populations, where one strain will always be out-competed, bacteriocin-producing and bacteriocin-sensitive strains can coexist in biofilm communities, clearly demonstrating major differences between biofilm and planktonic competition. This paper highlights the importance of bacteriocin production in the development of biofilm communities.     

Searching for bacteriocin-producing lactic acid bacteria in soil

A survey was conducted on the isolation and characterization of bacteriocin-producing lactic acid bacteria in soil. Forty-two acid-producing bacterial strains were isolated from 55 soil samples collected in Yamanashi prefecture, Japan. Investigation of antibacterial activities of isolates revealed that three isolates, Lactobacillus animalis C060203, Enterococcus durans C102901 and Leuconostoc mesenteroides subsp. mesenteroides C060204, showed antibacterial activities against the indicator strain of Lactobacillus sakei JCM 1157T. Bacteriocin from Enterococcus durans C102901 showed different characteristics from the known durancin L28-1A, produced by Enterococcus durans L28-1. Furthermore, this is the first report of a bacteriocin being produced by Lactobacillus animalis. Viewing from the species, bacteriocins from strains C102901 and C060203 showed high possibilities for the novel substances. These significant findings suggest that soil may be a common source for the isolation of novel bacteriocin-producing lactic acid bacteria.     

Bacteriocin production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> in complex growth media

Objective

To test if the production of bacteriocins by Streptococcus thermophilus is influenced when grown in various complex media commonly used for the culturing of lactic acid bacteria.

Results

Forty-one strains of S. thermophilus were screened for the production of bacteriocins in tryptone/yeast extract/lactose (TYL), M17-lactose (M17L), M17-glucose (M17G) and MRS media. Two strains, ST144 and ST145, were identified as novel bacteriocin producers, with constitutive production observed only in M17G. Strains ST110, ST114 and ST134 constitutively produced bacteriocins in all growth media but ST114 required growth in MRS for its antimicrobial activity to persist in a 24 h culture. The addition of a synthetic quorum sensing peptide (BlpC) induced bacteriocin production by ST106 in all media tested; and by ST118 in TYL and M17L. Strain ST109, which constitutively produced a bacteriocin in TYL and M17 broths, required BlpC induction when grown in MRS. Real-time PCR analysis showed that the natural expression of blpC in ST109 was lower when grown in MRS, suggesting that something in medium interfered with the blp quorum sensing system.

Conclusion

As the choice of growth medium influences both bacteriocin production and peptide stability, several types of production media should be tested when screening for novel bacteriocin-producing strains of S. thermophilus.

     

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.     

益生菌产生的细菌素及其功能机制

细菌素是细菌核糖体合成的具有抑菌活性的小肽.细菌素的产生是益生菌重要的益生特性,它们天然无毒,不仅对食品腐败菌和人体致病菌有很好的抑菌活性,还具有有助益生菌定殖和调节肠道菌群等益生特性.本文综述了益生菌产生的细菌素的种类、条件性合成、益生功能及其作用机理等,以期为深入认识益生菌的益生功能及其作用方式,研究开发对人体有益...     

Detection of bacteriocins of lactic acid bacteria isolated from foods and comparison with pediocin and nisin

A total of 663 533 colonies from 72 dairy and meat sources showed a detection rate of 0·2% for bacteriocin producers using direct plating techniques. A further 83 000 colonies from 40 fish and vegetable sources showed a detection rate of 3·4% for bacteriocin producers using selective enrichment procedures. A collection of seven purified isolates showing a different host spectrum of bacteriocin activity and with the ability to produce bacteriocins in broth culture were compared with nisin and pediocin (with respect to their inhibitory activity, determined by the critical dilution method), against various indicator bacteria in agar and broth. The sensitivity of Listeria species to various bacteriocins was influenced by the agar and broth test systems used. A Lactobacillus curvatus strain was found to be the most suitable indicator for quantitating antimicrobial effects of all the bacteriocins investigated in both agar and broth test systems. The bacteriocin-producing isolates were characterized by biochemical reactions and DNA restriction enzyme profiles and taxonomic identification revealed species of Lactobacillus , Carnobacterium and Lactococcus assigned on the basis of 16S rDNA sequences.     

Bacteriocin production by members of the genusKlebsiella

Bacteriocin production was tested in 36Klebsiella and 3Enterobacter aerogenes strains. Bacteriocins produced byK. pneumoniae were found to be active on most strains ofK. edwardsi, K. aerogenes, K. rhinoscleromatis andE. aerogenes. The bacteriocin produced byE. aerogenes 37 is also active onK. pneumoniae andK. ozaenae. The bacteriocins produced byK. rhinoscleromatis, K. edwardsi andK. aerogenes are active on only a few strains. The activity spectra of the bacteriocins of a number of strains were similar. The method of classification used for colicins could not be applied to these bacteriocins as mutants resistant to one bacteriocin were nearly always resistant to all other bacteriocins. One mutant, though resistant, still adsorbed the bacteriocin to which it was resistant and it is very likely that the same applies for all other resistant mutants. The hypothesis is made that allKlebsiella bacteriocins have the same biochemical target, or more likely, possess a common transmission mechanism.     

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.     

Bacteriocinogenic LAB Strains for Fermented Meat Preservation: Perspectives,Challenges, and Limitations

Over the last decades, much research has focused on lactic acid bacteria (LAB) bacteriocins because of their potential as biopreservatives and their action against the growth of spoilage microbes. Meat and fermented meat products are prone to microbial contamination, causing health risks, as well as economic losses in the meat industry. The use of bacteriocin-producing LAB starter or protective cultures is suitable for fermented meats. However, although bacteriocins can be produced during meat processing, their levels are usually much lower than those achieved during in vitro fermentations under optimal environmental conditions. Thus, the direct addition of a bacteriocin food additive would be desirable. Moreover, safety and technological characteristics of the bacteriocinogenic LAB must be considered before their widespread applications. This review describes the perspectives and challenges toward the complete disclosure of new bacteriocins as effective preservatives in the production of safe and “healthy” fermented meat products.     

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.