Rapid and Accurate Species Identification of Mitis Group Streptococci Using the MinION Nanopore Sequencer.

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 (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) 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 (n = 514), clinical isolates (n = 31), and reference strains (n = 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 (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 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 S. infantis, S. australis, S. peroris, and S. sinensis. 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.

Similar genomic patterns of clinical infective endocarditis and oral isolates of Streptococcus sanguinis and Streptococcus gordonii.

Streptococcus gordonii and Streptococcus sanguinis belong to the Mitis group streptococci, which mostly are commensals in the human oral cavity. Though they are oral commensals, they can escape their niche and cause infective endocarditis, a severe infection with high mortality. Several virulence factors important for the development of infective endocarditis have been described in these two species. However, the background for how the commensal bacteria, in some cases, become pathogenic is still not known. To gain a greater understanding of the mechanisms of the pathogenic potential, we performed a comparative analysis of 38 blood culture strains, S. sanguinis (n = 20) and S. gordonii (n = 18) from patients with verified infective endocarditis, along with 21 publicly available oral isolates from healthy individuals, S. sanguinis (n = 12) and S. gordonii (n = 9). Using whole genome sequencing data of the 59 streptococci genomes, functional profiles were constructed, using protein domain predictions based on the translated genes. These functional profiles were used for clustering, phylogenetics and machine learning. A clear separation could be made between the two species. No clear differences between oral isolates and clinical infective endocarditis isolates were found in any of the 675 translated core-genes. Additionally, random forest-based machine learning and clustering of the pan-genome data as well as amino acid variations in the core-genome could not separate the clinical and oral isolates. A total of 151 different virulence genes was identified in the 59 genomes. Among these homologs of genes important for adhesion and evasion of the immune system were found in all of the strains. Based on the functional profiles and virulence gene content of the genomes, we believe that all analysed strains had the ability to become pathogenic.

Genomic, Phenotypic, and Virulence Analysis of Streptococcus sanguinis Oral and Infective-Endocarditis Isolates.

Streptococcus sanguinis, an abundant and benign inhabitant of the oral cavity, is an important etiologic agent of infective endocarditis (IE), particularly in people with predisposing cardiac valvular damage. Although commonly isolated from patients with IE, little is known about the factors that make any particular S. sanguinis isolate more virulent than another or, indeed, whether significant differences in virulence exist among isolates. In this study, we compared the genomes of a collection of S. sanguinis strains comprised of both oral isolates and bloodstream isolates from patients diagnosed with IE. Oral and IE isolates could not be distinguished by phylogenetic analyses, and we did not succeed in identifying virulence genes unique to the IE strains. We then investigated the virulence of these strains in a rabbit model of IE using a variation of the Bar-seq (barcode sequencing) method wherein we pooled the strains and used Illumina sequencing to count unique barcodes that had been inserted into each isolate at a conserved intergenic region. After we determined that several of the genome sequences were misidentified in GenBank, our virulence results were used to inform our bioinformatic analyses, identifying genes that may explain the heterogeneity in virulence. We further characterized these strains by assaying for phenotypes potentially contributing to virulence. Neither strain competition via bacteriocin production nor biofilm formation showed any apparent relationship with virulence. Increased cell-associated manganese was, however, correlated with blood isolates. These results, combined with additional phenotypic assays, suggest that S. sanguinis virulence is highly variable and results from multiple genetic factors.

The MiiA motif is a common marker present in polytopic surface proteins of oral and urinary tract invasive bacteria.

Many surface virulence factors of bacterial pathogens show mosaicism and confounding phylogenetic origin. The Streptococcus gordonii platelet-binding GspB protein, the Streptococcus sanguinis SrpA adhesin and the Streptococcus pneumoniae DiiA protein, share an imperfect 27-residue motif. Given the disparate domain architectures of these proteins and its association to invasive disease, this motif was named MiiA from Multiarchitecture invasion-involved motif A. MiiA is predicted to adopt a beta-sheet folding, probably related to the Ig-like fold, with a symmetrical positioning of two conserved aspartic residues. A specific hidden Markov model profiling MiiA was built, which specifically detected the motif in proteins from 58 species, mainly in cell-wall proteins from Gram-positive bacteria. These proteins contained one to ten MiiA motifs, which were embedded within larger repeat units of 70-82 residues. MiiA motifs combined to other domains and elements such as coiled-coils and low-complexity regions. The species carrying MiiA-proteins included commensals from the urogenital tract and the oral cavity, which can cause opportunistic endocarditis and sepsis. Intra-protein MiiA repeats showed a complex mixture of orthologal, paralogal and inter-species relationships, suggestive of a multistep origin. Presence of these repeats in proteins involved in oligosaccharide recognition and lifestyle of species suggest a putative function for MiiA repeats in sugars binding, probably those present in receptors of epithelial and blood cells. MiiA modules appear to have been transferred horizontally between species co-habiting in the same niche to create their own MiiA-containing determinants. The present work provides a global study and a catalog of potential MiiA virulence factors that should be analyzed experimentally.

[Arbitrary primed polymerase chain reaction for the genotypic identification of Streptococcus sanguis group].

OBJECTIVE: To find out an ideal method used in identification of Streptococcus sanguis group (SSG) strains by arbitrary primed polymerase chain reaction (AP-PCR). METHODS: AP-PCR was used to distinguish SSG strains by designing 25 bp arbitrary primer 5' AAG AGA GGA GCT AGC TCT TCT TGG A 3'. RESULTS: There were great differences in the main band of DNA polymorphism among SSG species. The similar band can be obtained from the different DNA extractions in the same species. CONCLUSION: AP-PCR may be useful in the identification and classification of SSG species.

Identification of Streptococcus sanguinis with a PCR-generated species-specific DNA probe.

The objective of the present study was to design a PCR-generated DNA probe and determine the specificity of the probe for the identification of clinical isolates of Streptococcus sanguinis. To do this, we examined over 200 arbitrarily primed PCR (AP-PCR) amplicon patterns obtained with DNA from clinical isolates of S. sanguinis. A 1.6-kb DNA amplicon that was common to all AP-PCR profiles was extracted from agarose gels and then cloned and sequenced. A search for a similar sequence in the GenBank database with the BLASTN program revealed that the 1.6-kb DNA fragment comprised an intergenic region between two housekeeping genes, uncC (proton-translocating ATPase) and murA (UDP-N-acetylglucosamine enolpyruvyl transferase). Three digoxigenin-labeled DNA probes were synthesized on the basis of the sequence of the 1.6-kb fragment: the sequence of probe SSA-1 contained the proton-translocating ATPase (uncC) and the entire intergenic region, the sequence of probe SSA-2 contained only the intergenic region, and the sequence of probe SSA-3 contained an internal region of the murA gene. Dot blot hybridization showed that the three probes displayed signals for hybridization to both S. sanguinis strain ATCC 10556 and the S. sanguinis clinical isolates. Probe SSA-1, however, hybridized to DNA from S. oralis and S. mitis. Probe SSA-3 hybridized to DNA from S. gordonii, S. mitis, S. oralis, S. parasanguinis, and S. vestibularis. The probe SSA-2-specific intergenic region appeared to be specific for S. sanguinis. The results from this study suggest that probe SSA-2 may serve as a species-specific DNA probe for the identification of clinical isolates of S. sanguinis.

Prevalence of Csh-like fibrillar surface proteins among mitis group oral streptococci.

The prevalence of Csh-like fibrillar surface proteins among oral streptococci was investigated by ELISA and by immunoelectron microscopy using antiserum raised to recombinant fragments of CshA of Streptococcus gordonii DL1. The majority of S. gordonii, Streptococcus sanguis and Streptococcus oralis strains tested elaborated short (ca. 50-80 nm long) surface fibrils and reacted with antiserum to the amino acid repeat region of CshA, demonstrating the widespread nature of Csh-like proteins among these species. In contrast, reactivity with antiserum raised to the adhesion-mediating non-repetitive region of CshA was more restricted. On the basis of the ELISA results, several isolates were selected for immunogold analysis using CshA antisera. Immunogold-negative staining showed a surface distribution of 10 nm gold particles consistent with antibody binding to short fibrils. Long fibrils (>150 nm long), where present, were not significantly labelled with gold. The results suggest that some of the short peritrichous fibrils on many mitis group streptococci comprise Csh-like fibrillar protein. Further, the data are consistent with our hypothesis that the antigenically conserved amino acid repeat region of Csh-like proteins forms a scaffold for cell-distal presentation of the amino-terminal non-repetitive region that, at least in S. gordonii DL1, functions as an adhesin.

Subgingival microbiota of indigenous Indians of Central America.

OBJECTIVE: To define the subgingival microbial profiles of adult subjects from a previously identified rural community of indigenous Indians in Guatemala, Central America. MATERIALS AND METHODS: A full-mouth periodontal examination was performed in 114 adult subjects from 45 families. Plaque samples were collected from both deep and shallow periodontal pockets and checkerboard DNA-DNA hybridization was employed to identify 17 species previously associated with periodontitis or health. RESULTS: Plaque deposits and gingivitis were universal and widespread, and periodontal pocketing > or =5 mm was highly prevalent (84% of subjects). Streptococcus sanguis, Actinomyces naeslundii genospecies 2 and Fusobacterium nucleatum were significantly more prevalent in shallow sites. At the subject level, Actinomyces naeslundii and Peptostreptococcus micros were significantly more prevalent in periodontally-healthy subjects. Actinobacillus actinomycetemcomitans was not detected in any sample. CONCLUSION: There was no association between periodontal disease status and presence of suspected periodontal pathogens. These latter results conflict somewhat with those from treated populations. However, in this population where extensive plaque deposits and gingivitis are universal, the presence of putative pathogens may be more reflective of the local environment.

A protocol for polymerase chain reaction detection of Enterococcus faecalis and Enterococcus faecium from the root canal.

AIM: The present study was set up to develop a protocol for detection of Enterococcus faecalis and Enterococcus faecium from the root canal. METHODOLOGY: A collection of type strains and clinical isolates ol E. faecalis and faecium was used. Specific polymerase chain reaction (PCR) primers targeted against the 16S/23S rDNA intergenic region were used and PCR reactions were set up. PCR products were run on TBE-agarose gel and analysed. The sensitivity of the PCR systems was studied using serial dilutions of (i) bacterial DNA and (ii) bacterial cells from E. faecalis. The specificity of the identification was tested against closely related species. RESULTS: All strains of E. faecalis and E. faecium produced identical amplicon profiles composed of two major bands corresponding to sizes of 320 and 420 bp. When amplifying DNA of higher purity, a third band of 600 bp became evident as well. Closely related species demonstrated single bands of various sizes and were easily distinguished from enterococci. The detection level of DNA from serial dilutions of DNA was 10(-13) g. The DNA extraction protocol from bacterial cell suspensions resulted in a detection level of 10 bacterial cells per sample. CONCLUSIONS The present study demonstrated a good potential for using PCR technology in the detection of F. faecalis and E. faecium from root canal samples. With a high specificity the methodology was able to detect 10 cells of E. faecalis.

Endocarditis caused by penicillin-resistant viridans streptococci: 2 cases and controversies in therapy.

Although penicillin-resistant viridans streptococci have been isolated from samples from the mouth, blood, and wounds in increasing numbers, viridans streptococci isolated from patients with endocarditis have remained sensitive to penicillin for the past 5 decades. We report the cases of 2 patients with penicillin-resistant viridans streptococcal endocarditis, review 6 other cases from the literature, and summarize 2 studies that used an animal model of penicillin-resistant viridans streptococcal endocarditis.

Phenotypic and genotypic diversity of Streptococcus sanguis in infants.

Streptococcus sanguis comprises a heterogeneous group of oral streptococci indigenous to the oral cavity of humans. A total of 289 isolates from an infant population (n=37) were tentatively identified as S. sanguis on the basis of the distinctive colony morphology as shown on MM10-sucrose non-selective medium. These isolates were divided into four biovars of S. sanguis as determined by an extended panel of biochemical attributes. Chromosomal DNA was extracted from each isolate, and an AP-PCR fingerprint profile was obtained to allow study of the diversity within and among the infants. In this study, all four biovars of S. sanguis were detected in the infants. A wide genotypic diversity of S. sanguis was observed among these isolates; on average, each infant harbored 2.7 unique amplitypes as shown by the AP-PCR fingerprints. To explore the phylogenic relationship among these S. sanguis isolates, 20 strains representing the four biovars were selected at random for sequencing of their 16S rDNA and 16S-23S rDNA intergenic spacer chromosomal loci. Two major sequence patterns were identified within the 16S rDNA sequences. A phylogenic analysis showed that members from each of the four biovars of S. sanguis bore close relationship with the type-strain ATCC 10556 sequence, and that all of the isolates representing the four biovars could be clustered into two main phylotypes. The biovars were distributed throughout the phylotypes, indicating no correlation between the genetic and phenotypic groupings.

Recommended conservation of the names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, and seven other names included in the Approved Lists of Bacterial Names. Request for an opinion.

With reference to the first Principle of the International Code of Nomenclature of Bacteria, which emphasizes stability of names, it is proposed that the original names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, Erwinia ananas, Eubacterium tarantellus, Lactobacillus sake, Nitrosococcus oceanus, Pseudomonas betle, Rickettsia canada and Streptomyces rangoon, all included in the Approved Lists of Bacterial Names, be conserved. Request for an Opinion.

[Pyruvate oxidase gene from Streptococcus sanguis: molecular cloning and sequence analysis of the gene].

OBJECTIVE: To clone and sequence the gene of pyruvate oxidase (Sopox) from Streptococcus sanguis. METHODS: The PCR primers for Sopox gene were designed and synthesized according to the sequence of pyruvate oxidase (spxB) gene of S. pneumonia. The amplified PCR product was cloned into pUC18 and then subcloned into M13mp18 and M13mp19. The DNA sequence of the gene was analyzed. RESULTS: Sopox gene was successfully amplified from S. sanguis ATCC10557. The nucleotide sequence of the whole gene was revealed to be 1788 base pairs with one open reading frame coding pyruvate oxidase with 591 amino acid residuals. CONCLUSION: The clone and DNA sequence of Sopox gene were obtained which could serve as a foundation on which to elucidate the molecular mechanisms of hydrogen peroxide production and its regulation by oral streptococci.

Similarity of bacteriocin activity profiles of mutans streptococci within the family when the children acquire the strains after the age of 5.

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 > or =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.

Taxonomic study of "tufted mitior" strains of streptococci (Streptococcus sanguinis biotype 11); recognition of a new genospecies.

The taxonomic position of tufted strains of streptococci, phenotypically resembling Streptococcus mitis and previously referred to as 'tufted mitior' was investigated. By 16S rRNA sequence analysis, it was clear that the "tufted mitior" strains belonged to the mitis group of species within the genus Streptococcus. It was confirmed that these strains were taxonomically independent at the species level, sharing less than 43%, DNA-DNA similarity with all established species of the mitis group. However biochemical test data obtained, using three commercial identification kits (Rapid ID32 Strep, STREPTOGRAM, and Biolog GP-plate) together with in-house biochemical tests employing 4-MUF-linked fluorogenic substrates did not reveal sufficient differential tests with which to identify the "tufted mitior" strains unequivocally. From these data, we conclude that these "tufted mitior" strains represent a new taxon within the mitis group of the genus Streptococcus, and propose that they should be considered as a genospecies until differential phenotypic characteristics are found for their identification.

Natural history of Streptococcus sanguinis in the oral cavity of infants: evidence for a discrete window of infectivity.

The heterogeneous group of oral bacteria within the sanguinis (sanguis) streptococci comprise members of the indigenous biota of the human oral cavity. While the association of Streptococcus sanguinis with bacterial endocarditis is well described in the literature, S. sanguinis is thought to play a benign, if not a beneficial, role in the oral cavity. Little is known, however, about the natural history of S. sanguinis and its specific relationship with other oral bacteria. As part of a longitudinal study concerning the transmission and acquisition of oral bacteria within mother-infant pairs, we examined the initial acquisition of S. sanguinis and described its colonization relative to tooth emergence and its proportions in plaque and saliva as a function of other biological events, including subsequent colonization with mutans streptococci. A second cohort of infants was recruited to define the taxonomic affiliation of S. sanguinis. We found that the colonization of the S. sanguinis occurs during a discrete "window of infectivity" at a median age of 9 months in the infants. Its colonization is tooth dependent and correlated to the time of tooth emergence; its proportions in saliva increase as new teeth emerge. In addition, early colonization of S. sanguinis and its elevated levels in the oral cavity were correlated to a significant delay in the colonization of mutans streptococci. Underpinning this apparent antagonism between S. sanguinis and mutans streptococci is the observation that after mutans streptococci colonize the infant, the levels of S. sanguinis decrease. Children who do not harbor detectable levels of mutans streptococci have significantly higher levels of S. sanguinis in their saliva than do children colonized with mutans streptococci. Collectively, these findings suggest that the colonization of S. sanguinis may influence the subsequent colonization of mutans streptococci, and this in turn may suggest several ecological approaches toward controlling dental caries.

[A comparative study on the capacities of different strains of Streptococcus sanguis for P-aminobenzoic acid production].

This study was intended to compare the capacities of different strains of Strep, sanguis for P-Aminobenzoic acid (PABA) production. The synthesis of PABA during the growth of four strains of Strep. sanguis was measured by the reversed-phase high-performance liquid chromatographic method. The results showed that the concentrations of PABA synthesized by S. sanguis 10556, S. sanguis 10557, S. sanguis S34 and S. sanguis H7-4. Were 1.979 +/- 0.081 micrograms/ml, 1.383 +/- 0.193 micrograms/ml, 1.983 +/- 0.052 micrograms/ml and 1.032 +/- 0.229 micrograms/ml, respectively, and in term of PABA concentration, S. sanguis 10556 was significantly different from S. sanguis 10557 and S. sanguis H7-4; S. sanguis S34 was significantly different from S. sanguis 10557 and S. sanguis H7-4. No significant difference was found between S. sanguis 10556 and S. sanguis S34, nor between S. sanguis 10557 and S. sanguis H7-4, either. In conclusion, the method is simple, rapid and accurate. S. sanguis did synthesize PABA, and the difference in ability for PABA formation existed among the four strains of S. sanguis. This study is helpful to researches on the symbiosis between S. sanguis and S. muntans and to determination of their role in the microbial homeostasis of dental plaque.

Identification of oral mitis group streptococci by arbitrarily primed polymerase chain reaction.

"Mitis group" streptococci are commensal but may play some role in dental caries, septicemia or endocarditis. Rapid genotypic identification would aid studies of dental plaque ecology, or diagnostic use. AP-PCR with 58 unpaired arbitrary primers was used to characterize 7 Streptococcus gordonii, 11 Streptococcus sanguis, 2 Streptococcus crista, 5 Streptococcus parasanguis, 18 Streptococcus oralis, and 36 Streptococcus mitis (22 biovar 1 and 14 biovar 2). S. parasanguis 16S rRNA variable region primer RR2 produced species-specific bands with all S. gordonii and S. sanguis. Human V beta 1 T-cell receptor primer 434 yielded concordant genotypic identification of all phenotypically defined S. crista and S. parasanguis, 83% of S. oralis, and 74% of S. mitis biovar 1. Amplicon patterns for S. mitis biovar 2 were heterogeneous. Findings suggest that primers RR2 and 434 in succession will allow rapid identification of genotypic groups corresponding closely to mitis group species established by phenotype.

Whole-cell protein electrophoretic analysis of viridans streptococci: evidence for heterogeneity among Streptococcus mitis biovars.

One hundred reference strains representing all species belonging to the different phylogenetic lineages of the viridans streptococci were examined by means of one-dimensional whole-organism protein electrophoresis. For most of the species examined, multiple strains characterized by DNA-DNA hybridization were included and, wherever described, representatives of different biochemical variants were analysed. Most species were clearly differentiated. The data support the viewpoint that members of the Streptococcus anginosus group constitute a single species and indicate that Streptococcus mitis biovar 2 is a heterogeneous taxon comprising strains from several streptococcal species.