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Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS ( ) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner \"What's in My Pot?\" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes ( = 514), clinical isolates ( = 31), and reference strains ( = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates ( = 8, = 17 and = 6) and 4 reference strains ( = 1, = 1, = 1, and = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except , and . The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

BackgroundDifferentiation of Streptococcus pneumoniae from other viridans group streptococci is well known to be challenging in clinical laboratories. Matrix assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF MS) had been reported to be a good alternative for Streptococcus species level identification. However, differentiation of S. pneumoniae from other Streptococcus mitis group organisms was found to be problematic using the Bruker MALDI Biotyper system.MethodsThis study used the Bruker MALDI Biotyper system in addition to a mass spectra model analysis generated by 10 reference strains of S. pneumoniae, 8 strains of S. mitis and 2 strains of S. oralis in the ClinProTools to identify 28 clinical isolates of S. pneumoniae and 47 isolates of S. mitis/oralis. The results were compared with those generated by the MALDI Biotyper system alone.ResultsThe percentages of correct species level identification using the MALDI Biotyper system alone and the direct transfer and extraction method were 66.7% (50/75) and 70.7% (53/75), respectively. With the additional ClinProTools mass spectra analysis, the percentages of correct identification by the direct transfer and extraction method increased to 85.3% (64/75) and 100% (75/75), respectively. This new workflow significantly improved the accuracy of S. pneumoniae and S. mitis/oralis identification.ConclusionsThe additional ClinProTools mass spectra analysis with extraction method after MALDI Biotyper identification significantly improved the accuracy of identification among S. pneumoniae, S. oralis and S. mitis. The extra 15 min processing time of spectra analysis should be affordable in most clinical laboratories. We suggest that the same approach could be further explored in handling other bacterial species with high similarities.

subspecies is explored as an anti-cariogenic probiotic. Here, subjecting freshly stimulated saliva samples of 35 healthy volunteers, six epidemiologically unrelated and two related strains were isolated (prevalence around 20%) applying a newly developed three-step procedure. Furthermore, the probiotic strain 7746 (AB-Dentisanium®) was tested under a variety of environmental conditions for its inhibitory effect on six , two , 15 other oral or intestinal streptococci, 15 strains, and six representatives of other species including periodontopathogens. All except one of the strains were inhibited by 7746 colonies or culture supernatant concentrate but only if either the test cell number was low or the producer or its bacteriocin concentration, respectively, was high. OMI 332, OMI 315, OMI 335, OMI 238, and the intestinal OMI 339 were not inhibited, while the other 10 streptococcal strains (especially OMI 334 and intestinal OMI 326) showed a certain degree of inhibition. From the panel of other bacterial species only was slightly inhibited. With the exception of OMI 285 and OMI 291 that possessed a 7746 bacteriocin-like gene cluster, all strains and especially type strain 7747 were strongly inhibited by 7746. In conclusion, probiotic strain 7746 might antagonize the initiation and progression of dental caries by reducing if not too abundant. strains inhibit each other, but strains with similar bacteriocin-related gene clusters, including immunity genes, are able to co-exist due to cross-resistance. In addition, development of resistance and adaptation to 7746-bacteriocins was observed during our study and needs attention. Hence, mechanisms underlying such processes need to be further investigated using omics-approaches. On the manufacturing level, probiotic strains should be continuously tested for function. Further clinical studies investigating inhibition of by AB-Dentisanium® are required that should also monitor the impact on the oral microbiome composition including resident strains.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. Our aim was to identify conserved targets in pneumococci that showed positive prediction for lipoprotein and extracellular subcellular location using bioinformatics programs and verify the distribution and the degree of conservation of these targets in pneumococci. These targets can be considered potential vaccine candidate to be evaluated in the future. A set of 13 targets were analyzed and confirmed the presence in all pneumococci tested. These 13 genes were highly conserved showing around >96 % of amino acid and nucleotide identity, but they were also present and show high identity in the closely related species Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. S. oralis clusters away from S. pneumoniae, while S. pseudopneumoniae and S. mitis cluster closer. The divergence between the selected targets was too small to be observed consistently in phylogenetic groups between the analyzed genomes of S. pneumoniae. The proteins analyzed fulfill two of the initial criteria of a vaccine candidate: targets are present in a variety of different pneumococci strains including different serotypes and are conserved among the samples evaluated.

It has been shown that there is a window of infectivity for mutans streptococci between the ages of 19 and 31 months, when many children acquire mutans streptococci transmitted from their mothers. Part of the children that escape this window acquire mutans streptococci at a later age. In this group, maternal transmission is expected to be less prevalent. The present study compared the bacteriocin activity profiles of mutans streptococci isolated from mothers, fathers and children when the children acquired the mutans streptococci between the ages of 5 and 11. Twelve families were randomly selected from a group of 11–year–old children who were known to have acquired mutans streptococci during this age period. From the saliva of the mothers (n = 12), fathers (n = 8) and children (n = 12) approximately 30 mutans streptococci strains were isolated. All isolates were tested twice for bacteriocin activity against 21 indicator strains with a double–layer technique. Bacteriocin activity of strains was considered to be different when the number of strains against which bacteriocin was produced differed >1 or when the width of one or more inhibition zones differed ≥4 mm. In 7/12 mother–child pairs similar profiles were found. In the 8 father–child pairs similar profiles were only found on 2 occasions. In these 2 families, all 3 ( mother, father and child) harboured strains with a similar profile. In 4/8 father–mother pairs similar profiles were found. There was no correlation between the prevalence of mutans streptococci strains, the number of indicator strains against which the strains made bacteriocin, nor the mean size of the inhibition zones and the presence of similarity of bacteriocin activity profiles of mutans streptococci within the family members. The results show that even when a child acquires mutans streptococci after the age of 5, there may be similarity between mutans streptococci in mother, father and child, indicating that transmission between the family members occurs.

Streptococcus pneumoniae is one of the most important human pathogens but is closely related to Streptococcus mitis , with which humans live in harmony. The fact that the two species evolved from a common ancestor provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. By detailed comparisons of genomes of the two species and other related streptococci, we identified 224 genes associated with virulence and 25 genes unique to the mutualistic species. The exclusive presence of the virulence factors in S. pneumoniae enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. Successful adaptation of S. mitis and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms. From a common ancestor, Streptococcus pneumoniae and Streptococcus mitis evolved in parallel into one of the most important pathogens and a mutualistic colonizer of humans, respectively. This evolutionary scenario provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. We performed detailed comparisons of 60 genomes of S. pneumoniae , S. mitis , Streptococcus pseudopneumoniae , the three Streptococcus oralis subspecies oralis , tigurinus , and dentisani , and Streptococcus infantis . Nonfunctional remnants of ancestral genes in both S. pneumoniae and in S. mitis support the evolutionary model and the concept that evolutionary changes on both sides were required to reach their present relationship to the host. Confirmed by screening of >7,500 genomes, we identified 224 genes associated with virulence. The striking difference to commensal streptococci was the diversity of regulatory mechanisms, including regulation of capsule production, a significantly larger arsenal of enzymes involved in carbohydrate hydrolysis, and proteins known to interfere with innate immune factors. The exclusive presence of the virulence factors in S. pneumoniae enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. In addition to loss of these virulence-associated genes, adaptation of S. mitis to a mutualistic relationship with the host apparently required preservation or acquisition of 25 genes lost or absent from S. pneumoniae . Successful adaptation of S. mitis and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms. IMPORTANCE Streptococcus pneumoniae is one of the most important human pathogens but is closely related to Streptococcus mitis , with which humans live in harmony. The fact that the two species evolved from a common ancestor provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. By detailed comparisons of genomes of the two species and other related streptococci, we identified 224 genes associated with virulence and 25 genes unique to the mutualistic species. The exclusive presence of the virulence factors in S. pneumoniae enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. Successful adaptation of S. mitis and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms.

Background Streptococcus pneumoniae (pneumococcus), S. mitis , and S. oralis are closely related bacteria that colonize the human upper respiratory tract. While pneumococcus is a leading cause of global morbidity and mortality, S. mitis and S. oralis are generally considered commensals, rarely causing disease in immunocompetent hosts. Here, we characterized the genomes of seven commensal streptococcal strains ( S. oralis A22 and S. mitis B22–G22) identified as potential biotherapeutics due to their bacteriocin-mediated antipneumococcal activity. Results Comparative genomic analyses revealed key differences between these commensals and pneumococci. Commensal strains encode diverse adhesin-like proteins, absent in pneumococci, and lack key virulence factors such as pilus islets and pneumococcal surface proteins. They also possess extensive restriction-modification and type II toxin-antitoxin systems, alongside novel prophages, suggesting roles in genetic stability and phage defense. Metabolic adaptations in commensals indicate a “cheater” strategy, relying on extracellular metabolites from other microorganisms, particularly in the nutrient-scarce nasopharynx. Additionally, commensal strains exhibit distinct teichoic acid compositions, with galactose-rich lipoteichoic acids potentially enhancing niche adaptation. Capsular diversity was also observed, with some strains encoding unique polysaccharides. These findings highlight genomic features that likely enhance commensal colonization and survival in the upper respiratory tract, while distinguishing them from pneumococci. Conclusions This study highlights the genomic characteristics of the seven commensal streptococcal strains with broad anti-pneumococcal activity recently described and provides insights into species-specific traits that could inform targeted strategies for pneumococcal control.

In the present research, the MALDI-TOF MS technique was applied as a tool to rapidly identify the salivary microbiome. In this fact, it has been monitored the changes occurred in molecular profiles under different antibiotic therapy. Significant changes in the composition of the salivary microbiota were noticed not only in relation to the non antibiotic (non-AT) and antibiotic treatment (AT) groups, but also to the used media, the antibiotic therapy and co-existed microbiota. Each antibiotic generates specific changes in molecular profiles. The highest number of bacterial species was isolated in the universal culture medium (72%) followed by the selective medium (48% and 38%). In the case of non-AT patients, the prevalence of Streptococcus salivarius (25%), Streptococcus vestibularis (19%), Streptococcus oralis (13%), and Staphylococcus aureus (6%) was identified while in the case of AT, Streptococcus salivarius (11%), Streptococcus parasanguinis (11%), Staphylococcus epidermidis (12%), Enterococcus faecalis (9%), Staphylococcus hominis (8%), and Candida albicans (6%) were identified. Notable to specified that the Candida albicans was noticed only in AT samples, indicating a negative impact on the antibiotic therapy.The accuracy of the MALDI-TOF MS technique was performed by the 16S rRNA gene sequencing analysis—as a reference method. Conclusively, such an approach highlighted in the present study can help in developing the methods enabling a faster diagnosis of disease changes at the cellular level before clinical changes occur. Once the MALDI tool allows for the distinguishing of the microbiota of non-AT and AT, it may enable to monitor the diseases treatment and develop a treatment regimen for individual patients in relation to each antibiotic.Key pointsThe salivary microbiota of antibiotic-treated patients was more bacteria varietyMALDI-TOF MS is a promising tool for recording of reproducible molecular profilesOur data can allow to monitor the treatment of bacterial diseases for patients

Streptococcus oralis is an oral commensal and opportunistic pathogen that can enter the bloodstream and cause bacteremia and infective endocarditis. Here, we investigated the mechanisms of S. oralis binding to oral mucins using clinical isolates, isogenic mutants and glycoconjugates. S. oralis bound to both MUC5B and MUC7, with a higher level of binding to MUC7. Mass spectrometry identified 128 glycans on MUC5B, MUC7 and the salivary agglutinin (SAG). MUC7/SAG contained a higher relative abundance of Lewis type structures, including Lewis b/y, sialyl-Lewis a/x and α2,3-linked sialic acid, compared to MUC5B. S. oralis subsp. oralis binding to MUC5B and MUC7/SAG was inhibited by Lewis b and Lacto-N-tetraose glycoconjugates. In addition, S. oralis binding to MUC7/SAG was inhibited by sialyl Lewis x. Binding was not inhibited by Lacto-N-fucopentaose, H type 2 and Lewis x conjugates. These data suggest that three distinct carbohydrate binding specificities are involved in S. oralis subsp. oralis binding to oral mucins and that the mechanisms of binding MUC5B and MUC7 differ. Efficient binding of S. oralis subsp. oralis to MUC5B and MUC7 required the gene encoding sortase A, suggesting that the adhesin(s) are LPXTG-containing surface protein(s). Further investigation demonstrated that one of these adhesins is the sialic acid binding protein AsaA .

We report 2 adult cases of invasive disease in Japan caused by Streptococcus oralis that expressed the serotype 3 pneumococcal capsule and formed mucoid colonies. Whole-genome sequencing revealed that the identical serotype 3 pneumococcal capsule locus and hyl fragment were recombined into the genomes of 2 distinct S. oralis strains.