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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.

Streptococcus pneumoniae’s polysaccharide capsule is an important virulence factor; vaccine-induced immunity to specific capsular polysaccharide effectively prevents disease. Serotype 1 S . pneumoniae is rarely found in healthy persons, but is highly invasive and a common cause of meningitis outbreaks and invasive disease outside of the United States. Here we show that genes for polysaccharide capsule similar to those expressed by pneumococci were commonly detected by polymerase chain reaction among upper respiratory tract samples from older US adults not carrying pneumococci. Serotype 1-specific genes were predominantly detected. In five oropharyngeal samples tested, serotype 1 gene belonging to S . mitis expressed capsules immunologically indistinct from pneumococcal capsules. Whole genome sequencing revealed three distinct S . mitis clones, each representing a cps1 operon highly similar to the pneumococcal cps1 reference operon. These findings raise important questions about the contribution of commensal streptococci to natural immunity against pneumococci, a leading cause of mortality worldwide.

Streptococcus mitis is the closest relative of the major human pathogen S. pneumoniae. The 2,15 Mb sequence of the Streptococcus mitis B6 chromosome, an unusually high-level beta-lactam resistant and multiple antibiotic resistant strain, has now been determined to encode 2100 genes. The accessory genome is estimated to represent over 40%, including 75 mostly novel transposases and IS, the prophage phiB6 and another seven phage related regions. Tetracycline resistance mediated by Tn5801, and an unusual and large gene cluster containing three aminoglycoside resistance determinants have not been described in other Streptococcus spp. Comparative genomic analyses including hybridization experiments on a S. mitis B6 specific microarray reveal that individual S. mitis strains are almost as distantly related to the B6 strain as S. pneumoniae. Both species share a core of over 900 genes. Most proteins described as pneumococcal virulence factors are present in S. mitis B6, but the three choline binding proteins PcpA, PspA and PspC, and three gene clusters containing the hyaluronidase gene, ply and lytA, and the capsular genes are absent in S. mitis B6 and other S. mitis as well and confirm their importance for the pathogenetic potential of S. pneumoniae. Despite the close relatedness between the two species, the S. mitis B6 genome reveals a striking X-alignment when compared with S. pneumoniae.

Streptococcus mitis has emerged as one of the leading causes of bacterial endocarditis and is related to Streptococcus pneumoniae. Antibiotic resistance has also increased among strains of S. mitis and S. pneumoniae. Phages are being reinvestigated as alternatives to antibiotics for managing infections. In this study, the two virulent phages Cp-1 (Podoviridae) and Dp-1 (Siphoviridae), previously isolated from S. pneumoniae, were found to also infect S. mitis. Microbiological assays showed that both pneumophages could not only replicate in S. mitis but also produced more visible plaques on this host. However, the burst size and phage adsorption data were lower in S. mitis as compared to S. pneumoniae. A comparison of the genomes of each phage grown on both hosts produced identical nucleotide sequences, confirming that the same phages infect both bacterial species. We also discovered that the genomic sequence of podophage Cp-1 of the Félix d'Hérelle collection is different than the previously reported sequence and thus renamed SOCP.

Identification of Mitis group streptococci (MGS) to the species level is challenging for routine microbiology laboratories. Correct identification is crucial for the diagnosis of infective endocarditis, identification of treatment failure, and/or infection relapse. Eighty MGS from Danish patients with infective endocarditis were whole genome sequenced. We compared the phylogenetic analyses based on single genes ( recA, sodA , gdh) , multigene (MLSA), SNPs, and core-genome sequences. The six phylogenetic analyses generally showed a similar pattern of six monophyletic clusters, though a few differences were observed in single gene analyses. Species identification based on single gene analysis showed their limitations when more strains were included. In contrast, analyses incorporating more sequence data, like MLSA, SNPs and core-genome analyses, provided more distinct clustering. The core-genome tree showed the most distinct clustering.

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.

To define the relationship between salivary SIgA antibodies and commensal oral bacteria, we examined the reactivity of SIgA antibodies from the saliva of four infants with their own colonizing Streptococcus mitis biovar 1 ( S. mitis bv 1) clones (ribotypes). Immunoblot analysis was used to examine reactivity of these antibodies with persistent ribotypes isolated from the mouths of the infants over the first year postpartum . Results showed that the pattern of SIgA antibody reactivity with the majority of clones increased in complexity after colonization but that most additional bands were common to other clones, indicating that they represented shared antigens. However, unique bands were identified in 75% of the selected persistent clones. We hypothesized that if strain-specific SIgA antibody was induced in response to colonization of a particular clone and contributed to its elimination from the mouth, then the appearance of unique bands would immediately precede the disappearance of the strain. Seventy-three percent of all unique bands identified in the study fulfilled this criterion. Because the mouth is an open, dynamic environment and multiple factors are believed to play a role in the immune response at mucosal surfaces, it may not be possible to conclusively define the relationship between SIgA antibody and commensal bacteria. However, our data provide evidence that SIgA antibody, reactive with unique antigens of their own colonizing strains, is produced in infants and may point to a role of this antibody in regulating colonization by S. mitis bv 1.

Background: High-throughput technologies for typing caries or health-associated bacterial populations including PCR, DNA microarrays and next-generation sequencing techniques require significant amounts of bacterial DNA. In clinical settings, the amount of sampled DNA is often limited and amplification is therefore essential. Protocols should be able to reproducibly amplify sequences in order to maintain initial sequence ratios and should not bias the representation of particular DNA sequence types. Methods: A linear amplification protocol using DNA polymerase I was modified to permit the amplification and subsequent analysis of small amounts of bacterial DNA. The protocol was tested on human oral bacterial biofilms from different sources, including carious dentine and plaque, and compared to amplification by degenerate PCR of 16S rDNA sequences. Real-time quantitative PCR of 24 bacterial species was used as a readout system to test amplified DNA against unamplified DNA. Results: The amplification protocol reliably yielded 5–10 µg DNA from as little as 12.5 ng of template DNA. Correlation coefficients between real-time quantitative PCR results from amplified and unamplified DNA were between 0.78 and 0.98. Conclusion: The optimized protocol consistently produced amplification products from minute amounts of bacterial DNA from caries and plaque; the amplification products are suitable for downstream genetic analyses.

Current understanding of dental caries considers this disease a demineralization of the tooth tissues due to the acid produced by sugar-fermenting microorganisms. Thus, caries is considered a diet- and pH-dependent process. We present here the first metagenomic analysis of the bacterial communities present at different stages of caries development, with the aim of determining whether the bacterial composition and biochemical profile are specific to the tissue affected. The data show that microbial composition at the initial, enamel-affecting stage of caries is significantly different from that found at subsequent stages, as well as from dental plaque of sound tooth surfaces. Although the relative proportion of Streptococcus mutans increased from 0.12% in dental plaque to 0.72% in enamel caries, Streptococcus mitis and Streptococcus sanguinis were the dominant streptococci in these lesions. The functional profile of caries-associated bacterial communities indicates that genes involved in acid stress tolerance and dietary sugar fermentation are overrepresented only at the initial stage (enamel caries), whereas other genes coding for osmotic stress tolerance as well as collagenases and other proteases enabling dentin degradation are significantly overrepresented in dentin cavities. The results support a scenario in which pH and diet are determinants of the disease during the degradation of enamel, but in dentin caries lesions not only acidogenic but also proteolytic bacteria are involved. We propose that caries disease is a process of varying etiology, in which acid-producing bacteria are the vehicle to penetrate enamel and allow dentin degrading microorganisms to expand the cavity.