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Recently, it has been reported that eriC and crcB are involved in bacterial fluoride resistance. However, the fluoride-resistance mechanism in oral streptococci remains unclear. BLAST studies showed that two types of eriCs (eriC1 and eriC2) and two types of crcBs (crcB1 and crcB2) are present across 18 oral streptococci, which were identified in ≥ 10% of 166 orally healthy subjects with ≥ 0.01% of the mean relative abundance. They were divided into three groups based on the distribution of these four genes: group I, only eriC1; group II, eriC1 and eriC2; and group III, eriC2, crcB1, and crcB2. Group I consisted of Streptococcus mutans, in which one of the two eriC1s predominantly affected fluoride resistance. Group II consisted of eight species, and eriC1 was responsible for fluoride resistance, but eriC2 was not, in Streptococcus anginosus as a representative species. Group III consisted of nine species, and both crcB1 and crcB2 were crucial for fluoride resistance, but eriC2 was not, in Streptococcus sanguinis as a representative species. Based on these results, either EriC1 or CrcBs play a role in fluoride resistance in oral streptococci. Complementation between S. mutans EriC1 and S. sanguinis CrcB1/CrcB2 was confirmed in both S. mutans and S. sanguinis. However, neither transfer of S. sanguinis CrcB1/CrcB2 into wild-type S. mutans nor S. mutans EriC1 into wild-type S. sanguinis increased the fluoride resistance of the wild-type strain. Co-existence of different F- channels (EriC and CrcB) did not cause the additive effect on fluoride resistance in oral Streptococcus species.

Proteins harboring intrinsically disordered regions (IDRs) lacking stable secondary or tertiary structures are abundant across the three domains of life. These regions have not been systematically studied in prokaryotes. Here, our genome-wide analysis identifies extracytoplasmic serine/threonine-rich IDRs in several biologically important membrane-associated proteins in streptococci. We demonstrate that these IDRs are glycosylated with glucose by glycosyltransferases GtrB and PgtC2 in Streptococcus pyogenes and Streptococcus pneumoniae , and with N-acetylgalactosamine by a Pgf-dependent mechanism in Streptococcus mutans . The absence of glycosylation leads to a defect in biofilm formation under ethanol-stressed conditions in S. mutans . We link this phenotype to the C-terminal IDR of the post-translocation chaperone PrsA. Our data reveal that O -linked glycosylation protects the IDR-containing proteins from proteolytic degradation and is critical for the biological function of PrsA in biofilm formation. Here, the authors identify mechanisms of glycosylation of intrinsically disordered regions present in streptococci membrane proteins, uncovering a functional role for glycosylation in Streptococcus mutans chaperone PrsA.

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.

The Centers for Disease Control and Prevention's Arctic Investigations Program evaluated whole-genome sequencing (WGS) workflows and bioinformatics pipelines developed by the Centers' Streptococcus Laboratory. We compared WGS-based antimicrobial drug resistance predictions with phenotypic testing for group B (n = 130) and group A (n = 217) Streptococcus and Streptococcus pneumoniae (n = 293). Isolates were collected in Alaska during January 2019-February 2021. We also included a historical phenotypically nonsusceptible subset. Concordances between phenotypic testing and WGS predictions were 99.9% (895/896) for group B Streptococcus, 100% (1,298/1,298) for group A Streptococcus, and 99.98% (3,516/3,517) for S. pneumoniae. Common resistance determinants were ermTR, ermB, and mef for macrolides, tetM for tetracyclines, and gyrA and parC for levofloxacin. S. pneumoniae trimethoprim/sulfamethoxazole nonsusceptibility was associated with folP gene insertions and folA mutations. In 2022, the Arctic Investigations Program transitioned Streptococcus spp. workflows to WGS, enabling more rapid monitoring and prevention of invasive disease.

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.

The numerous species that make up the oral microbiome are now understood to play a key role in establishment and maintenance of oral health. The ability to taxonomically identify community members at the species level is important to elucidating its diversity and association to health and disease. We report the overall ecological effects of using a toothpaste containing enzymes and proteins compared to a control toothpaste on the plaque microbiome. The results reported here demonstrate that a toothpaste containing enzymes and proteins can augment natural salivary defences to promote an overall community shift resulting in an increase in bacteria associated with gum health and a concomitant decrease in those associated with periodontal disease. Statistical analysis shows significant increases in 12 taxa associated with gum health including Neisseria spp. and a significant decrease in 10 taxa associated with periodontal disease including Treponema spp. The results demonstrate that a toothpaste containing enzymes and proteins can significantly shift the ecology of the oral microbiome (at species level) resulting in a community with a stronger association to health.

Prophages (viral genomes integrated within a host bacterial genome) can confer various phenotypic traits to their hosts, such as enhanced pathogenicity. Here we analyse >1300 genomes of 70 different Streptococcus species and identify nearly 800 prophages and satellite prophages (prophages that do not encode their own structural components but rely on the bacterial host and another helper prophage for survival). We show that prophages and satellite prophages are widely distributed among streptococci in a structured manner, and constitute two distinct entities with little effective genetic exchange between them. Cross-species transmission of prophages is not uncommon. Furthermore, a satellite prophage is associated with virulence in a mouse model of Streptococcus pneumoniae infection. Our findings highlight the potential importance of prophages in streptococcal biology and pathogenesis. Prophages are viral genomes integrated within bacterial genomes. Here, Rezaei Javan et al. identify nearly 800 prophages and satellite prophages in > 1300 Streptococcus genomes, and show that a satellite prophage is associated with virulence in a mouse model of pneumococcal infection.

Streptococcus consists of ecologically diverse species, some of which are important pathogens of humans and animals. We sought to quantify and compare the frequencies and characteristics of within-species recombination in the pan-genomes of Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus suis . We used 1081, 1813 and 1204 publicly available genome sequences of each species, respectively. Based on their core genomes, S. agalactiae had the highest relative rate of recombination to mutation (11.5743) compared to S. pyogenes (1.03) and S. suis (0.57). The proportion of the species pan-genome that have had a history of recombination was 12.85%, 24.18% and 20.50% of the pan-genomes of each species, respectively. The composition of recombining genes varied among the three species, and some of the most frequently recombining genes are implicated in adhesion, colonization, oxidative stress response and biofilm formation. For each species, a total of 22.75%, 29.28% and 18.75% of the recombining genes were associated with prophages. The cargo genes of integrative conjugative elements and integrative and mobilizable elements contained genes associated with antimicrobial resistance and virulence. Homologous recombination and mobilizable pan-genomes enable the creation of novel combinations of genes and sequence variants, and the potential for high-risk clones to emerge.

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.

tRNA modifications are central to bacterial translational control. Here, we integrated genetics, mass spectrometry, epitranscriptomics and comparative genomics to map the tRNA modification genes of the Gram-positive pathogens Streptococcus mutans and Streptococcus pneumoniae. Both species show a marked loss of modifications dependent on Fe–S enzymes, consistent with a broader trend of Fe–S enzyme reduction in Streptococcus central metabolism. In addition, the D, m1A, m7G, t6A and i6A modifications were mapped in S. pneumoniae tRNAs, and we confirmed that a unique DusB1 enzyme is responsible for the insertion of all the detectable D modifications. We uncovered differences in queuosine (Q) metabolism: while S. mutans synthesizes Q de novo, S. pneumoniae instead salvages preQ₁ and accumulates the epoxy-Q precursor, a strategy shared with multiple other streptococci as revealed by analysis of Q pathways in 1599 sequenced streptococcal genomes. Comparative essentiality profiling of modification genes revealed notable differences, including the essentiality of the N⁶-threonylcarbamoyladenosine (t⁶A) synthesis enzyme TsaE in S. pneumoniae but not in S. mutans, which was confirmed by genetic studies. We found that suppressor mutations in asnS encoding asparaginyl-tRNA synthetase (AsnRS) restored viability to ∆tsaE mutants, albeit with reduced growth. Our finding highlights the functional importance of modifications in the recognition of tRNAs by aminoacyl-tRNA synthetases.