Streptococcus Mitis Group Research Articles

Crystalline keratopathy: an infrequent corneal infection produced by the Streptococcus mitis group

The objective of this report is to describe a case of crystalline keratopathy caused by the Streptococcus mitis group corresponding to a patient who attended hospital for discomfort in her right eye. The ophthalmological examination showed an interrupted stitch of 10-0 nylon suture without tension and with attached mucus secretions. The loose suture was removed under aseptic conditions. Moxifloxacin 0.5 % eye drops were topically indicated. The treated eye successfully epithelialized and evolved favorably. However, after 15 days, a white tree-shaped infiltrate developed. A corneal sample was taken in the operating room, threading the intrastromal path of the removed stitch with a 7-0 vicryl suture. Vancomycin 50 mg/ml drops were indicated. The infiltrate, which was stable for 45 days, later increased its size and tissue necrosis occurred with danger of corneal perforation. A bipedicle conjunctival flap was performed in the affected corneal area, which evolved favorably. After spontaneous conjunctival flap retraction, only corneal scarring and neovascularization outside the visual axis were observed.

The pathogenic persona of community‐associated oral streptococci

The mitis group streptococci (MGS) are widespread in the oral cavity and are traditionally associated with oral health. However, these organisms have many attributes that contribute to the development of pathogenic oral communities. MGS adhere rapidly to saliva-coated tooth surfaces, thereby providing an attachment substratum for more overtly pathogenic organisms such as Porphyromonas gingivalis, and the two species assemble into heterotypic communities. Close physical association facilitates physiologic support, and pathogens such as Aggregatibacter actinomycetemcomitans display resource partitioning to favour carbon sources generated by streptococcal metabolism. MGS exchange information with community members through a number of interspecies signalling systems including AI-2 and contact dependent mechanisms. Signal transduction systems induced in P. gingivalis are based on protein dephosphorylation mediated by the tyrosine phosphatase Ltp1, and converge on a LuxR-family transcriptional regulator, CdhR. Phenotypic responses in P. gingivalis include regulation of hemin uptake systems and gingipain activity, processes that are intimately linked to the virulence of the organism. Furthermore, communities of S. gordonii with P. gingivalis or with A. actinomycetemcomitans are more pathogenic in animal models than the constituent species alone. We propose that MGS should be considered accessory pathogens, organisms whose pathogenic potential only becomes evident in the context of a heterotypic microbial community.

Streptococcus lactarius sp. nov., isolated from breast milk of healthy women

Three strains of a hitherto-unknown, Gram-stain-positive coccus were recovered from the milk of three non-related healthy women. The isolates shared 99 % 16S rRNA gene sequence similarity with sequences from uncultured members of the Lactobacillales and Streptococcus. The closest sequence corresponding to a defined species was that of Streptococcus peroris GTC 848(T), with a similarity of 98 %. A partial sequence (488 bp) of the tuf gene also showed 97 % similarity with that of S. peroris CCUG 39814(T). The combined 16S rRNA/tuf-based phylogeny revealed that all the isolates grouped in a statistically well-supported cluster separate from S. peroris. Enzyme activity profiles as well as fermentation patterns differentiated the novel bacteria from other members of the Streptococcus mitis group. Finally, phenotypic, genotypic and phylogenetic data supported the proposal of a novel species of the genus Streptococcus, for which the name Streptococcus lactarius sp. nov. is proposed. The type strain is MV1(T) ( = CECT 7613(T) = DSM 23027(T)).

ST-ELEVATION MYOCARDIAL INFARCTION IS OFTEN DUE TO INFECTION WITH PATHOGENIC BACTERIA ASSOCIATED WITH PERIODONTAL DISEASE

Methods: Our aim was to study the possible role of bacteria in patients with acute MI. Consecutive patients with ST-elevation MI treated with primary PCI and macroscopic thrombus aspirated during the intervention were included. Control blood sample was obtained for every patient from the arterial sheath at the end of the intervention. Ortopantomographic analyses have been performed in a subgroup of patients. Results: This preliminary analysis includes 22 patients. The patients were treated within 24 hours from symptom onset. The culprit vessel was LAD in 45%, LCX in 32% and RCA in 23%. Final TIMI3 low was achieved in 91%. The macroscopic aspirate contained red thrombus in 77%, mixed thrombi in 14% and white thrombus in 9%. Detailed DNA analysis of the thrombi revealed common oral pathogens (mainly mitis-group streptococci) in 19 (86%) of the patients. Other common bacteria (for example Chl. Pneumoniae) were not found. Control blood samples showed no evidence of ongoing bacteremia or contamination. Conclusions: Periodontal pathogens are often found in coronary thrombi of patients with acute MI. This suggests that periodontal bacteria may have a direct causative role in triggering acute MI. Patient recruitment and data analyses are ongoing and we expect to present data involving approximately 100 patients at the ACC.

Genome of Streptococcus oralis Strain Uo5

Streptococcus oralis, a commensal species of the human oral cavity, belongs to the Mitis group of streptococci, which includes one of the major human pathogens as well, S. pneumoniae. We report here the first complete genome sequence of this species. S. oralis Uo5, a high-level penicillin- and multiple-antibiotic-resistant isolate from Hungary, is competent for genetic transformation under laboratory conditions. Comparative and functional genomics of Uo5 will be important in understanding the evolution of pathogenesis among Mitis streptococci and their potential to engage in interspecies gene transfer.

Prevalence of clustered regulatory interspaced short palindromic repeat (CRISPR)-like sequences in mitis-group streptococci

Clustered regulatory interspaced short palindromic repeats (CRISPRs) have been discovered in many bacteria and archaea. Many CRISPR-like sequences have been identified in an increasing number of studies on the function of CRISPRs. One CRISPR-like sequence of approximately 240 base pairs has been found to be highly conserved within 11 genome sequences of Streptococcus pneumoniae. A specific CRISPR-like polymerase chain reaction (PCR) assay was designed with the novel primers CRISPR 5F (forward primer) 5’-CTA ATY TCA TAA CCA TAR GAA TC-3’ and CRISPR 3R (reverse primer) 5’-GAT AAR ATC CTY TAA WCT TCT AG-3’ to detect the presence of this CRISPR-like sequence in pneumococci, as well as in viridans-group streptococci (VGS). This study investigates the prevalence of this CRISPR-like sequence in S. pneumoniae and 12 viridans-group streptococcal species and shows its existence to be shared by the majority of S. pneumoniae and, to a lesser extent, S. mitis. This CRISPR-like sequence was also found in S. australis and it is highly conserved among these strains, suggesting possible biological functional differences from true CRISPR because this CRISPR-like sequence has relatively few repeat numbers, and adjacent homology of CRISPR-associated (cas) genes was absent. The sharing of this CRISPR-like sequence between pneumococci, the mitis group and other VGS, as well as its high sequence homology, may suggest close evolutionary emergence of this sequence between these species.

Differentiation of α-hemolytic streptococci by direct mass spectrometric profiling

The modern phenotypic and genetic methods except for Multi Locus Sequence Typing do not allow the reliable differentiation within Mitis group of α-hemolytic streptococci. During this study the MALDI mass spectra were acquired for 28 clinical isolates initially identified as S. pneumoniae by routine bacteriological tests. Due to Multi Locus Sequence Typing these isolates were found to belong to two closely related species - S. pneumoniae (n = 22) and S. mitis (n = 6). Distribution of those isolates in accordance with cluster analysis of collected mass spectra matched to Multi Locus Sequence Typing data. The diagnostic model based on Genetic Algorithm classifier demonstrated the differentiation of α-hemolytic streptococci with 100% sensitivity and 94.6% accuracy. Statistical analysis of MS peak areas revealed 2 peaks which are different for S. mitis and S. pneumoniae groups.

Investigation of Supragingival Plaque Microbiota in Different Caries Status of Chinese Preschool Children by Denaturing Gradient Gel Electrophoresis

This study aimed to detect differences in the richness of total supragingival plaque microbiota as well as the species composition of oral streptococci involved in the different stages of dental caries. Forty-five plaque samples were collected from caries-moderate (CM, 4 ≤ dmfs ≤ 6), caries-susceptible (CS, dmfs ≥ 10), and age-matched caries-free children separately. Total DNA was isolated directly from each sample, and polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analyses using universal and primers specific for oral streptococci were carried out. Using 16S rDNA PCR-DGGE, 34 different species of bacteria were identified in a culture-independent manner and classified into 11 genera according to phylogenetic analysis. Among them, Mitis group streptococci and Campylobacter, which were present in health status, no longer appeared in caries-susceptible samples. In addition, Capnocytophaga, Burkholderia, and Prevotella were found significantly less frequently in the CS group samples (P < 0.05), while there were no significant differences among the prevalence of Neisseria, Leptotrichia, Haemophilus, Mutans group streptococci, Corynebacterium, and Actinomyces in the three groups. Further DGGE analysis of rnpB gene amplicons obtained with oral streptococci species-specific primers showed that a total of 23 species of oral streptococci were identified. Streptococcus sanguinis, Streptococcus mitis, and Streptococcus oralis showed a significantly higher prevalence in healthy children (P < 0.05), while that of Streptococcus mutans and Streptococcus sobrinus did not vary among the three groups. Overall, these results suggest that supragingival plaque microbiota as a whole undergoes a more complicated shift in the caries process.

Comparison of MALDI TOF with conventional identification of clinically relevant bacteria

A completely new approach to diagnose microbial agents at least one day earlier based on mass spectrometric analysis becomes possible in the microbiology laboratory: MALDI TOF: matrix-assisted laser desorption/ionisation time-of-flight. Comparison between results of the new procedure with those obtained by conventional testing is mandatory. 204 clinical isolates grown on agar plates were analysed both, by the MALDI TOF Bruker microflex apparatus and by conventional identification using the VITEK II and API systems, both from bioMérieux. Of the identified isolates, 72 were gram-positive and 130 gram-negative; 2 were yeasts (candida). Concordance was seen with 61/72 (85%) of the Gram-positive bacteria and with 115/130 (88%) of the Gram-negative bacteria. In 27 samples (13.2%), a discrepancy of the species and/or genus was obvious. The discrepancy appeared with 16 gram-negative (12.2%) and with 11 gram-positive germs (15.3%, n.s.). In the latter group, 6 samples showed discordance with Streptococcus pneumoniae (MALDI) and Streptococcus mitis/oralis (conventional identification) constellation. Among gram-negative samples, most differences occurred on the species level only, e.g. Enterobacter cloacae versus Enterobacter kobei. In 5 cases, discordance was major and appeared on the genus level: Enterobacter/Raoultella, Streptococcus/Gemella, Pseumdomonas/Burkholderia, Microbacter/Sphingomonas and Candida/Cryptococcus. The most outstanding difference was Microbacterium arborescens (MALDI TOF) and Sphingomonas paucimobilis (conventional). Molecular biological identification of two Streptococcus mitis group bacteria confirmed the erroneous diagnosis by MALDI TOF of Streptococcus pneumoniae. Good comparability between MALDI TOF analysis and conventional identification procedures (86.8%) but special caution is needed when identifying streptococcal species.

Identification of horizontal gene transfer and recombination of PsaA gene in streptococcus mitis group

Pneumococcal surface adhesin A (psaA) gene is universally confirmed as one of the Streptococcus pneumoniae adhesion genes, but it is disputed whether the psaA gene is a Streptococcus pneumoniae species-specific gene. In the present study, the presence of the psaA gene in 34 streptococcus mitis group isolates was identified by the PCR approach and a comparison of sequencing PCR products (Streptococcus pneumoniae R6 as the control strain). Also, the evolutionary scenarios of these psaA genes in these streptococcus mitis group isolates were analyzed by a phylogenetic tree based on the housekeeping genes (sodA and rnpB) and psaA genes. As a result, a high degree of conservation of open reading frame sequences in all six Streptococcus pneumoniae strains (100% similarity) and in the other species of the streptococcus mitis group (92.6-100% similarity) was revealed. Further genetics research based on housekeeping genes and psaA gene phylogenies showed that the psaA gene was of vertical inheritance only in Streptococcus pneumoniae; however, high-frequency horizontal psaA gene transfer and recombination occurred in the other species of the streptococcus mitis group. These findings confirmed that the psaA gene was not a Streptococcus pneumoniae species-specific gene, and high-frequency HGT and recombination events may explain the presence of the psaA gene in the other species of the streptococcus mitis group.

Phenotypic and genomic characterization of pneumococcus-like streptococci isolated from HIV-seropositive patients

Accurate differentiation between pneumococci and other viridans streptococci is essential given their differences in clinical significance. However, classical phenotypic tests are often inconclusive, and many examples of atypical reactions have been reported. In this study, we applied various phenotypic and genotypic methods to discriminate between a collection of 12 streptococci isolated from the upper respiratory tract of HIV-seropositive individuals in 1998 and 1999. Conventional phenotypic characterization initially classified these streptococci as Streptococcus pneumoniae, as they were all sensitive to optochin and were all bile soluble. However, they did not agglutinate with anti-pneumococcal capsular antibodies and were also far more resistant to antimicrobial agents than typeable pneumococci isolated in the same period. Genotypic characterization of these isolates and control isolates by both multilocus sequence analysis (MLSA) and comparative genomic hybridization (CGH) showed that only a single isolate was genetically considered to be a true S. pneumoniae isolate, and that the remaining 11 non-typable isolates were indeed distinct from true pneumococci. Of these, 10 most closely resembled a subgroup of Streptococcus mitis isolates genetically, while one strain was identified as a Streptococcus pseudopneumoniae isolate. CGH also showed that a considerable part of the proposed pneumococcal core genome, including many of the known pneumococcal virulence factors, was conserved in the non-typable isolates. Sequencing of part of the 16S rRNA gene and investigation for the presence of ply by PCR corroborated these results. In conclusion, our findings confirm the close relationship between streptococci of the Mitis group, and show that both MLSA and CGH enable pneumococci to be distinguished from other Mitis group streptococci.

Evolution of Streptococcus pneumoniae and Its Close Commensal Relatives

Streptococcus pneumoniae is a member of the Mitis group of streptococci which, according to 16S rRNA-sequence based phylogenetic reconstruction, includes 12 species. While other species of this group are considered prototypes of commensal bacteria, S. pneumoniae is among the most frequent microbial killers worldwide. Population genetic analysis of 118 strains, supported by demonstration of a distinct cell wall carbohydrate structure and competence pheromone sequence signature, shows that S. pneumoniae is one of several hundred evolutionary lineages forming a cluster separate from Streptococcus oralis and Streptococcus infantis. The remaining lineages of this distinct cluster are commensals previously collectively referred to as Streptococcus mitis and each represent separate species by traditional taxonomic standard. Virulence genes including the operon for capsule polysaccharide synthesis and genes encoding IgA1 protease, pneumolysin, and autolysin were randomly distributed among S. mitis lineages. Estimates of the evolutionary age of the lineages, the identical location of remnants of virulence genes in the genomes of commensal strains, the pattern of genome reductions, and the proportion of unique genes and their origin support the model that the entire cluster of S. pneumoniae, S. pseudopneumoniae, and S. mitis lineages evolved from pneumococcus-like bacteria presumably pathogenic to the common immediate ancestor of hominoids. During their adaptation to a commensal life style, most of the lineages gradually lost the majority of genes determining virulence and became genetically distinct due to sexual isolation in their respective hosts.

Establishing a genetic system for ecological studies ofStreptococcus oligofermentans

Streptococcus oligofermentans is a newly characterized species belonging to the mitis group of oral streptococci. So far no correlation has been demonstrated between S. oligofermentans and dental caries. Furthermore, a reverse correlation has been observed between the number of S. oligofermentans and the number of Streptococcus mutans, a major cariogenic pathogen, in the oral cavity. These properties suggest that S. oligofermentans may have a potential to be used as a 'probiotics' for caries prevention. In this study, we aim to establish a genetic system in S. oligofermentans to further study the biology of this new species. Using homologous regions of the comCDE locus in other streptococci, the comC gene was isolated and sequenced. A synthetic competence-stimulating peptide (CSP) was synthesized and shown to be able to effectively induce competence in S. oligofermentans. This CSP-induced transformation system in S. oligofermentans was used to construct green fluorescent protein (gfp) and luciferase (luc) reporter systems, both of which are driven by the lactate dehydrogenase (ldh) promoter. These reporter systems were further shown to be highly expressed in planktonic and biofilm cells, suggesting that these reporter systems can be used in future ecological studies of S. oligofermentans.

Interactions of mitis group streptococci with sialic acid receptors

The serine-rich repeat glycoprotein Hsa in Streptococcus gordonii Challis mediates bacterial cell interactions with fetuin, glycocalicin, fibronectin and human platelets. Different strains of S. gordonii vary markedly in their adhesion levels to sialylated proteins, while other mitis group streptococci have distinct patterns of recognition of sialylated or desialylated receptor oligosaccharides.

Clinical, phenotypic, and genotypic evidence for Streptococcus sinensis as the common ancestor of anginosus and mitis groups of streptococci

In 2002, we reported the discovery of a novel species of viridans streptococcus, Streptococcus sinensis. Recently, we reported the isolation of two more strains of S. sinensis. Clinically, S. sinensis is a definite cause of infective endocarditis, a characteristic mainly pertaining to the mitis group of streptococci. Phenotypically, two of the three S. sinensis isolates were Lancefield group F, a characteristic of the anginosus group. However, none of the three strains possess the caramel smell typical of this group of streptococci. Biochemically, S. sinensis was identified in 56% of the time as members of the anginosus group, and in 33% of the time as members of the mitis group. These clinical and phenotypic properties should be governed by the presence/absence or expressivity of particular genes in the S. sinensis genome. Genotypically, phylogenetic analysis using 16S rRNA gene sequences showed that S. sinensis branched out as the first branch in the anginosus group, implying that it is the ancestor of the other members of this group. However, the bootstrap value for S. sinensis clustered with members of the anginosus group is only 47%, meaning that it is often not clustered with members of this group, but the mitis group. Furthermore, the differences in the 16S rRNA gene sequences between S. sinensis and Streptococcus intermedius (3.7%) and those between S. sinensis and Streptococcus gordonii (3.6%) are almost the same. All these indicated that it is very likely that S. sinensis is the common ancestor of the anginosus and mitis groups of streptococci. Complete genome sequencing of S. sinensis and comparative genomics studies on the S. sinensis genome and genomes of members in the anginosus and mitis groups should reveal clues to the underlying genotypic differences that govern the different phenotypic properties of the two groups of streptococci, such as why streptococci of the anginosus group are prone to cause abscess formation but not infective endocarditis as compared to other viridans streptococci.

Detection of heat shock proteins but not superantigen by isolated oral bacteria from patients with Behcet's disease

We isolated bacteria from periodontal sites and mixed saliva in eight patients with Behcet's disease and surveyed them to determine whether they produced heat shock proteins (HSPs) and superantigen. Cultivable bacterial compositions from periodontal sites and saliva were examined by anaerobic culture using blood agar plates. Gram-negative anaerobic rods such as Prevotella intermedia, Fusobacterium nucleatum, and Capnocytophaga species were predominant in the isolates from the subgingival plaque samples. The Streptococcus mitis group was the most common type isolated from the saliva samples. To detect the production of HSPs, Western blot analyses were performed using a polyclonal rabbit antibody to Escherichia coli DnaK and a monoclonal antibody to Helicobacter pylori Gro-EL. Sonic extracts of 27 of the strains (79.4%) reacted with the antibody against E. coli DnaK. Nine of these 34 strains (26.5%) were found to produce HSPs that reacted with antibody to H. pylori Gro-EL. A total of 54 isolates were examined for superantigen activity against human peripheral leukocytes. Twenty-five gram-negative clinical strains isolated from chronic periodontitis lesions and 20 ATCC strains of microorganisms were also examined. We could not detect any superantigen activity in 500x diluted supernatant of the strains isolated from the eight patients with Behcet's disease. The present study indicates that the anaerobic strains isolated from the oral cavity of these patients produce HSPs, the production being related to Bechet's disease.

Method for rapid identification of oral streptococci by PCR using 16S-23S ribosomal RNA intergenic spacer gene

Mutans group streptococci play an important role in the formation of dental caries, while mitis group streptococci are considered to be associated with subacute bacterial endocarditis. Both mitis and salivarius group streptococci, which are early colonizers in the human oral cavity, have an effect on the infection and colonization of mutans streptococci. In the present study, a rapid and exact PCR method to detect those oral streptococci was developed using the 16S-23S ribosomal RNA intergenic spacer gene, which is present between 16S rRNA and 23S rRNA within the RNA operon. The PCR analysis was able to correctly identify mutans and mitis group streptococcal species. Our results indicate that the combined PCR analysis method established in the present study is useful for detecting oral streptococcal species, as well as in clinical applications to predict and prevent dental caries and etiological studies of streptococcal infective endocarditis.

Effect of acidic pH on expression of surface-associated proteins of Streptococcus oralis.

Streptococcus oralis, a member of the mitis group of oral streptococci, is implicated in the pathogenesis of infective endocarditis and is the predominant aciduric non-mutans-group streptococcus in dental plaque. We undertook to identify the most abundant surface-associated proteins of S. oralis and to investigate changes in protein expression when the organism was grown under acidic culture conditions. Surface-associated proteins were extracted from cells grown in batch culture, separated by two-dimensional gel electrophoresis, excised, digested with trypsin, and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Putative functions were assigned by homology to a translated genomic database of Streptococcus pneumoniae. A total of 27 proteins were identified; these included a lipoprotein, a ribosome recycling factor, and the glycolytic enzymes phosphoglycerate kinase, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and enolase. The most abundant protein, phosphocarrier protein HPr, was present as three isoforms. Neither lactate dehydrogenase nor pyruvate oxidase, dominant intracellular proteins, were present among the proteins on the gels, demonstrating that proteins in the surface-associated pool did not arise as a result of cell lysis. Eleven of the proteins identified were differentially expressed when cells were grown at pH 5.2 versus pH 7.0, and these included superoxide dismutase, a homologue of dipeptidase V from Lactococcus lactis, and the protein translation elongation factors G, Tu, and Ts. This study has extended the range of streptococcal proteins known to be expressed at the cell surface. Further investigations are required to ascertain their functions at this extracellular location and determine how their expression is influenced by other environmental conditions.

Identification of streptococcus mitis group species by RFLP of the PCR-amplified 16S-23S rDNA intergenic spacer

Mitis group streptococci are pioneer colonizers of tooth surfaces and are implicated in various pathologies. Thus, accurate identification of oral mitis group strains would be valuable for studies of plaque ecology and dental caries and for diagnostic use in endocarditis or sepsis patients. The aim of this study was to evaluate the usefulness of PCR-RFLP analysis of the 16S-23S intergenic spacer for differentiating and identifying streptococcus mitis group species. The 16S-23S rDNA spacer regions of 27 type and reference Streptococcus strains, representing 8 species, were studied by PCR-mediated amplification by using oligonucleotide primers FGPS1490-72 and FGPL 132′-38. PCR products were digested, independently, with 14 restriction enzymes. Only AluI, MboI, CfoI, HinfI and MaeII distinguished some species, particularly AluI and CfoI, but not all the species. Eight clusters were clearly generated, corresponding to currently recognized species, but only with the addition of five ITS restriction patterns, generated by AluI + MboI + CfoI + HinfI + MaeII, then clustered by UPGMA, on a distance consensus matrix. The combination of these five ITS RFLP tests allowed a relatively conclusive genomic group differentiation of mitis group species. Despite this observation, more strains of each species will need to be analyzed, particularly clinical isolates, before arriving at general conclusions about the utility of ITS restrictions for identification of strains at the species level. An ITS PCR-RFLP-based identifying method for streptococcus mitis group species would provide significant advantages over other molecular taxonomic methods which require DNA extraction and DNA–DNA hybridization.

Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov., new members of the Streptococcus mitis group, isolated from human clinical specimens.

Taxonomic studies were performed on eight strains of alpha-haemolytic streptococci that showed very low DNA-DNA hybridization similarity values with all established members of the mitis group of the genus Streptococcus. These strains were isolated from the tooth surface and pharynx of humans. 16S rRNA gene sequence analysis showed that these strains belonged to the mitis group, but that they fell into two new branches. DNA-DNA hybridization demonstrated two new similarity groups. From the results of the present study, the names Streptococcus peroris sp. nov. and Streptococcus infantis sp. nov. are proposed for these new groups. The type strains are O-66T (= GTC 848T = JCM 10158T) and O-122T (= GTC 849T = JCM 10157T), respectively.