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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.

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 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.

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.

Abstract Timely surveillance of bacteremia is important for identifying emerging pathogens and to implement effective antimicrobial strategies and public health measures. Organisms in the genera Streptococcus and Enterococcus are often the cause of bacteremia and merit particular attention. The objective of this study was to determine the prevalence of bacteremia caused by several clinically significant Streptococcus species (S. pyogenes, S. agalactiae, S. pneumoniae, S. dysgalactiae/equisimilis, the S. anginosus group (S. anginosus, S. constellatus, S. intermedius), and Enterococcus species (E. faecium and E. faecalis) over a 5 year period (2017-2022). Bacteria isolated from blood culture bottles were identified by MALDI-TOF mass spectrometry (VITEK® MS). Data were obtained from a Laboratory Results Repository in Epic Beaker to include these organisms, isolated in blood cultures (BCs), patient gender, patient age, and the collection date. The data were then consolidated to yield one unique organism per patient per episode. The prevalence of organisms as a percent of the total positive BCs for the overall time period and for each year was determined. Species from the Enterococcus genus were the two most prevalent organisms for each year except for 2017. E. faecalis was the most prevalent overall (57.4%, n = 534) and for each year except for 2020. Combining all analyzed organisms, S. agalactiae was the third most prevalent organism for each year except for 2017 when it was the second and 2020-2021 when it was the fourth. However, within the Streptococcus genus alone, S. agalactiae was the most prevalent (27.5%, n = 281). S. pneumoniae, demonstrating seasonality, was the second most prevalent from 2018-2019, however, it was outranked by S. dysgalactiae/equisimilis from 2020-2022 when it was the fourth and by the S. anginosus group in 2017 and 2020-2022. S. pyogenes was the fourth most prevalent organism in 2017 and the third most in 2018; however, it decreased in subsequent years and was surpassed by all other organisms. Stratified by decade of life, patients in their seventh decade of life (60-69 yr) accounted for the most positive BCs for every organism, except for E. faecalis, followed frequently by patients in their sixth (50-59 yr) and eighth (70-79 yr) decades of life. Patients in their first decade of life (0-9 yr) accounted for a preponderance of positive BCs for E. faecalis, S. agalactiae (highest prevalence <7 days of life), and S. pneumoniae. This study highlighted the relative abundance of bacteremia caused by Enterococcus species. Additionally, it demonstrated the importance of Streptococcus agalactiae bacteremia, especially in the first and seventh decades of life. Finally, this study showed the increasing prevalence of organisms within the Streptococcus anginosus group and Streptococcus dysgalactiae/equisimilis. This analysis demonstrated the dynamics of bacteremia and prevalence among several species within the Enteroccocus and Streptococcus genera.

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.

Purpose Streptococcal bloodstream infections (BSIs) are common, yet prognostic factors are poorly investigated. We aimed to investigate the mortality according to streptococcal species and seasonal variation. Methods Patients with streptococcal BSIs from 2008 to 2017 in the Capital Region of Denmark were investigated, and data were crosslinked with nationwide registers for the identification of comorbidities. A multivariable logistic regression analysis was performed to assess mortality according to streptococcal species and season of infection. Results Among 6095 patients with a streptococcal BSI (mean age 68.1 years), the 30-day mortality was 16.1% and the one-year mortality was 31.5%. With S. pneumoniae as a reference, S. vestibularis was associated with a higher adjusted mortality both within 30 days (odds ratio (OR) 2.89 [95% confidence interval (CI) 1.20–6.95]) and one year (OR 4.09 [95% CI 1.70–9.48]). One-year mortality was also higher in S. thermophilus , S. constellatus , S. parasanguinis , S. salivarius , S. anginosus , and S. mitis/oralis . However, S. mutans was associated with a lower one-year mortality OR 0.44 [95% CI 0.20–0.97], while S. gallolyticus was associated with both a lower 30-day (OR 0.42 [95% CI 0.26–0.67]) and one-year mortality (OR 0.66 [95% CI 0.48–0.93]). Furthermore, with infection in the summer as a reference, patients infected in the winter and autumn had a higher association with 30-day mortality. Conclusions The mortality in patients with streptococcal BSI was associated with streptococcal species. Further, patients with streptococcal BSIs infected in the autumn and winter had a higher risk of death within 30 days, compared with patients infected in the summer.

Background: Streptococcus suis is a zoonotic pathogen that infects pigs and can occasionally cause serious infections in humans. S. suis infections occur sporadically in human Europe and North America, but a recent major outbreak has been described in China with high levels of mortality. The mechanisms of S. suis pathogenesis in humans and pigs are poorly understood. Methodology/Principal Findings: The sequencing of whole genomes of S. suis isolates provides opportunities to investigate the genetic basis of infection. Here we describe whole genome sequences of three S. suis strains from the same lineage: one from European pigs, and two from human cases from China and Vietnam. Comparative genomic analysis was used to investigate the variability of these strains. S. suis is phylogenetically distinct from other Streptococcus species for which genome sequences are currently available. Accordingly, approximately 40% of the approximately 2 Mb genome is unique in comparison to other Streptococcus species. Finer genomic comparisons within the species showed a high level of sequence conservation; virtually all of the genome is common to the S. suis strains. The only exceptions are three approximately 90 kb regions, present in the two isolates from humans, composed of integrative conjugative elements and transposons. Carried in these regions are coding sequences associated with drug resistance. In addition, small-scale sequence variation has generated pseudogenes in putative virulence and colonization factors. Conclusions/Significance: The genomic inventories of genetically related S. suis strains, isolated from distinct hosts and diseases, exhibit high levels of conservation. However, the genomes provide evidence that horizontal gene transfer has contributed to the evolution of drug resistance.

Many commensal oral streptococci generate H 2 O 2 via pyruvate oxidase (SpxB) to inhibit the growth of competing bacteria like Streptococcus mutans , a major cariogenic species. In Streptococcus sanguinis SK36 (SK36) and Streptococcus gordonii DL1 (DL1), spxB expression and H 2 O 2 release are subject to carbon catabolite repression by the catabolite control protein A (CcpA). Surprisingly, ccpA deletion mutants of SK36 and DL1 fail to inhibit S. mutans despite their production of otherwise inhibitory levels of H 2 O 2 . Using H 2 O 2 -deficient spxB deletion mutants of SK36 and DL1, it was subsequently discovered that both strains confer protection in trans to other bacteria when H 2 O 2 is added exogenously. This protective effect depends on the direct detoxification of H 2 O 2 by the release of pyruvate. The pyruvate dependent protective effect is also present in other spxB -encoding streptococci, such as the pneumococcus, but is missing from spxB -negative species like S. mutans . Targeted and transposon-based mutagenesis revealed Nox (putative H 2 O-forming NADH dehydrogenase) as an essential component required for pyruvate release and oxidative protection, while other genes such as sodA and dps play minor roles. Furthermore, pyruvate secretion is only detectable in aerobic growth conditions at biofilm-like cell densities and is responsive to CcpA-dependent catabolite control. This ability of spxB -encoding streptococci reveals a new facet of the competitive interactions between oral commensals and pathobionts and provides a mechanistic basis for the variable levels of inhibitory potential observed among H 2 O 2 -producing strains of commensal oral streptococci.

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.