Salivarius Group Research Articles

Emergence of a Cell Wall Protease in the Streptococcus thermophilus Population

Streptococcus thermophilus is perceived as a recently emerged food bacterium that evolved from a commensal ancestor by loss and gain of functions. Here, we provide data allowing a better understanding of this evolutionary scheme. A multilocus sequence typing approach that we developed showed that S. thermophilus diverges significantly from its potential ancestors of the salivarius group and displays a low level of allelic variability, confirming its likely recent emergence. An analysis of the origin and dissemination of the prtS gene was carried out within this evolutionary scheme. This gene encodes a protease that allows better growth in milk by facilitating casein breakdown to supply amino acids. The S. thermophilus protease exhibits 95% identity to the animal Streptococcus suis protein PrtS. Genomic analysis showed that prtS is part of an island flanked by two tandem insertion sequence elements and containing three other genes which present the best identities and synteny with the S. suis genome. These data indicate a potential origin for this "ecological" island in a species closely related to S. suis. The analysis of the distribution of the prtS gene in S. thermophilus showed that the gene is infrequent in historical collections but frequent in recent industrial ones. Moreover, this "ecological" island conferring an important metabolic trait for milk adaptation appears to have disseminated by lateral transfer in the S. thermophilus population. Taken together, these data support an evolutionary scheme of S. thermophilus where gene acquisition and selection by food producers are determining factors. The source and impact of genes acquired by horizontal gene transfer on the physiology and safety of strains should be addressed.

Evolutionary relationships among salivarius streptococci as inferred from multilocus phylogenies based on 16S rRNA-encoding, recA, secA, and secY gene sequences.

BackgroundStreptococci are divided into six phylogenetic groups, i.e, anginosus, bovis, mitis, mutans, pyogenic, and salivarius, with the salivarius group consisting of only three distinct species. Two of these species, Streptococcus salivarius and Streptococcus vestibularis, are members of the normal human oral microflora whereas the third, Streptococcus thermophilus, is found in bovine milk. Given that S. salivarius and S. vestibularis share several physiological characteristics, in addition to inhabiting the same ecosystem, one would assume that they would be more closely related to each other than to S. thermophilus. However, the few phylogenetic trees published so far suggest that S. vestibularis is more closely related to S. thermophilus. To determine whether this phylogenetic relationship is genuine, we performed phylogenetic inferences derived from secA and secY, the general secretion housekeeping genes, recA, a gene from a separate genetic locus that encodes a major component of the homologous recombinational apparatus, and 16S rRNA-encoding gene sequences using other streptococcal species as outgroups.ResultsThe maximum likelihood (ML) and maximum parsimony (MP) phylogenetic inferences derived from the secA and recA gene sequences provided strong support for the S. vestibularis/S. thermophilus sister-relationship, whereas 16S rRNA-encoding and secY-based analyses could not discriminate between alternate topologies. Phylogenetic analyses derived from the concatenation of these sequences unambiguously supported the close affiliation of S. vestibularis and S. thermophilus.ConclusionOur results corroborated the sister-relationship between S. vestibularis and S. thermophilus and the concomitant early divergence of S. salivarius at the base of the salivarius lineage.

Lactobacillus aquaticus sp. nov., isolated from a Korean freshwater pond

A Lactobacillus strain, IMCC1736T, was isolated recently from a Korean freshwater pond following an extensive study of the microbial community in this ecosystem. Its 16S rRNA gene was sequenced and phylogenetic analysis placed this strain within the Lactobacillus salivarius group, closely related to Lactobacillus satsumensis NRIC 0604T, with 97.9% sequence similarity. In the present work, the taxonomic status of strain IMCC1736T has been re-evaluated. It was characterized phylogenetically, genotypically and phenotypically and, based on DNA-DNA hybridization values, this strain represents a novel Lactobacillus species. Strain IMCC1736T can be differentiated genotypically from its closest relatives by randomly amplified polymorphic DNA analysis and ribotyping patterns; phenotypically, it can be distinguished by its inability to grow in 5% NaCl and at pH 3.3 and by certain carbohydrate fermentations. Strain IMCC1736T is Gram-positive, catalase-negative and microaerophilic. Cells are motile rods and show homofermentative metabolism. The name Lactobacillus aquaticus sp. nov. is proposed, with strain IMCC1736T (=CECT 7355T=DSM 21051T) as the type strain.

Screening of isolated lactic acid bacteria as potential beneficial strains for fermented liquid pig feed production

In this paper the selection of potential probiotic strains for producing fermented liquid feed (FLF) for pigs is presented. One hundred and forty six strains of lactic acid bacteria were isolated from ileal digesta of piglets, cecal digesta of slaughter pigs and FLF from a preliminary trial at our laboratory. The strains were identified with repetitive-element PCR and partial 16S rRNA sequencing and screened for acidification potential. Ten of the strains were selected and their culture supernatant was checked for antibacterial activity against Salmonella spp. Also, they were tested in vitro in batch fermentations at 30 °C for 72 h, for their capacity to produce FLF (water:feed ratio of 4:1) with a concentration of acetic acid less than 40 mmol/L and of lactic acid above 100 mmol/L, resulting in a pH below 4.5. The Lactobacillus strains MB 3123 ( L. johnsonii), MB 3182 ( L. salivarius group) and MB 3083 ( L. plantarum) were found to be very effective for the production of FLF. The FLF produced by including this strain had a pH drop from 5.9 to 4.2, 4.3 and 4.4 after 24 h and to 4.1 after 48 h of fermentation. Also, these three strains showed high antimicrobial activity against Salmonella spp. and produced more than 100 mmol/L lactic acid after 48 h fermentation. The MB 3170 strain increased the lactic acid concentration above 100 mmol/L after 48 h, but at 24 h the pH was only 4.5. The results indicate that it is possible to screen for probiotic strains capable of fermenting feeds and of producing high quantities of lactic acid with possible antimicrobial effects in the gastro-intestinal tract of pigs.

Phylogenetic analysis of type strains of the Salivarius group of the genus Streptococcus based on their 16S rRNA gene sequences

Phylogenetic analysis of type strains of the Salivarius group of the genus Streptococcus based on their 16S rRNA gene sequences

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.

Identification of 43 Streptococcus Species by Pyrosequencing Analysis of the rnpB Gene

Pyrosequencing technology was evaluated for identification of species within the Streptococcus genus. Two variable regions in the rnpB gene, which encodes the RNA subunit of endonuclease P, were sequenced in two reactions. Of 43 species, all could be identified to the species level except strains of the species pairs Streptococcus anginosus/S. constellatus and S. infantis/S. peroris. A total of 113 blood culture isolates were identified by pyrosequencing analysis of partial rnpB sequences. All but eight isolates could be unambiguously assigned to a specific species when the first 30 nucleotides of the two regions were compared to an rnpB database comprising 107 streptococcal strains. Principal coordinate analysis of sequence variation of strains from viridans group streptococci resulted in species-specific clusters for the mitis and the salivarius groups but not for the anginosus group. The identification capacity of pyrosequencing was compared to the biochemical test systems VITEK 2 and Rapid ID 32 Strep. The concordance between pyrosequencing and VITEK 2 was 75%, and for Rapid ID 32 Strep the corresponding figure was 77%. Isolates with discrepant identifications in the three methods were subjected to entire rnpB DNA sequence analysis that confirmed the identifications by pyrosequencing. In conclusion, pyrosequencing analysis of the rnpB gene can reliably identify Streptococcus species with high resolution.

Method for rapid identification of oral streptococci by PCR using 16S-23S ribosomal RNA intergenic spacer gene

Mutans group streptococci play an important role in the formation of dental caries, while mitis group streptococci are considered to be associated with subacute bacterial endocarditis. Both mitis and salivarius group streptococci, which are early colonizers in the human oral cavity, have an effect on the infection and colonization of mutans streptococci. In the present study, a rapid and exact PCR method to detect those oral streptococci was developed using the 16S-23S ribosomal RNA intergenic spacer gene, which is present between 16S rRNA and 23S rRNA within the RNA operon. The PCR analysis was able to correctly identify mutans and mitis group streptococcal species. Our results indicate that the combined PCR analysis method established in the present study is useful for detecting oral streptococcal species, as well as in clinical applications to predict and prevent dental caries and etiological studies of streptococcal infective endocarditis.

Mannosidase production by viridans group streptococci.

The production of mannosidase activity by all currently recognized species of human viridans group streptococci was determined using an assay in which bacterial growth was dependent on the degradation of the high-mannose-type glycans of RNase B and subsequent utilization of released mannose. RNase B is an excellent substrate for the demonstration of mannosidase activity since it is a glycoprotein with a single glycosylation site which is occupied by high-mannose-type glycoforms containing five to nine mannose residues. Mannosidase activity was produced only by some members of the mitis group (Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus cristatus, Streptococcus infantis, Streptococcus parasanguinis, and Streptococcus pneumoniae) and Streptococcus intermedius of the anginosus group. None of the other species within the salivarius and mutans groups or Streptococcus peroris and Streptococcus sanguinis produced mannosidase activity. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, it was demonstrated that the Man(5) glycan alone was degraded while Man(6) to Man(9), which contain terminal alpha(1-->2) mannose residues in addition to the alpha(1-->3), alpha(1-->6), and beta(1-->4) residues present in Man(5), remained intact. Investigations on mannosidase production using synthetic (4-methylumbelliferone- or p-nitrophenol-linked) alpha- or beta-mannosides as substrates indicated that there was no correlation between degradation of these substrates and degradation of the Man(5) glycan of RNase B. No species degraded these alpha-linked mannosides, while degradation of the beta-linked synthetic substrates was restricted to strains within the Streptococcus anginosus, S. gordonii, and S. intermedius species. The data generated using a native glycoprotein as the substrate demonstrate that mannosidase production within the viridans group streptococci is more widely distributed than had previously been considered.

Automated fluorescent in situ hybridization for the specific detection and quantification of oral streptococci in dental plaque

Our aim was to develop a rapid fluorescent in situ hybridization (FISH) assay for the identification of different oral groups of streptococci in dental plaque and to combine it with digital image analysis for the automated enumeration of target cells. Cy3-labeled oligonucleotide probes specific for 16S rRNA gene sequences of the anginosus, mitis, mutans, and salivarius groups of streptococci were hybridized under stringent conditions with bacterial cultures or supragingival plaque samples that had been permeabilized with lysozyme. Probe specificity was determined with strains from 30 different species, mainly of oral origin. Results showed that probes ANG541, MIT447, SSP001, and SAL090 with specificity for the anginosus, mitis, mutans, and salivarius groups, respectively, the pan-reactive streptococcal probe STR405, the S. mutans specific probe MUT590, and the S. sobrinus specific probe SOB174 were well-suited for the identification of cultured streptococci. Probes STR405, MIT447 and SSP001 were then successfully applied to enumerate automatically bacteria of the recognized taxa in 144 supragingival plaque samples. On the average, total streptococci accounted for 8.2%, streptococci of the mitis and mutans groups for 3.9 and 1.7%, respectively, of the plaques. The combined application of FISH and automated image analysis provides an objective time-saving alternative to culture or PCR for the enumeration of selected oral streptococci in dental plaque.

Identification of clinically relevant viridans streptococci by analysis of transfer DNA intergenic spacer length polymorphism.

The utility of PCR analysis of transfer DNA intergenic spacer length polymorphism (tDNA-ILP) for the identification to the species level of clinically relevant viridans streptococci was evaluated with a collection of reference strains of 15 species of the salivarius, anginosus, mitis and mutans rRNA homology groups. PCR products generated by using fluorescent, outwardly directed, consensus tDNA primers were analysed by electrophoresis on denaturating polyacrylamide gels and by laser fluorescence scanning. Eleven species showed specific and distinct tDNA patterns: Streptococcus cristatus, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis, Streptococcus pneumoniae, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus anginosus, Streptococcus mutans, Streptococcus criceti and Streptococcus ratti. Indistinguishable patterns were obtained among two groups of species: Streptococcus vestibularis and Streptococcus salivarius on the one hand and Streptococcus constellatus and Streptococcus intermedius on the other. S. mitis strains produced heterogeneous patterns that could be separated into three groups: a group containing S. mitis biovar 1 and two S. mitis biovar 2 groups, one of which clustered with S. parasanguinis strains while the other showed patterns unrelated to other species. These results agree in part with protein electrophoretic analysis showing that S. mitis biovar 2 strains belong to several streptococcal taxa. In conclusion, PCR analysis of tDNA-ILP holds promise for rapid identification of viridans streptococci that are difficult to identify by phenotypic tests.

Homofermentative Lactobacillus species predominantly isolated from canine feces.

Lactobacillus salivarius group biovars VIa and VIb, which are homofermentative Lactobacillus, were predominantly isolated from canine feces. Identification and biotyping of L. salivarius group biovars VIa and VIb were originally done by Mitsuoka et al. (1976). These strains were tentatively identified as L. salivarius by their phenotypic characteristics. They are, however, more similar to those of some new Lactobacillus species, particularly L. animalis and L. murinus, than those of L. salivarius and have now been identified as L. animalis by DNA-DNA homology.

Microbial induction of dentinal caries in human teeth in vitro

The ability of Lactobacillus casei, Streptococcus sobrinus, Actinomyces viscosus, and Streptococcus salivarius to induce dental caries was determined in vitro. A class I cavity (depth: 2mm) was prepared in extracted human caries-free premolars to make dentin blocks. The blocks were inoculated with these four bacterial strains in a monoinfective fashion and were incubated under anaerobic conditions. In addition to the monoinfection groups, mixed-infection groups of L. Casei with S. sobrinus or A. viscosus were also prepared. Half of the culture medium was renewed every 3 days, and the pH of the medium was measured. After 4 or 12 wk, these dentin blocks were prepared by Brown-Brenn staining and by contact microradiography for light microscopic observation and for immunohistochemical staining. The final pH of the S. salivarius group was the highest among the experimental groups, at approximately 5.1; that of the others was approximately 4.3. Bacterial invasion into the dentinal tubules was observed in all but the S. salivarius group. Among the monoinfection groups, the S. sobrinus group showed the highest invasion rate, followed by the A. Viscosus group and the L. casei group. The invasion rate was also high in the mixed-infection groups. Immunohistochemical staining revealed invasion only by L. casei, and not by S. sobrinus and A. viscosus. The invasion rate by L. casei was higher in the mixed-infection group with either S. sobrinus or A. viscosus than in the monoinfection groups. These findings suggest that lactobacillus might play an important role in the initiation and progress of dentinal caries, and that this bacterial species might exhibit a cooperative cariogenicity when it coexists with other bacterial species that surpasses its individual cariogenicity.

Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus.

We determined the 16S rRNA sequences of the type strains of Streptococcus mitis and Streptococcus gordonii and calculated the phylogenetic distances between those organisms and other members of the genus Streptococcus. The viridans group streptococci were separated into five phylogenetic groups; we named these groups the anginosus group, the mitis group, the salivarius group, the bovis group, and the mutans group. S. mitis and S. gordonii clustered in the mitis group together with Streptococcus pneumoniae, Streptococcus oralis, Streptococcus sanguis, and Streptococcus parasanguis at levels of sequence homology of more than 96%. Within this group, S. mitis, S. oralis, and S. pneumoniae exhibited more than 99% sequence homology with each other, although the DNA-DNA similarity values for their total chromosome DNAs were less than 60%.