<Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> NK02: a Potent Probiotic for Clinical Application in Mouthwash

A specific strain of naturally occurring oral lactobacilli was isolated and identified based on morphological, biochemical, and 16S rRNA gene sequencing. The phylogenetic affiliation of the isolate confirmed that the NK02 strain had close association with the Lactobacillus salivarius. An effective mouthwash was developed for treatment of periodontitis and suppression of the indicator bacterium Aggregatibacter actinomycetemcomitans which is an obvious pathogen of periodontal disease. The mouthwash containing L. salivarius NK02 was tested at a dose level of 10⁸ (colony forming units (CFU) ml^?1), monitoring over a period of 4 weeks. The study was a randomized double-blind placebo control trial, and the patients were treated in two groups of control and test by using scaling and root planing (SRP) + placebo and scaling and root planing (SRP) + probiotic, respectively. It appeared that the probiotic mouthwash was able to inhibit the bacterial growth on both saliva and sub-gingival crevice and exhibited antibacterial activity against A. actinomycetemcomitans. The results also showed that SRP+ probiotic treatment led to a significant decrease of gingival index (GI) and bleeding on probing (BOP) compared with that of SRP + placebo for the probiotic group. The rate of decrease in pocket depth was displayed in the group with SRP + probiotic treatment equal to 1/2 mm, and probing pocket depth (PPD) value was decreased in the probiotic bacteria treatment group that can explain the decrease in inflammation in gingiva. Our findings suggest that probiotic mouthwash is healthy for daily use as an alternative for maintaining dental and periodontal health.     

L. plantarum,L. salivarius,and L. lactis Attenuate Th2 Responses and Increase Treg Frequencies in Healthy Mice in a Strain Dependent Manner

Many studies on probiotics are aimed at restoring immune homeostasis in patients to prevent disease recurrence or reduce immune-mediated pathology. Of equal interest is the use of probiotics in sub-clinical situations, which are characterized by reduced immune function or low-grade inflammation, with an increased risk of infection or disease as a consequence. Most mechanistic studies focus on the use of probiotics in experimental disease models, which may not be informative for these sub-clinical conditions. To gain better understanding of the effects in the healthy situation, we investigated the immunomodulatory effects of two Lactobacillus probiotic strains, i.e. L. plantarum WCFS1 and L. salivarius UCC118, and a non-probiotic lactococcus strain, i.e. L. lactis MG1363, in healthy mice. We studied the effect of these bacteria on the systemic adaptive immune system after 5 days of administration. Only L. plantarum induced an increase in regulatory CD103⁺ DC and regulatory T cell frequencies in the spleen. However, all three bacterial strains, including L. lactis, reduced specific splenic T helper cell cytokine responses after ex vivo restimulation. The effect on IFN-γ, IL5, IL10, and IL17 production by CD4⁺ and CD8⁺ T cells was dependent on the strain administered. A shared observation was that all three bacterial strains reduced T helper 2 cell frequencies. We demonstrate that systemic immunomodulation is not only observed after treatment with probiotic organisms, but also after treatment with non-probiotic bacteria. Our data demonstrate that in healthy mice, lactobacilli can balance T cell immunity in favor of a more regulatory status, via both regulatory T cell dependent and independent mechanisms in a strain dependent manner.     

Probiotics Can Generate FoxP3 T-Cell Responses in the Small Intestine and Simultaneously Inducing CD4 and CD8 T Cell Activation in the Large Intestine

Most studies on probiotics aim to restore intestinal homeostasis to reduce immune-pathology in disease. Of equal importance are studies on how probiotics might prevent or delay disease in healthy individuals. However, knowledge on mechanisms of probiotic actions in healthy individuals is scarce. To gain more insight in how different bacterial strains may modulate the healthy intestinal immune system, we investigated the effect of the food derived bacterial strains L. plantarum WCFS1, L. salivarius UCC118, and L. lactis MG1363, on the intestinal regulatory immune phenotype in healthy mice. All three bacterial strains induced an upregulation of activity and numbers of CD11c⁺ MHCII⁺ DCs in the immune-sampling Peyer’s Patches. Only L. salivarius UCC118 skewed towards an immune regulatory phenotype in the small intestinal lamina propria (SILP). The effects were different in the large intestine lamina propria. L. salivarius UCC118 induced activation in both CD4 and CD8 positive T-cells while L. plantarum WCFS1 induced a more regulatory phenotype. Moreover, L. plantarum WCFS1 decreased the Th1/Th2 ratio in the SILP. Also L. lactis MG1363 had immunomodulatory effects. L. lactis MG1363 decreased the expression of the GATA-3 and T-bet in the SILP. As our data show that contradictory effects may occur in different parts of the gut, it is recommended to study effects of probiotic in different sites in the intestine. Our strain-specific results suggest that unspecified application of probiotics may not be very effective. Our data also indicate that selection of specific probiotic strain activities on the basis of responses in healthy mice may be a promising strategy to specifically stimulate or suppress immunity in specific parts of the intestine.     

Persistence of the Oral Probiotic Streptococcus salivarius M18 Is Dose Dependent and Megaplasmid Transfer Can Augment Their Bacteriocin Production and Adhesion Characteristics

Bacteriocin-producing probiotic Streptococcus salivarius M18 offers beneficial modulatory capabilities within the oral microbiome, apparently through potent inhibitory activity against potentially deleterious bacteria, such as Streptococcus pyogenes. The oral cavity persistence of S. salivarius M18 was investigated in 75 subjects receiving four different doses for 28 days. Sixty per cent of the subjects already had some inhibitor-producing S. salivarius in their saliva prior to probiotic intervention. Strain M18’s persistence was dependent upon the dose, but not the period of administration. Culture analysis indicated that in some individuals the introduced strain had almost entirely replaced the indigenous S. salivarius, though the total numbers of the species did not increase. Selected subjects showing either high or low probiotic persistence had their salivary populations profiled using Illumina sequencing of the V6 region of the 16S rRNA gene. Analysis indicated that while certain bacterial phenotypes were markedly modulated, the overall composition of the oral microbiome was not modified by the probiotic treatment. Megaplasmids encoding bacteriocins and adhesion factors were transferred in vitro to generate a transconjugant S. salivarius exhibiting enhanced antimicrobial production and binding capabilities to HEp-2 cells. Since no widespread perturbation of the existing indigenous microbiota was associated with oral instillation and given its antimicrobial activity against potentially pathogenic streptococci, it appears that application of probiotic strain M18 offers potential low impact alternative to classical antibiotic prophylaxis. For candidate probiotic strains having relatively poor antimicrobial or adhesive properties, unique derivatives displaying improved probiotic performance may be engineered in vitro by megaplasmid transfer.     

Functional role of <Emphasis Type="Italic">oppA</Emphasis> encoding an oligopeptide-binding protein from <Emphasis Type="Italic">Lactobacillus salivarius</Emphasis> Ren in bile tolerance

Lactobacillus salivarius is a member of the indigenous microbiota of the human gastrointestinal tract (GIT), and some L. salivarius strains are considered as probiotics. Bile tolerance is a crucial property for probiotic bacteria to survive the transit through the GIT and exert their beneficial effects. In this work, the functional role of oppA encoding an oligopeptide transporter substrate-binding protein from L. salivarius Ren in bile salt tolerance was investigated. In silico analysis revealed that the oppA gene encodes a 61.7-kDa cell surface-anchored hydrophilic protein with a canonical lipoprotein signal peptide. Homologous overexpression of OppA was shown to confer 20-fold higher tolerance to 0.5 % oxgall in L. salivarius Ren. Furthermore, the recombinant strain exhibited 1.8-fold and 3.6-fold higher survival when exposed to the sublethal concentration of sodium taurocholate and sodium taurodeoxycholate, respectively, while no significant change was observed when exposed to sodium glycocholate and sodium glycodeoxycholate (GDCA). Our results indicate that OppA confers specific resistance to taurine-conjugated bile salts in L. salivarius Ren. In addition, the OppA overexpression strain also showed significant increased resistance to heat and salt stresses, suggesting the protective role of OppA against multiple stresses in L. salivarius Ren.     

Putative Microbial Population Shifts Attributable to Nasal Administration of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a

Changes in bacterial composition of nasal microbiota may alter the host’s susceptibility to several infectious and allergic diseases such as chronic rhinosinusitis and allergic rhinitis. The aim of this study was to evaluate the effects of 1-week administration of a probiotic product, composed by a combination of Streptococcus salivarius 24SMBc and Streptococcus oralis 89a, on the nostril microbiota. Differences in the nasal microbiota composition were investigated by using a next-generation sequencing approach. A strong and significant decrease in Staphylococcus aureus abundance was detected immediately after the bacterial administration. Moreover, comparing the microbial networks of nostril microbiota before and 1 month after the end of treatment, we detected an increase in the total number of both bacterial nodes and microbial correlations, with particular regard to the beneficial ones. Furthermore, a less abundance of microbial genera commonly associated to potential harmful bacteria has been observed. These results suggest a potential ability of S. salivarius 24SMBc and S. oralis 89a to regulate and reorganize the nasal microbiota composition, possibly favoring those microorganisms that may be able to limit the overgrowth of potential pathogens.

     

Fibrinogen-binding and platelet-aggregation activities of a Lactobacillus salivarius septicaemia isolate are mediated by a novel fibrinogen-binding protein

The marketplace for probiotic foods is burgeoning, measured in billions of euro per annum. It is imperative, however, that all bacterial strains are fully assessed for human safety. The ability to bind fibrinogen is considered a potential pathogenicity trait that can lead to platelet aggregation, serious medical complications, and in some instances, death. Here we examined strains from species frequently used as probiotics for their ability to bind human fibrinogen. Only one strain (CCUG 47825), a Lactobacillus salivarius isolate from a case of septicaemia, was found to strongly adhere to fibrinogen. Furthermore, this strain was found to aggregate human platelets at a level comparable to the human pathogen Staphylococcus aureus. By sequencing the genome of CCUG 47825, we were able to identify candidate genes responsible for fibrinogen binding. Complementing the genetic analysis with traditional molecular microbiological techniques enabled the identification of the novel fibrinogen receptor, CCUG_2371. Although only strain CCUG 47825 bound fibrinogen under laboratory conditions, homologues of the novel fibrinogen binding gene CCUG_2371 are widespread among L. salivarius strains, maintaining their potential to bind fibrinogen if expressed. We highlight the fact that without a full genetic analysis of strains for human consumption, potential pathogenicity traits may go undetected.     

Protective effect of Lactobacillus salivarius Li01 on thioacetamide-induced acute liver injury and hyperammonaemia

The gut microbiota plays pivotal roles in liver disease onset and progression. The protective effects of Lactobacillus salivarius Li01 on liver diseases have been reported. In this study, we aimed to detect the protective effect of L. salivarius Li01 on thioacetamide (TAA)-induced acute liver injury and hyperammonaemia. C57BL/6 mice were separated into three groups and given a gavage of L. salivarius Li01 or phosphate-buffered saline for 7 days. Acute liver injury and hyperammonaemia were induced with an intraperitoneal TAA injection. L. salivarius Li01 decreased mortality and serum transaminase levels and improved histological liver damage caused by TAA. Serum inflammatory cytokine and chemokine and lipopolysaccharide-binding protein (LBP) concentrations, nuclear factor κB (NFκB) pathway activation and macrophage and neutrophil infiltration into the liver were significantly alleviated by L. salivarius Li01. L. salivarius Li01 also reinforced gut barrier and reshaped the perturbed gut microbiota by upregulating Bacteroidetes and Akkermansia richness and downregulating Proteobacteria, Ruminococcaceae_UCG_014 and Helicobacter richness. Plasma and faecal ammonia levels declined noticeably in the Li01 group, accompanied by improvements in cognitive function, neuro-inflammation and relative brain-derived neurotrophic factor (BDNF) gene expression. Our results indicated that L. salivarius Li01 could be considered a potential probiotic in acute liver injury and hepatic encephalopathy (HE).     

Live and heat-inactivated lactobacilli from feces inhibit <Emphasis Type="Italic">Salmonella typhi</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> adherence to caco-2 cells

A quantitative approach has been proposed to evaluate the competitive inhibition of Escherichia coli and Salmonella typhi by live and heat-inactivated laboratory isolated Lactobacillus sp. on adhesion to monolayer of Caco-2 cells. Three species of Lactobacillus (L. casei, L. acidophilus, L. agilis) isolated from human neonate feces and two commercial probiotic strains (L. casei, L. acidophilus) have been compared for probiotic activity. All lactobacilli were able to attach to the Caco-2 cells, however, the degree of adhesion was bacterial strain-dependent. The adhesion indices of the two commercial probiotic strains were not significantly different from the values obtained for the other two similar fecal strains (p > 0.01). The inhibition of attachment of the pathogenic bacteria by inactivated cells of fecal L. acidophilus was examined and compared to the results of live bacteria. The inhibition pattern was similar for live and heat-inactivated L. acidophilus (p > 0.01). The number of attached pathogenic bacteria to the Caco-2 cells decreased when the number of L. acidophilus increased from 10⁶ to 10⁹ CFU/mL. The heat-inactivated L. acidophilus displayed similar probiotic activity compared to the live bacteria.     

Gut immune stimulation by non pathogenic Gram(+) and Gram(-) bacteria. Comparison with a probiotic strain

We analyzed the gut immune stimulation induced by Gram-positive bacteria: non probiotic Lactobacillus acidophilus CRL 1462 and Lactobacillus acidophilus A9; two potentially probiotic strains: L. acidophilus CRL 924 and Lactobacillus delbrueckii subsp. bulgaricus CRL 423; comparatively with a probiotic strain: Lactobacillus casei CRL 431. We also studied Gram-negative bacteria: Escherichia coli 129 and E. coli 13-7 in BALB/c mice. All the strains increased the number of IgA+ cells. We analyzed the cytokines IFNγ, TNFα, IL-17, IL-12, IL-6 and MIP-1α. The Gram(+) strains increased the number of IL-10+ cells. Gram(−) strains did not increase IL-10+ cells, but they increased the number of IL-12+ cells. The probiotic strain increased mainly IFNγ and TNFα. In the study of the receptors TLR-2, TLR-4 and CD-206, we demonstrated that only the probiotic strain increased the number of CD-206+ cells. All the Gram(+) strains increased the number of TLR-2+ cells and the Gram(−) strains of the TLR-4+ cells. The probiotic strain induced the release of IL-6 by a preparation enriched in intestinal epithelial cells (IEC). Gram(+) and Gram(−) bacteria activated different immune receptors and induced a different cytokine profile. The probiotic strain showed a great activity on the immune cells and the enriched population in IEC, activating mainly cells of the innate immune system.     

Screening for lactobacilli with probiotic properties in the infant gut microbiota

Lactobacilli are believed to be beneficial for the human hosts and are currently being evaluated as potentially probiotic bacteria. In this study, Lactobacillus strains were isolated from infant faeces and were examined in vitro for potential probiotic properties. Faecal specimens from 63 healthy, full-term infants were collected at 4, 30 and 90 days after delivery. Seventy-four Lactobacillus strains were isolated and one or more different phenotypes from each infant (n = 44) were selected for further testing. The bacterial isolates were identified mainly as L. gasseri, L. crispatus, Lactobacillus paracasei, L. salivarius, L. fermentum after amplification and sequencing of 16s rRNA gene. The strains were examined for acid and bile tolerance, adhesion to Caco-2 cells, antibiotic susceptibility and antimicrobial activity against selected enteric pathogens. The great majority of the isolated lactobacilli were susceptible to ampicillin, amoxicillin/clavulanic acid, tetracycline, erythromycin, cephalothin, chloramphenicol and rifampicin. Resistance to vancomycin or bacitracin was detected to 34% of the strains. Twenty strains out of forty-four exhibited significant tolerance to bile salts. Those strains were subsequently tested for resistance to low pH conditions (pH 2 and 3). Interestingly, 85% (17 strains) of the tested lactobacilli remained unaffected at pH 3 after 3 h of incubation, 6 strains were found resistant at pH 2 after 1.5 h and only 2 strains found resistant after 3 h of incubation. Two of the strains were able to adhere to Caco-2 cells. In conclusion, two isolates fulfilled the in vitro probiotic criteria and are good candidates for further in vivo evaluation.     

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.     

Unusual genome complexity in Lactobacillus salivarius JCM1046

Background

Lactobacillus salivarius strains are increasingly being exploited for their probiotic properties in humans and animals. Dissemination of antibiotic resistance genes among species with food or probiotic-association is undesirable and is often mediated by plasmids or integrative and conjugative elements. L. salivarius strains typically have multireplicon genomes including circular megaplasmids that encode strain-specific traits for intestinal survival and probiotic activity. Linear plasmids are less common in lactobacilli and show a very limited distribution in L. salivarius. Here we present experimental evidence that supports an unusually complex multireplicon genome structure in the porcine isolate L. salivarius JCM1046.

Results

JCM1046 harbours a 1.83 Mb chromosome, and four plasmids which constitute 20% of the genome. In addition to the known 219 kb repA-type megaplasmid pMP1046A, we identified and experimentally validated the topology of three additional replicons, the circular pMP1046B (129 kb), a linear plasmid pLMP1046 (101 kb) and pCTN1046 (33 kb) harbouring a conjugative transposon. pMP1046B harbours both plasmid-associated replication genes and paralogues of chromosomally encoded housekeeping and information-processing related genes, thus qualifying it as a putative chromid. pLMP1046 shares limited sequence homology or gene synteny with other L. salivarius plasmids, and its putative replication-associated protein is homologous to the RepA/E proteins found in the large circular megaplasmids of L. salivarius. Plasmid pCTN1046 harbours a single copy of an integrated conjugative transposon (Tn6224) which appears to be functionally intact and includes the tetracycline resistance gene tetM.

Conclusion

Experimental validation of sequence assemblies and plasmid topology resolved the complex genome architecture of L. salivarius JCM1046. A high-coverage draft genome sequence would not have elucidated the genome complexity in this strain. Given the expanding use of L. salivarius as a probiotic, it is important to determine the genotypic and phenotypic organization of L. salivarius strains. The identification of Tn6224-like elements in this species has implications for strain selection for probiotic applications.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-771) contains supplementary material, which is available to authorized users.     

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.     

Antagonistic activity of Lactobacillus acidophilus RY2 isolated from healthy infancy feces on the growth and adhesion characteristics of enteroaggregative Escherichia coli

Enteroaggregative Escherichia coli (EAggEC) infection is an important cause of acute diarrhea, affecting children in developing countries and travelers visiting tropical or subtropical areas. Three probiotics can exert bacteriostatic or bactericidal effects on human and animal intestinal pathogens, the efficiency of probiotics on EAggEC infection remains unclear. In this study, the antagonistic activity of probiotic bacteria isolated from infant faeces was examined against several EAggEC stains. While three isolates, Lactobacillus acidophilus RY2, Lactobacillus salivarius MM1 and Lactobacillus paracasei En4 were shown to significantly inhibit the growth of EAggEC. In addition, the antagonistic activity of the Lactobacillus species was maintained despite heating (100 °C, 15 min) of cell free culture supernatant (CFCS). The antagonistic activity of the CFCS however, could be reduced following lactate dehydrogenase treatment and at pH 7.2. Furthermore, in an adhesion–inhibition assay, L. acidophilus RY2 was shown to be more effective than L. salivarius MM1 and L. paracasei EN4. This study suggests that L. acidophilus RY2 could be used as a probiotic organism against EAggEC.     

Lactobacillus salivarius CTC2197 Prevents Salmonella enteritidis Colonization in Chickens

A rifampin-resistant Lactobacillus salivarius strain, CTC2197, was assessed as a probiotic in poultry, by studying its ability to prevent Salmonella enteritidis C-114 colonization in chickens. When the probiotic strain was dosed by oral gavage together with S. enteritidis C-114 directly into the proventriculus in 1-day-old Leghorn chickens, the pathogen was completely removed from the birds after 21 days. The same results were obtained when the probiotic strain was also administered through the feed and the drinking water apart from direct inoculation into the proventriculus. The inclusion of L. salivarius CTC2197 in the first day chicken feed revealed that a concentration of 10⁵ CFU g⁻¹ was enough to ensure the colonization of the gastrointestinal tract of the birds after 1 week. However, between 21 and 28 days, L. salivarius CTC2197 was undetectable in the gastrointestinal tract of some birds, showing that more than one dose would be necessary to ensure its presence till the end of the rearing time. Freeze-drying and freezing with glycerol or skim milk as cryoprotective agents, appeared to be suitable methods to preserve the probiotic strain. The inclusion of the L. salivarius CTC2197 in a commercial feed mixture seemed to be a good way to supply it on the farm, although the strain showed sensitivity to the temperatures used during the feed mixture storage and in the chicken incubator rooms. Moreover, survival had been improved after several reinoculations in chicken feed mixture.     

Oral Administration of Lactobacillus Strains Isolated from Breast Milk as an Alternative for the Treatment of Infectious Mastitis during Lactation

In this study, 20 women with staphylococcal mastitis were randomly divided in two groups. Those in the probiotic group daily ingested 10 log₁₀ CFU of Lactobacillus salivarius CECT5713 and the same quantity of Lactobacillus gasseri CECT5714 for 4 weeks, while those in the control one only ingested the excipient. Both lactobacillus strains were originally isolated from breast milk. On day 0, the mean staphylococcal counts in the probiotic and control groups were similar (4.74 and 4.81 log₁₀ CFU/ml, respectively), but lactobacilli could not be detected. On day 30, the mean staphylococcal count in the probiotic group (2.96 log₁₀ CFU/ml) was lower than that of the control group (4.79 log₁₀ CFU/ml). L. salivarius CECT5713 and L. gasseri CECT5714 were isolated from the milk samples of 6 of the 10 women of the probiotic group. At day 14, no clinical signs of mastitis were observed in the women assigned to the probiotic group, but mastitis persisted throughout the study period in the control group women. In conclusion, L. salivarius CECT5713 and L. gasseri CECT5714 appear to be an efficient alternative for the treatment of lactational infectious mastitis during lactation.     

Role of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes in influencing faecal bacteria and biochemical parameters in human subjects

Aims: To evaluate the positive influence of the probiotic strain Lactobacillus paracasei LMGP22043 carried by artichokes into the human gut with special reference to faecal bacterial balance, short‐chain fatty acid concentrations and enzyme activities in a randomized, double‐blind human trial in comparison with probiotic‐free artichokes (control). Methods: Twenty subjects were randomized into two groups, which consumed daily 180 g of the artichoke product (probiotic or control) during two 15‐day study periods (periods 1 and 2) separated by a 15‐day washout in a crossover manner. Faecal samples were subjected to microbiological and biochemical analyses, and a strain‐specific PCR was performed to monitor the probiotic strain. Results: The probiotic strain, transported by the vegetable matrix, transiently colonized the gut of 17/20 subjects (median 6·87 log CFU g^?1 faeces), antagonized Escherichia coli and Clostridium spp. and increased the genetic diversity of lactic population based on REP‐PCR profiles, mainly after period 1. Conclusions: The probiotic L. paracasei LMGP22043 successfully colonized the human gut and positively influenced faecal bacteria and biochemical parameters. Significance and Impact of the Study: The association of the probiotic L. paracasei with a food carrier rich in fibre can represent a new strategy for favouring a daily supply of probiotics and attracting more consumers to vegetable food fortified with probiotic strains.     

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.     

Development of a Probiotic Cheddar Cheese Containing Human-Derived Lactobacillus paracasei Strains

Cheddar cheese was manufactured with either Lactobacillus salivarius NFBC 310, NFBC 321, or NFBC 348 or L. paracasei NFBC 338 or NFBC 364 as the dairy starter adjunct. These five strains had previously been isolated from the human small intestine and have been characterized extensively with respect to their probiotic potential. Enumeration of these strains in mature Cheddar cheese, however, was complicated by the presence of high numbers (>10⁷ CFU/g of cheese) of nonstarter lactic acid bacteria, principally composed of lactobacilli which proliferate as the cheese ripens. Attempts to differentiate the adjunct lactobacilli from the nonstarter lactobacilli based on bile tolerance and growth temperature were unsuccessful. In contrast, the randomly amplified polymorphic DNA method allowed the generation of discrete DNA fingerprints for each strain which were clearly distinguishable from those generated from the natural flora of the cheeses. Using this approach, it was found that both L. paracasei strains grew and sustained high viability in cheese during ripening, while each of the L. salivarius species declined over the ripening period. These data demonstrate that Cheddar cheese can be an effective vehicle for delivery of some probiotic organisms to the consumer.