Salivarius Strains Research Articles

Effect of Probiotics on the Treatment of Children with Atopic Dermatitis

BackgroundAtopic dermatitis, a chronic recurrent disease, is frequently encountered in clinical practice. In the last 30 years, the prevalence of atopic dermatitis has rapidly increased due to industrialization. Therefore, there have been attempts in recent years to find new ways of treating and preventing atopic dermatitis.ObjectiveIn this double-blind, randomized, placebo-controlled study, a combination of Bifidobacterium bifidum, Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus salivarius strains were evaluated in the treatment of atopic dermatitis in pediatric patients.MethodsForty pediatric patients (23 males and 17 females) aged 1~13 years were enrolled. One eligible individual who was approached declined to participate. The probiotic group was administered a probiotic complex containing B. bifidum, L. acidophilus, L. casei, and L. salivarius for 8 weeks. The placebo group, on the other hand, was administered skim milk powder and dextrose. All of the parameters including serum cytokines, eosinophil cationic protein), SCORing Atopic Dermatitis (SCORAD) index, and total serum immunoglobulin E (IgE) were measured in both the probiotic group and the placebo group at the end of 8 weeks.ResultsProbiotic intervention in pediatric atopic dermatitis patients effectively reduced the SCORAD index and serum cytokines interleukin (IL)-5, IL-6, interferon (IFN)-γ, and total serum IgE levels, but did not reduce levels of serum cytokines IL-2, IL-4, IL-10, ECP, or tumor necrosis factor-α (TNF-α) compared to the placebo group.ConclusionOur study found probiotics to be effective in reducing atopic dermatitis patients' SCORAD index, serum IL-5, IL-6, IFN-γ, and total serum IgE levels but not effective in reducing serum IL-2, IL-4, IL-10, ECP, or TNF-α levels.

Protein-carbohydrate interactions between Lactobacillus salivarius and pig mucins1

Adherence to the gastrointestinal tract is a key element desirable for many of the proposed beneficial health effects of probiotic bacteria. The aims of this study were to determine the amounts of adhesion of 3 Lactobacillus salivarius strains (Lb6, Lb9, and Lb10) to porcine small intestinal mucins and to determine whether adhesion is a function of lectin-like activities. Dot and Western blot assays were performed to investigate bacterial adhesion. Several carbohydrates and glycoproteins were evaluated to determine whether they interfered with adhesion of the Lactobacillus strains to intestinal mucins and to determine whether they had lectin-like activities. The Lb9 and Lb10 strains had greater association with piglet mucins than did those from 22- to 24-wk-old finishing pigs (P = 0.021 and 0.037, respectively), whereas the Lb6 strain adhered to both (P = 0.138). Western blot assays showed that bacterial adhesion detected piglet mucosa from the duodenum, jejunum, and ileum. In finishing pigs, the adhesion was variable throughout the gastrointestinal tract. Galactose and mannose diminished the interaction of the Lb9 and Lb10 strains in intestinal mucosa (P = 0.028 and 0.026, respectively), whereas pig gastric mucin reduced the adhesion of the Lb6 strain (P = 0.013). Adhesion of the Lb9 and Lb10 strains to intestinal mucosa was less after protease treatment (P = 0.023 and 0.018, respectively), which indicates that proteins are needed for the Lb9 and Lb10 strains to recognize mucin. The Lb6 strain also demonstrated diminished adhesion after periodate treatment (P = 0.038). From these results, we suggest that the nature of the bacterial lectin-like substance is a surface protein that loosely binds to the bacterial cell surface. All the tested strains adhered to specific targets in the small intestinal mucosa of piglets, and the bacteria had lectin-like proteins involved in this adhesion.

Salivaricin 9, a new lantibiotic produced by Streptococcus salivarius

Salivaricin 9 (Sal9) is a 2560 Da lantibiotic having just 46 % amino acid identity with its closest known homologue, the Streptococcus pyogenes lantibiotic SA-FF22. The Sal9 locus (designated siv) in Streptococcus salivarius strain 9 was partially sequenced and localized to an approximately 170 kb megaplasmid, which also harbours the locus for the lantibiotic salivaricin A4. The entire locus was fully characterized in the draft genome sequence of S. salivarius strain JIM8780 and shown to consist of eight genes, having the following putative functions: sivK, sensor kinase; sivR, response regulator; sivA, Sal9 precursor peptide; sivM, lantibiotic modification enzyme; sivT, ABC transporter involved in the export of Sal9 and concomitant cleavage of its leader peptide; and sivFEG, encoding lantibiotic self-immunity. Intriguingly, in contrast to strain 9, the siv locus was chromosomally located in strain JIM8780--the first lantibiotic locus shown not to be exclusively plasmid-associated in S. salivarius. Sal9-containing extracts specifically induced lantibiotic production in both strain 9 and strain JIM8780, indicating that Sal9 functions as a signal peptide for upregulation of its own biosynthesis. Screening representative strains of three streptococcal species (S. salivarius, S. pyogenes and S. mitis) for sivA indicated that it was present only in S. salivarius, with 12 of 28 tested S. salivarius positive. Since Sal9 was inhibitory to all tested S. pyogenes strains it appears to have potential as an important component of the bacteriocin armoury of S. salivarius probiotics intended to control S. pyogenes infections of the human oral cavity.

Identification of oral bacteria by 16S rRNA gene analysis in elderly persons requiring nursing care

After incubation of saliva from 58 semi-bedridden elderly persons, the cultures were identified based on the 16S rRNA gene base sequence to compare the identification by the conventional culture method. As a result, the 16S rRNA gene base sequence of 198 strains identified by the culture method showed 98.5% or more homology in some of the Human Oral Microbiome database, and the identification of bacterial species and genus was possible. When an organism identified by the 16S rRNA gene sequencing method was compared with that by the culture method, the concordance rates were 54.5% at the genus level and 35.9% at the species level. Streptococcus mitis strains most frequently isolated from saliva that were identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method (32/35), and all the 11 Streptococcus salivarius strains identified by the culture method were identified as the same species by the 16S rRNA gene sequencing method. All the strains identified as Streptococcus anginosus group by the culture method and 8 of the 9 strains identified as Prevotella species by the culture method were identified as the same group and genus by the 16S rRNA gene sequencing method. When an oral microbial flora test with saliva samples from elderly persons is performed, the 16S rRNA gene sequence identification enables us to identify major indigenous bacteria and pathogenic bacteria and is considered useful as a means of supplementing the conventional culture method.

Effect of fermented liquid feed on morpho-histological parameters in the piglet gut

Fermented liquid feed (FLF) is a feeding strategy which is said to ameliorate the gastro-intestinal health in pigs. However, the morpho-histological characteristics of the gut are mostly not assessed. Two experiments with weaned piglets were conducted in order to investigate the effect of FLF on villus height, crypt depth, villus height/crypt depth ratio and number of intra-epithelial lymphocytes (IELs). In experiment I, piglets (n = 6 per treatment) were fed a dry weaner feed or the same feed as FLF (with a feed:water ratio of 1:3; fermented with a Lactobacillus salivarius strain (MB 3083) at 30 °C). At day 9 the piglets were sacrificed and their gastro-intestinal tract was sampled at two places (3 m distal to the stomach = SI1 and 3 m proximal to the caecum = SI2). In experiment II, piglets (n = 9 per treatment) were fed the same dry weaner feed or as FLF (feed:water ratio of 1:2.5; fermented with Pediococcus acidilactici (Bactocell®, Lallemand, France) at 30 °C) and were sacrificed at day 28. In experiment I, the piglets on FLF had a significantly ( P < 0.05) lower villus height (414 vs. 517 μm) and villus/crypt ratio (1.52 vs. 1.65) in SI2 compared with the control piglets. In experiment II, there were no significant differences for these parameters between the two treatments. In both experiments no significant treatment effects were noted for IELs. In conclusion, no improvement of the morpho-histological parameters was seen when feeding a complete diet as FLF.

Detection of oral streptococci with collagen‐binding properties in saliva specimens from mothers and their children

Approximately 10-20% of Streptococcus mutans strains have been reported to possess collagen-binding properties, whereas other species in the oral cavity with those properties remain to be elucidated. Aim. To identify strains with collagen-binding properties and analyse their characteristics in comparison with S. mutans. A total of 110 expectorated saliva specimens were collected from 55 pairs of mothers and their children. Bacterial strains with collagen-binding properties were isolated and the species specified. In addition, strains with collagen-binding properties isolated from mother-child pairs were analysed using molecular biological approaches. The detection frequency of strains with collagen-binding properties was shown to be 40.9%, among which S. salivarius was the most frequently detected, followed by S. mutans. The collagen-binding activity of the S. mutans group was the highest, followed by S. salivarius. In addition, S. mutans and S. salivarius strains from 3 and 1 mother-child pairs, respectively, were shown to be the same clones. Our results indicate that S. mutans and S. salivarius are major species with collagen-binding properties in the oral cavity, and that strains with such properties may be related to mother-child transmission.

Novel Analysis of Bacterium−Substratum Bond Maturation Measured Using a Quartz Crystal Microbalance

Studies in flow displacement systems have shown that the reversibility of bacterial adhesion decreases within seconds to minutes after initial contact of a bacterium with a substratum surface. Atomic force microscopy (AFM) has confirmed that the forces mediating bacterial adhesion increase over a similar time span. The interfacial rearrangements between adhering bacteria and substratum surfaces responsible for this bond maturation have never been studied. Quartz crystal microbalance with dissipation (QCM-D) senses the interfacial region in real time and nondisruptively up to 250 nm from the sensor surface. In this paper, QCM-D is combined with real-time observation of bacterial adhesion in a flow displacement system, in order to analyze resident-time-dependent changes in dissipation. Three different Streptococcus salivarius strains showed a nonlinear relation between total dissipation shift (DeltaD) and number of adhering bacteria, whereas inert and rigid silica particles demonstrated a linear relation between DeltaD and the number of adhering particles. This suggests removal of interfacial water due to residence time dependent deformation of the nonrigid bacterium-substratum interface during bond maturation. Dissipation could be described by an exponentially decaying function, which combined with adhesion data allowed extraction of the dissipation shifts per bacterium upon initial contact (DeltaD(0)), after bond maturation (DeltaD(infinity)), as well as a characteristic time constant (tau(bm)). All bacterial strains showed significant bond maturation within one minute after their arrival at the substratum surface, which was not observed for silica particles. Dissipation analysis at the level of individually adhering bacteria would have been impossible without the simultaneous real-time analysis of bacterial adhesion numbers.

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.

Lactobacillus salivarius modulates cytokine induction and virulence factor gene expression in Helicobacter pylori

Human infection by the gastric pathogen Helicobacter pylori is characterized by a robust immune response which rarely prevents persistent H. pylori colonization. Emerging evidence suggests that lactobacilli may reduce H. pylori infection rates and associated inflammation. In this study, we measured the ability of two model strains of Lactobacillus salivarius (UCC118 and UCC119) to modulate gastric epithelial cell chemokine responses to H. pylori infection. Pre-treatment of AGS cells with either L. salivarius strain significantly decreased interleukin-8 (IL-8) production upon exposure to H. pylori, but not in cells stimulated with TNF-alpha. The production of the chemokines CCL20 and IP-10 by AGS cells infected with H. pylori was also altered following pre-treatment with UCC118 and UCC119. We showed that a greater reduction in IL-8 production with UCC119 was due to the production of more acid by this strain. Furthermore, UV-killed cells of both lactobacillus strains were still able to reduce H. pylori-induced IL-8 in the absence of acid production, indicating the action of a second anti-inflammatory mechanism. This immunomodulatory activity was not dependent on adhesion to epithelial cells or bacteriocin production. Real-time RT-PCR analysis showed that expression of eight of twelve Cag pathogenicity island genes tested was downregulated by exposure to L. salivarius, but not by cells of four other lactobacillus species. CagA accumulated in H. pylori cells following exposure to L. salivarius presumably as a result of loss of functionality of the Cag secretion system. These data identified a new mechanism whereby some probiotic bacteria have a positive effect on H. pylori-associated inflammation without clearing the infection.

Use of colony-based bacterial strain typing for tracking the fate of Lactobacillus strains during human consumption

BackgroundThe Lactic Acid Bacteria (LAB) are important components of the healthy gut flora and have been used extensively as probiotics. Understanding the cultivable diversity of LAB before and after probiotic administration, and being able to track the fate of administered probiotic isolates during feeding are important parameters to consider in the design of clinical trials to assess probiotic efficacy. Several methods may be used to identify bacteria at the strain level, however, PCR-based methods such as Random Amplified Polymorphic DNA (RAPD) are particularly suited to rapid analysis. We examined the cultivable diversity of LAB in the human gut before and after feeding with two Lactobacillus strains, and also tracked the fate of these two administered strains using a RAPD technique.ResultsA RAPD typing scheme was developed to genetically type LAB isolates from a wide range of species, and optimised for direct application to bacterial colony growth. A high-throughput strategy for fingerprinting the cultivable diversity of human faeces was developed and used to determine: (i) the initial cultivable LAB strain diversity in the human gut, and (ii) the fate of two Lactobacillus strains (Lactobacillus salivarius NCIMB 30211 and Lactobacillus acidophilus NCIMB 30156) contained within a capsule that was administered in a small-scale human feeding study. The L. salivarius strain was not cultivated from the faeces of any of the 12 volunteers prior to capsule administration, but appeared post-feeding in four. Strains matching the L. acidophilus NCIMB 30156 feeding strain were found in the faeces of three volunteers prior to consumption; after taking the Lactobacillus capsule, 10 of the 12 volunteers were culture positive for this strain. The appearance of both Lactobacillus strains during capsule consumption was statistically significant (p < 0.05).ConclusionWe have shown that genetic strain typing of the cultivable human gut microbiota can be evaluated using a high throughput RAPD technique based on single bacterial colonies. Validation of this strategy paves the way for future systematic studies on the fate and efficacy of bacterial probiotics during human clinical trials.

Antibiotic susceptibility of bacterial isolates from probiotic products available in Italy

Objective : An emerging issue of probiotic products is the antibiotic resistance of the strains used. The aim of this study was to determine the susceptibility of the isolates of 10 probiotic products available in Italy. Materials and methods : The susceptibility of 15 strains of Lactobacillus spp., 5 Streptococcus salivarius ssp. thermophilus, 1 Enterococcus faecium and 8 Bifidobacterium spp . to several groups of antibacterial agents was determined by E-test using MRS agar for Lactobacillus spp. and E. faecium and MH agar+5% sheep blood for S. thermophilus , with different conditions of incubation. For Lactobacillus , S. thermophilus and E. faecium the MICs obtained by E-test were compared to the MICs by broth microdilution test obtained following CLSI M45-A (2006) and CLSI M100-S17 (2007) guidelines. Results: The broth microdilution test resulted in MICs identical to those obtained with the E-test or in MICs with differences of 1 or 2 log dilution steps. All the strains of Lactobacillus were susceptible to ampicillin. Species-dependent antibiotic susceptibility was detected for cephalosporins; gentamicin and ciprofloxacin had variable activity. Intrinsic resistance to vancomycin was confirmed for L. paracasei, L. salivarius and L. plantarum . Atypical resistance to erythromycin was detected in one strain of L. salivarius . The strains of Bifidobacterium were susceptible to ampicillin, cefotaxime and erythromycin. The strains of E. faecium were susceptible to the tested antibiotics; the strain of S. thermophilus was resistant only to ciprofloxacin. The observed resistance in the strains used in the Italian probiotic products tested seemed to be intrinsic except for erythromycin in one L. salivarius strain. Key words: antibiotic susceptibility, probiotic, lactic acid bacteria, bifidobacteria

Purification of bacteriocin LS1 produced by human oral isolate Lactobacillus salivarius BGHO1

Lactobacillus salivarius BGHO1, a human oral isolate with antagonistic activity against growth of Streptococcus mutans, Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis, Micrococcus flavus, and Salmonella enteritidis, probably produces more than one proteinaceous antimicrobial substance. The objective of this study was the purification of a bacteriocin, named LS1, produced by L. salivarius BGHO1. A simple and fast procedure for bacteriocin purification was developed, consisting of reverse-phase chromatography of the ammonium sulfate precipitate of cell-free culture supernatant by fast protein liquid chromatography and high-performance liquid chromatography, followed by tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with the subsequent extraction of bacteriocin from the gel. The supernatant of L. salivarius BGHO1 culture retained its antimicrobial activity after boiling in a water bath for 15 min. Its antimicrobial activity was also maintained even after treatment for 20 min at 121 degrees C in an autoclave. Bacteriocin LS1 was purified to homogeneity. The molecular mass of bacteriocin LS1 was estimated to be approximately 10 kDa, based on tricine SDS-PAGE. During purification, another compound with antimicrobial activity, produced by L. salivarius BGHO1, was detected. The molecular mass of this compound was estimated to be approximately 5 kDa, based on tricine SDS-PAGE. Our results imply that LS1 is most probably a new bacteriocin, different from previously described bacteriocins produced by L. salivarius strains. The purification of bacteriocin LS1 enabled the further characterization of LS1 on both the molecular and genetic levels.

Strain-specific inhibition of Helicobacter pylori by Lactobacillus salivarius and other lactobacilli

To investigate the anti-Helicobacter pylori activity of 28 strains of Lactobacillus salivarius and 12 other lactobacilli, isolated from different sites and from different geographical regions. An in vitro agar plate diffusion assay was employed to assess the Lactobacillus anti-H. pylori activity. Nine out of 28 L. salivarius strains and 3/12 other Lactobacillus species tested inhibited H. pylori growth. There was no correlation between ecological niche/geographical location of isolation of the lactobacilli and their inhibitory capability. Further studies on strain L. salivarius UCC119 showed that this strain could inhibit growth of 6/6 clinical isolates of H. pylori, five of which were antibiotic-resistant. This inhibition was not due to acid production and was not mediated by a protein, but did require the presence of live cells. Growth inhibition of H. pylori by L. salivarius is strain-dependent and is not linked to any particular environmental niche or geographic location. Strains of L. salivarius showing highest anti-H. pylori activity may be useful as an adjunct in the treatment of strains that are resistant to conventional antibiotics.

Distribution of megaplasmids in Lactobacillus salivarius and other lactobacilli.

The genome of Lactobacillus salivarius UCC118 includes a 242-kb megaplasmid, pMP118. We now show that 33 strains of L. salivarius isolated from humans and animals all harbor a megaplasmid, which hybridized with the repA and repE replication origin probes of pMP118. Linear megaplasmids that did not hybridize with the pMP118 repA probe were also found in some strains of L. salivarius, showing for the first time that a lactic acid bacterium has multiple megaplasmids. Phylogenetic analysis of the repE and groEL sequences of 28 L. salivarius strains suggested similar evolutionary paths for the chromosome and megaplasmid. Although the replication origin of circular megaplasmids in L. salivarius was highly conserved, genotypic and phenotypic comparisons revealed significant variation between megaplasmid-encoded traits. Furthermore, megaplasmids of sizes ranging from 120 kb to 490 kb were present in seven strains belonging to six other Lactobacillus species from among 91 strains and 47 species tested. The discovery of the widespread presence of megaplasmids in L. salivarius, and restricted carriage by other Lactobacillus species, provides an opportunity to study the contribution of large extrachromosomal replicons to the biology of Lactobacillus.

CloacalLactobacillusIsolates from Broilers Often Display Resistance Toward Tetracycline Antibiotics

Lactobacillus crispatus, L. reuteri, L. amylovorus, L. gallinarum, and L. salivarius subsp. salivarius strains isolated from cloacal swabs of broiler chickens derived from 20 different farms in Belgium were tested for susceptibility to tetracycline and minocycline. Acquired resistance percentages to these antibiotics were extremely high for L. crispatus, L. reuteri, L. gallinarum, and L. salivarius subsp. salivarius (75%-100%). L. amylovorus on the contrary, displayed lower resistance percentages (25%) toward minocycline and tetracycline. In several strains, resistance against the tetracycline antibiotics was associated with the presence of the resistance genes tet(K), tet(L), tet(M), tet(W), and tet(Z). To our knowledge, this is the first report of tet(Z) in lactobacilli and tet(K), tet(L), and tet(W) in lactobacilli identified to species level. Our findings strengthen the evidence of intestinal Lactobacillus species acting as a pool of antimicrobial resistance genes urging the need for prudent use of tetracycline antibiotics in poultry production.

Extent of horizontal gene transfer in evolution of Streptococci of the salivarius group.

The phylogenetically closely related species Streptococcus salivarius and Streptococcus vestibularis are oral bacteria that are considered commensals, although they can also be found in human infections. The relationship between these two species and the relationship between strains isolated from carriers and strains responsible for invasive infections were investigated by multilocus sequence typing and additional sequence analysis. The clustering of several S. vestibularis alleles and the extent of genomic divergence at certain loci support the conclusion that S. salivarius and S. vestibularis are separate species. The level of sequence diversity in S. salivarius alleles is generally high, whereas that in S. vestibularis alleles is low at certain loci, indicating that the latter species might have evolved recently. Cluster analysis indicated that there has been genetic exchange between S. salivarius and S. vestibularis at three of the nine loci investigated. Horizontal gene transfer between streptococci belonging to the S. salivarius group and other oral streptococci was also detected at several loci. A high level of recombination in S. salivarius was revealed by allele index association and split decomposition sequence analyses. Commensal and infection-associated S. salivarius strains could not be distinguished by cluster analysis, suggesting that the pathogen isolates are opportunistic. Taken together, our results indicate that there is a high level of gene exchange that contributes to the evolution of two streptococcal species from the human oral cavity.

The effect of ingestion of milk supplemented with salivaricin A-producing Streptococcus salivarius on the bacteriocin-like inhibitory activity of streptococcal populations on the tongue

The colonization efficacies of salivaricin A (SalA)-producing Streptococcus salivarius strains 20P3 and 5 were compared when given in milk to 219 children, using either 2-day or 9-day dosing regimens. Colonization levels overall were superior for strain 5, and the 9-day dosing schedule resulted in higher levels of both initial colonization and strain persistence. The indigenous streptococcal tongue populations of 20 (10.9%) of the 189 children in the 2-day trial showed markedly increased SalA-like inhibitory activity following use of the S. salivarius-supplemented milk. All 20 of these children were found to have had relatively small (<5% of total S. salivarius) indigenous tongue populations of SalA-producing S. salivarius, and the relative proportions and/or inhibitory activity of these SalA producers on the childrens' tongues increased following ingestion of the S. salivarius-supplemented milk. Because SalA is known to be strongly inhibitory to Streptococcus pyogenes, an important implication of this study is that the consumption of SalA-producing probiotic S. salivarius could potentially help to effect a sustained increase in SalA-mediated protection against S. pyogenes infection.

Polyphasic analysis indicates that Lactobacillus salivarius subsp. salivarius and Lactobacillus salivarius subsp. salicinius do not merit separate subspecies status

Lactobacillus salivarius Rogosa et al. 1953 was described as a homofermentative lactobacillus with two varieties: salivarius, typified inter alia by the ability to ferment rhamnose, and salicinius, characterized by the ability to ferment the glucoside salicin. These varieties have become accepted as subspecies divisions. We have examined the relatedness of 32 L. salivarius strains by a polyphasic approach. Carbohydrate fermentation profile analysis did not support clear distinction of the two subspecies. L. salivarius UCC118 was shown to be facultatively heterofermentative, confirming in silico genome analysis. 16S rRNA gene sequences and 16S-23S rRNA intergenic spacer region sequences provided no discrimination between any of the strains or subspecies. Broad subdivisions were distinguishable by pulsed-field gel genomic digest patterns, but they did not allow subspecific or phenotypic distinctions. A phylogeny based upon groEL gene sequences was discordant with rhamnose or salicin fermentation data for many taxa, and no reliable phenotypic correlations could be established. In the absence of meaningful taxonomic criteria, we therefore propose that Lactobacillus salivarius comprises a single species with no infraspecific taxa. Based on the present study and literature data, an emended description of the species Lactobacillus salivarius is provided.

Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is inhibitory to Campylobacter jejuni in the chicken gastrointestinal system.

We evaluated anti-Campylobacter jejuni activity among >1,200 isolates of different lactic acid bacteria. Lactobacillus salivarius strain NRRL B-30514 was selected for further study. The cell-free, ammonium sulfate precipitate from the broth culture was termed the crude antimicrobial preparation. Ten microliters of the crude preparation created a zone of C. jejuni growth inhibition, and growth within the zone resumed when the crude preparation was preincubated with proteolytic enzymes. Bacteriocin OR-7, derived from this crude preparation, was further purified using ion-exchange and hydrophobic-interaction chromatography. The determined amino acid sequence was consistent with class IIa bacteriocins. Interestingly, OR-7 had sequence similarity, even in the C-terminal region, to acidocin A, which was previously identified from L. acidophilus and had activity only to gram-positive bacteria, whereas OR-7 had activity to a gram-negative bacterium. Bacteriocin activity was stable following exposure to 90 degrees C for 15 min, also consistent with these types of antibacterial peptides. The purified protein was encapsulated in polyvinylpyrrolidone and added to chicken feed. Ten day-of-hatch chicks were placed in each of nine isolation units; two groups of birds were challenged with each of four C. jejuni isolates (one isolate per unit). At 7 days of age, one group of birds was treated with bacteriocin-emended feed for 3 days, and one group was left untreated. At 10 days of age, the birds were sacrificed and the challenge strain was enumerated from the bird cecal content. Bacteriocin treatment consistently reduced colonization at least one millionfold compared with levels found in the untreated groups. Nonchallenged birds were never colonized by C. jejuni. Bacteriocin from L. salivarius NRRL B-30514 appears potentially very useful to reduce C. jejuni in poultry prior to processing.

Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius

Streptococcus salivarius strains commonly produce bacteriocins as putative anti-competitor 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.