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Multidrug-resistant Streptococcus pneumoniae strains were isolated from blood and sputum of a patient with disseminated intravascular coagulation in Sapporo city, Japan. These antibiograms were only susceptible to vancomycin, linezolid, daptomycin, some carbapenems, and some fluoroquinolones. Identical antibiograms, serotypes (19F), and sequence types (ST10017) suggested a shared origin of these isolates. Only one ST10017 strain has been isolated in the same city in Japan previously (2014), and the 2014 isolate is still susceptible to macrolides. The whole genome of the blood-derived isolate was sequenced. The strain harbored resistance mutations in parC, gyrA, pbp1a, pbp2a, pbp2b, and pbp2x, and harbored the resistance genes, ermB and tetM. The nucleotide sequences of parC and pbp2x genes of strain MDRSPN001 were clearly different from those of other S. pneumoniae strains and were similar to those of oral streptococci strains. These findings suggest that strain MDRSPN001 has been rapidly and drastically evolving multidrug resistance by gene replacement and accumulation of genes originating from other strains, such as oral streptococci, Streptococcus mitis.

A 57-year-old man presented with abdominal pain, hematemesis, and melena. He reported taking high-dose ibuprofen for back pain and drinking several 24-ounce beers daily. Examination was remarkable for icteric sclera, poor dentition, tachycardia, and crescendo-decrescendo murmur at right upper sternal border, radiating to the carotids. Labs revealed leukocytosis, anemia, thrombocytopenia, and elevated liver function tests and INR. Endoscopy demonstrated antral ulcers, duodenitis, and esophagitis. Blood cultures were obtained and broad-spectrum antibiotics started; cultures later grew Streptococcus mitis, and antibiotic coverage was narrowed. Transthoracic echocardiogram (TTE) demonstrated aortic stenosis and regurgitation, but no vegetation. Repeat blood cultures were negative; however, the patient developed neurological symptoms concerning for cauda equina syndrome, and MRI revealed epidural abscess. Emergent decompression could not be performed as the patient developed hematemesis and required intubation. Transesophageal echocardiogram (TEE), initially deferred due to friable esophageal mucosa, was performed and revealed small aortic valve vegetation. Poor oral hygiene was felt to be the probable source of the patient’s S. mitis bacteremia, epidural abscess, and infective endocarditis. The patient’s neurological symptoms resolved without intervention and remaining teeth were extracted. This case demonstrates that Streptococcus mitis can result in clinically significant bacteremia, particularly in immunocompromised patients, including chronic heavy alcohol users.

PURPOSE: Streptococcus oralis and Streptococcus mitis belong to the Mitis group, which are mostly commensals in the human oral cavity. Even though S. oralis and S. mitis are oral commensals, they can be opportunistic pathogens causing infective endocarditis. A recent taxonomic re-evaluation of the Mitis group has embedded the species Streptococcus tigurinus and Streptococcus dentisani into the species S. oralis as subspecies. In this study, the distribution of virulence factors that contribute to bacterial immune evasion, colonization and adhesion was assessed in clinical strains of S. oralis (subsp. oralis, subsp. tigurinus and subsp. dentisani) and S. mitis.METHODOLOGY: Forty clinical S. oralis (subsp. oralis, subsp. dentisani and subsp. tigurinus) and S. mitis genomes were annotated with the pipeline PanFunPro and aligned against the VFDB database for assessment of virulence factors.Results/Key findings. Three homologues of pavA, psaA and lmb, encoding adhesion proteins, were present in all strains. Seven homologues of nanA, nanB, ply, lytA, lytB, lytC and iga, of importance regarding survival in blood and modulation of the human immune system, were variously present in the genomes. Few S. oralis subspecies specific differences were observed. iga homologues were identified in S. oralis subsp. oralis, whereas lytA homologues were identified in S. oralis subsp. oralis and subsp. tigurinus.CONCLUSION: Differences in the presence of virulence factors among the three S. oralis subspecies were observed. The virulence gene profiles of the 40 S. mitis and S. oralis (subsp. oralis, subsp. dentisani and subsp. tigurinus) contribute with important new knowledge regarding these species and new subspecies.

Streptococcus mitis colonizes all niches of the human oral cavity from early infancy and throughout life. Monocytes patrol blood vessels, lymphoid and non-lymphoid tissues and migrate into infected tissue where they participate in the inflammatory cascade and immune regulation. Here, we studied the effect of S. mitis on monocytes. Transcriptome analysis of monocytes exposed to S. mitis (SmMo) revealed increased transcription of chemotactic factors (CCL2, CCL3, CCL20, CXCL1, CXCL2) and cytokines (IL1A, IL1B, IL6, IL23, IL36G, TNF), indicating that S. mitis may trigger recruitment of leucocytes and initiate inflammation. Increased transcription in SmMo of IL1B, IL6 and IL23 indicated that S. mitis may participate in the induction of Th17 responses and agreed with our earlier findings of S. mitis-mediated memory Th17 reactivity. Furthermore, S. mitis inhibited tetanus toxoid-specific CD4 T cell proliferation. This can be due to the increased secretion of IL-10 and expression of PD-L1 that was observed in SmMo. PGE2 can modulate IL-10 and PD-L1 expression, concomitant with that of CCR7, IL-12 and IL-23 that also were changed. This, along with increased SmMo transcription of PTGS2 (COX2) and PTGER4 (EP4), pointed to a role of PGE2. Measurement of PGE2 secretion by SmMo showed indeed a marked increase, and chemical inhibition of PGE2 production lowered the PD-L1 expression on SmMo. In conclusion, our findings show that S. mitis may trigger immune modulation by recruiting immune cells to the site of infection, while at the same time dampening the severity of the response through expression of IL-10, PGE2 and PD-L1.

Streptococcus pneumoniae is responsible for 240,000 - 460,000 deaths in children under 5 years of age each year. Accurate identification of pneumococcal serotypes is important for tracking the distribution and evolution of serotypes following the introduction of effective vaccines. Recent efforts have been made to infer serotypes directly from genomic data but current software approaches are limited and do not scale well. Here, we introduce a novel method, SeroBA, which uses a hybrid assembly and mapping approach. We compared SeroBA against real and simulated data and present results on the concordance and computational performance against a validation dataset, the robustness and scalability when analysing a large dataset, and the impact of varying the depth of coverage in the cps locus region on sequence-based serotyping. SeroBA can predict serotypes, by identifying the cps locus, directly from raw whole genome sequencing read data with 98% concordance using a k-mer based method, can process 10,000 samples in just over 1 day using a standard server and can call serotypes at a coverage as low as 10x. SeroBA is implemented in Python3 and is freely available under an open source GPLv3 license from: https://github.com/sanger-pathogens/seroba.DATA SUMMARYThe reference genome Streptococcus pneumoniae ATCC 700669 is available from National Center for Biotechnology Information (NCBI) with the accession number: FM211187Simulated paired end reads for experiment 2 have been deposited in FigShare: https://doi.org/10.6084/m9.figshare.5086054.v1Accession numbers for all other experiments are listed in Supplementary Table S1 and Supplementary Table S2.I/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. ⊠IMPACT STATEMENTThis article describes SeroBA, a A-mer based method for predicting the serotypes of Streptococcus pneumoniae from Whole Genome Sequencing (WGS) data. SeroBA can identify 92 serotypes and 2 subtypes with constant memory usage and low computational costs. We showed that SeroBA is able to reliably predict serotypes at a depth of coverage as low as 10x and is scalable to large datasets.

ObjectivesAmong viridans group streptococcal infective endocarditis (IE), the Streptococcus mitis group is the most common aetiological organism. Treatment of IE caused by the S. mitis group is challenging due to the high frequency of β-lactam resistance, drug allergy and intolerability of mainstay antimicrobial agents such as vancomycin or gentamicin. Daptomycin has been suggested as an alternative therapeutic option in these scenarios based on its excellent susceptibility profile against S. mitis group strains . However, the propensity of many S. mitis group strains to rapidly evolve stable, high-level daptomycin resistance potentially limits this approach.MethodsWe evaluated the activity of 6 mg/kg/day daptomycin alone or in combination with gentamicin, ceftriaxone or ceftaroline against two daptomycin-susceptible S. mitis group strains over 96 h in a pharmacokinetic/pharmacodynamic model of simulated endocardial vegetations.ResultsDaptomycin alone was not bactericidal and high-level daptomycin resistance evolved at 96 h in both organisms. Combinations of daptomycin + ceftriaxone and daptomycin + ceftaroline demonstrated enhanced killing activity compared with each antibiotic alone and prevented emergence of daptomycin resistance at 96 h. Use of gentamicin as an adjunctive agent neither improved the efficacy of daptomycin nor prevented the development of daptomycin resistance.ConclusionsAddition of ceftriaxone or ceftaroline to daptomycin improves the bactericidal activity against S. mitis group strains and prevents daptomycin resistance emergence. Further investigation with combinations of daptomycin and β-lactams in a large number of strains is warranted to fully elucidate the clinical implications of such combinations for treatment of S. mitis group IE.

Penicillin-resistant Streptococcus pneumoniae strains are found at high rates in Romania and Iran. The mosaic structure of PBP2x was investigated in 9 strains from Iran and in 15 strains from Romania to understand their evolutionary history. Mutations potentially important for β-lactam resistance were identified by comparison of the PBP2x sequences with the sequence of the related PBP2x of reference penicillin-sensitive S. mitis strains. Two main PBP2x mosaic gene families were recognized. Eight Iranian strains expressed PBP2x variants in group 1, which had a mosaic block highly related to PBP2x of the Spain23F-1 clone, which is widespread among international penicillin-resistant S. pneumoniae clones. A second unique PBP2x group was observed in Romanian strains; furthermore, three PBP2x single mosaic variants were found. Sequence blocks of penicillin-sensitive strain S. mitis 658 were common among PBP2x variants from strains from both countries. Each PBP2x group contained specific signature mutations within the transpeptidase domain, documenting the existence of distinct mutational pathways for the development of penicillin resistance.

Lactational mastitis constitutes one of the main causes of undesired weaning, depriving the mother–infant pair from the benefits of breastfeeding; therefore, this condition should be considered a relevant public health issue. The role of specific microorganisms remains unclear since human milk cultures and antibiotic susceptibility testing (AST) are not routinely performed, despite the fact that this would be key to ensure an early and effective diagnosis and treatment. The objective of this study was to describe the culturable microbial diversity in 647 milk samples from breastfeeding women with clinical symptoms of mastitis by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF) VITEK MS technology and to analyze the antimicrobial susceptibility profiles of a collection of isolates from these samples by the VITEK 2 AST system. Staphylococcus epidermidis was the most common species isolated from mastitis samples (87.6%), while Staphylococcus aureus was detected in 22.1%. Streptococci constituted the second (68.6%) most prevalent bacterial group, with Streptococcus mitis/oralis, Streptococcus salivarius, and Streptococcus parasanguinis detected with frequencies of 40.8, 36.8, and 14.4%, respectively. The antibiotic susceptibility profiles of 642 staphylococcal isolates indicated a remarkable resistance to benzylpenicillin (88.3%) and erythromycin (67.3%) with differences between species. A high percentage of Staphylococcus isolates were resistant to at least one antibiotic (Staphylococcus hominis, 100%; S. epidermidis, 98.2%; S. aureus, 92.9%; Staphylococcus lugdunensis, 90.5%) and the percentage of multidrug-resistance (MDR) isolates was noticeable (S. hominis, 81%; S. epidermidis, 64.4%; S. aureus, 11.5%; S. lugdunensis, 10.5%). In relation to streptococcal isolates (n = 524), AST revealed high or moderate percentages of resistance to erythromycin (68.7%), benzylpenicillin (63.7%), ampicillin (51.5%), and tetracycline (30.8%). Antibiotic resistance to at least one antibiotic was detected in 97.6% of S. parasanguinis, 92.6% of S. salivarius, 83.3% of S. mitis/oralis, and 72.4% of Streptococcus vestibularis isolates. A significant number of MDR streptococcal isolates was also found (S. parasanguinis, 51.2%; S. salivarius, 39.3%; S. mitis/oralis, 34.6%; and S. vestibularis, 19%). The results highlight the important role of coagulase-negative staphylococci and streptococci as human mastitis-causing agents. Moreover, the high rates of antimicrobial resistance among these microorganisms must be contemplated as an issue of clinical relevance in relation to treatment options.

Streptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor., Streptococcus pneumoniae isolates of serotype 23F with intermediate penicillin resistance were recovered on seven occasions over a period of 37 months from a cystic fibrosis patient in Berlin. All isolates expressed the same multilocus sequence type (ST), ST10523. The genome sequences of the first and last isolates, D122 and D141, revealed the absence of two phage-related gene clusters compared to the genome of another ST10523 strain, D219, isolated earlier at a different place in Germany. Genomes of all three strains carried the same novel mosaic penicillin-binding protein (PBP) genes, pbp2x, pbp2b, and pbp1a; these genes were distinct from those of other penicillin-resistant S. pneumoniae strains except for pbp1a of a Romanian S. pneumoniae isolate. All PBPs contained mutations that have been associated with the penicillin resistance phenotype. Most interestingly, a mosaic block identical to an internal pbp2x sequence of ST10523 was present in pbp2x of Streptococcus mitis strain B93-4, which was isolated from the same patient. This suggests interspecies gene transfer from S. pneumoniae to S. mitis within the host. Nearly all genes expressing surface proteins, which represent major virulence factors of S. pneumoniae and are typical for this species, were present in the genome of ST10523. One exception was the hyaluronidase gene hlyA, which contained a 12-nucleotide deletion within the promoter region and an internal stop codon. The lack of a functional hyaluronidase might contribute to the ability to persist in the host for an unusually long period of time. IMPORTANCE Streptococcus pneumoniae is a common resident in the human nasopharynx. However, carriage can result in severe diseases due to a unique repertoire of pathogenicity factors that are rare in closely related commensal streptococci. We investigated a penicillin-resistant S. pneumoniae clone of serotype 23F isolated from a cystic fibrosis patient on multiple occasions over an unusually long period of over 3 years that was present without causing disease. Genome comparisons revealed an apparent nonfunctional pneumococcus-specific gene encoding a hyaluronidase, supporting the view that this enzyme adds to the virulence potential of the bacterium. The 23F clone harbored unique mosaic genes encoding penicillin resistance determinants, the product of horizontal gene transfer involving the commensal S. mitis as donor species. Sequences identical to one such mosaic gene were identified in an S. mitis strain from the same patient, suggesting that in this case S. pneumoniae played the role of donor.

This study aimed to study whether the Sortase A (srtA) gene helps mediate coaggregation and co-adherence between Streptococcus mutans (S. mutans) and other salivary bacteria. S. mutans UA159 and srtA-deficient mutant served as "bait" in classical co-aggregation assays and membrane-based co-adherence assays were used to examine interactions of S. mutans with Fusobacterium nucleatum (F. nucleatum), Streptococcus mitis (S. mitis), Streptococcus gordonii (S. gordonii), Streptococcus sanguis (S. sanguis), Actinomyces naeslundii (A. naeslundii) and Lactobacillus. Co-adherence assays were also performed using unfractionated saliva from healthy individuals. Co-adhering partners of S. mutans were sensitively detected using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Both UA159 and its srtA-deficient mutant bound to F. nucleatum but not to any of the other five salivary bacteria. The srtA-deficient mutant showed lower co-adherence with F.nucleatum. The two S. mutans strains also showed similar co-adherence profiles against unfractionated salivary bacteria, except that UA159 S. mutans but not the srtA-deficient bound to a Neisseria sp. under the same conditions. Deleting srtA reduces the ability of S. mutans to bind to F.nucleatum, but it does not appear to significantly affect the binding profile of S. mutans to bulk salivary bacteria.

The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies’ samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.

Studies on the possible association between bacteria and oral squamous cell carcinoma (OSCC) remain inconclusive, largely due to methodological variations/limitations. The objective of this study was to characterize the species composition as well as functional potential of the bacteriome associated with OSCC. DNA obtained from 20 fresh OSCC biopsies (cases) and 20 deep-epithelium swabs (matched control subjects) was sequenced for the V1-V3 region using Illumina’s 2 × 300 bp chemistry. High quality, non-chimeric merged reads were classified to species level using a prioritized BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp. polymorphum was the most significantly overrepresented species in the tumors followed by Pseudomonas aeruginosa and Campylobacter sp. Oral taxon 44, while Streptococcus mitis, Rothia mucilaginosa and Haemophilus parainfluenzae were the most significantly abundant in the controls. Functional prediction showed that genes involved in bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors while those responsible for DNA repair and combination, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, ribosome biogenesis and glycolysis/gluconeogenesis were significantly associated with the controls. This is the first epidemiological evidence for association of F. nucleatum and P. aeruginosa with OSCC. Functionally, an “inflammatory bacteriome” is enriched in OSSC.

Background This study of the oropharyngeal microbiome complements the previously published AZIthromycin in Severe ASThma (AZISAST) clinical trial, where the use of azithromycin was assessed in subjects with exacerbation-prone severe asthma. Here, we determined the composition of the oropharyngeal microbial community by means of deep sequencing of the amplified 16S rRNA gene in oropharyngeal swabs from patients with exacerbation-prone severe asthma, at baseline and during and after 6 months treatment with azithromycin or placebo. Results A total of 1429 OTUs were observed, of which only 59 were represented by more than 0.02% of the reads. Firmicutes, Bacteroidetes, Fusobacteria, Proteobacteria and Actinobacteria were the most abundant phyla and Streptococcus and Prevotella were the most abundant genera in all the samples. Thirteen species only accounted for two thirds of the reads and two species only, i.e. Prevotella melaninogenica and Streptococcus mitis/pneumoniae, accounted for one fourth of the reads. We found that the overall composition of the oropharyngeal microbiome in patients with severe asthma is comparable to that of the healthy population, confirming the results of previous studies. Long term treatment (6 months) with azithromycin increased the species Streptococcus salivarius approximately 5-fold and decreased the species Leptotrichia wadei approximately 5-fold. This was confirmed by Boruta feature selection, which also indicated a significant decrease of L. buccalis/L. hofstadtii and of Fusobacterium nucleatum. Four of the 8 treated patients regained their initial microbial composition within one month after cessation of treatment. Conclusions Despite large diversity of the oropharyngeal microbiome, only a few species predominate. We confirm the absence of significant differences between the oropharyngeal microbiomes of people with and without severe asthma. Possibly, long term azithromycin treatment may have long term effects on the composition of the oropharygeal microbiome in half of the patients. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-1022-6) contains supplementary material, which is available to authorized users.

Among the viridans group streptococci, the Streptococcus mitis group is the most common cause of infective endocarditis. These bacteria have a propensity to be β-lactam resistant, as well as to rapidly develop high-level and durable resistance to daptomycin (DAP). We compared a parental, daptomycin-susceptible (DAP s ) S. mitis/S. oralis strain and its daptomycin-resistant (DAP r ) variant in a model of experimental endocarditis in terms of (i) their relative fitness in multiple target organs in this model (vegetations, kidneys, spleen) when animals were challenged individually and in a coinfection strategy and (ii) their survivability during therapy with daptomycin-gentamicin (an in vitro combination synergistic against the parental strain). The DAP r variant was initially isolated from the cardiac vegetations of animals with experimental endocarditis caused by the parental DAP s strain following treatment with daptomycin. The parental strain and the DAP r variant were comparably virulent when animals were individually challenged. In contrast, in the coinfection model without daptomycin therapy, at both the 10 6 - and 10 7 -CFU/ml challenge inocula, the parental strain outcompeted the DAP r variant in all target organs, especially the kidneys and spleen. When the animals in the coinfection model of endocarditis were treated with DAP-gentamicin, the DAP s strain was completely eliminated, while the DAP r variant persisted in all target tissues. These data underscore that the acquisition of DAP r in S. mitis/S. oralis does come at an intrinsic fitness cost, although this resistance phenotype is completely protective against therapy with a potentially synergistic DAP regimen.

Background Our previous research on the diversity of microbiota in the endotracheal tubes (ETTs) of neonates in the neonatal intensive care unit found that Pseudomonas aeruginosa (P. aeruginosa) and Streptococcus mitis (S. mitis) were the dominant bacteria on the ETT surface and the existence of S. mitis could promote biofilm formation and pathogenicity of P. aeruginosa. Toll-like receptor 4 (TLR4), which has been widely detected on the surface of airway epithelial cells, is the important component of the innate immune system. Therefore, we hypothesized that the co-existence of these two bacteria might impact the host immune system through TLR4 signaling. Results S. mitis rarely caused inflammation, whereas P. aeruginosa caused the most severe inflammation accompanied by increases in the number of inflammatory cells, interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression, and total cell counts in BALF (p

Despite the benefits of MALDI-TOF MS technology (Matrix-Assisted Laser Desorption-Ionization Time-Of-Flight Mass Spectrometry) reported worldwide and the continuous improving of the available databases, discrimination between Streptococcus pneumoniae and closely related species within the Streptococcus mitis group (SMG) using this methodology has been suboptimal. However, the accurate identification at the species level of this group of microorganisms is important for the appropriate management of infected patients. In this study, 216 SMG isolates -101 S. pneumoniae and 115 corresponding to 7 non-pneumococcal species within this group- were analyzed. All the isolates had been previously identified by conventional methods (optochin and bile solubility tests) and non-pneumococcal isolates were confirmed by sequence analysis (sodA and plys genes) when required. The isolates were also identified with the MALDI Biotyper 3.1 (Bruker Daltonics, Bremen, Germany) using an updated library containing 6,903 Main Spectra Profiles (MSPs). All the analyzed S. pneumoniae were correctly identified with MALDI-TOF MS at species level using the most updated database and all the non-pneumococcal SMG isolates were also identified at the group level. Several peaks (4,964.32, 6,888.90, and 9,516.46 m/z) have been found to be specific of S. pneumoniae, whilst a different set of peaks have proved to be present only in S. mitis (6,839.07 m/z) and S. oralis (5,297.61, 5822.53, and 6,839.07 m/z). Peak analysis allowed correct species assignment of 101/101 S. pneumoniae isolates (100%) and 102/105 S. mitis/oralis isolates (97.1%). Thus, the implementation of MALDI-TOF MS plus peak analysis for the identification of this group of microorganisms may provide precise species-level information that will allow the early implementation of directed antibiotic therapy.

Synthesis and integrity of the cytoplasmic membrane are fundamental to cellular life. Experimental evolution studies have hinted at unique physiology in the Gram-positive bacteria Streptococcus mitis and S. oralis . These organisms commonly cause bacteremia and infectious endocarditis (IE) but are rarely investigated in mechanistic studies of physiology and evolution. Unlike in other Gram-positive pathogens, high-level (MIC ≥ 256 μg/ml) daptomycin resistance rapidly emerges in S. mitis and S. oralis after a single drug exposure. In this study, we found that inactivating mutations in cdsA are associated with high-level daptomycin resistance in S. mitis and S. oralis IE isolates. This is surprising given that cdsA is an essential gene for life in commonly studied model organisms. CdsA is the enzyme responsible for the synthesis of CDP-diacylglycerol, a key intermediate for the biosynthesis of all major phospholipids in prokaryotes and most anionic phospholipids in eukaryotes. Lipidomic analysis by liquid chromatography-mass spectrometry (LC-MS) showed that daptomycin-resistant strains have an accumulation of phosphatidic acid and completely lack phosphatidylglycerol and cardiolipin, two major anionic phospholipids in wild-type strains, confirming the loss of function of CdsA in the daptomycin-resistant strains. To our knowledge, these daptomycin-resistant streptococci represent the first model organisms whose viability is CdsA independent. The distinct membrane compositions resulting from the inactivation of cdsA not only provide novel insights into the mechanisms of daptomycin resistance but also offer unique opportunities to study the physiological functions of major anionic phospholipids in bacteria.

Streptococcus mitis (S. mitis) is a pioneer commensal bacterial species colonizing many of the surfaces of the oral cavity in healthy individuals. Yet, not much information is available regarding its interaction with the host. We used examination of its transcriptional regulation in oral keratinocytes to elucidate some of its potential roles in the oral cavity. Transcription factor analysis of oral keratinocytes predicted S. mitis-mediated activation of aryl hydrocarbon receptor (AhR). Activation and functionality of AhR was confirmed through nuclear translocation determined by immunofluorescence microscopy and real-time polymerase chain reaction with reverse transcription analysis of CYP1A1, the hallmark gene for AhR activation. Addition of Streptococcus mutans or Streptococcus gordonii did not induce CYP1A1 transcription in the keratinocyte cultures. Introduction of an AhR-specific inhibitor revealed that S. mitis-mediated transcription of CXCL2 and CXCL8 was regulated by AhR. Elevated levels of prostaglandin E2 (enzyme-linked immunosorbent assay) in supernatants from S. mitis-treated oral epithelial cells were also attenuated by inhibition of AhR activity. The observed AhR-regulated activities point to a contribution of S. mitis in the regulation of inflammatory responses and thereby to wound healing in the oral cavity. The concept that the oral commensal microbiota can induce AhR activation is important, also in view of the role that AhR has in modulation of T-cell differentiation and as an anti-inflammatory factor in macrophages.

Streptococcus mitis/oralis is an important pathogen, causing life-threatening infections such as endocarditis and severe sepsis in immunocompromised patients. The β-lactam antibiotics are the usual therapy of choice for this organism, but their effectiveness is threatened by the frequent emergence of resistance. The lipopeptide daptomycin (DAP) has been suggested for therapy against such resistant S. mitis/oralis strains due to its in vitro bactericidal activity and demonstrated efficacy against other Gram-positive pathogens. Unlike other bacteria, however, S. mitis/oralis has the unique ability to rapidly develop stable, high-level resistance to DAP upon exposure to the drug both in vivo and in vitro . Using isogenic DAP-susceptible and DAP-resistant S. mitis/oralis strain pairs, we describe a mechanism of resistance to both DAP and cationic antimicrobial peptides that involves loss-of-function mutations in cdsA (encoding a phosphatidate cytidylyltransferase). CdsA catalyzes the synthesis of cytidine diphosphate-diacylglycerol, an essential phospholipid intermediate for the production of membrane phosphatidylglycerol and cardiolipin. DAP-resistant S. mitis/oralis strains demonstrated a total disappearance of phosphatidylglycerol, cardiolipin, and anionic phospholipid microdomains from membranes. In addition, these strains exhibited cross-resistance to cationic antimicrobial peptides from human neutrophils (i.e., hNP-1). Interestingly, CdsA-mediated changes in phospholipid metabolism were associated with DAP hyperaccumulation in a small subset of the bacterial population, without any binding by the remaining larger population. Our results indicate that CdsA is the major mediator of high-level DAP resistance in S. mitis/oralis and suggest a novel mechanism of bacterial survival against attack by antimicrobial peptides of both innate and exogenous origins.

Studies on the possible association between bacteria and oral squamous cell carcinoma (OSCC) remain inconclusive, largely due to methodological variations/limitations. The objective of this study was to characterize the species composition as well as functional attributes of the bacteriome associated with OSCC. DNA obtained from 20 fresh OSCC biopsies (cases) and 20 deep-epithelium swabs (matched control subjects) were sequenced for the V1-V3 region using Illumina’s 2x300 bp chemistry. High quality, non-chimeric merged reads were classified to species level using a prioritized BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp. polymorphum was the most significantly overrepresented species in the tumors followed by Pseudomonas aeruginosa and Campylobacter sp. Oral taxon 44, while Streptococcus mitis, Rothia mucilaginosa and Haemophilus parainfluenzae were the most significantly abundant in the controls. Functionally, genes involved in bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors while those responsible for DNA repair and combination, purine metabolism, phenylalanine, tyrosine and tryptophan biosynthesis, ribosome biogenesis and glycolysis/gluconeogenesis were significantly associated with the controls. This is the first epidemiological evidence for association of F. nucleatum and P. aeruginosa with OSCC. Functionally, an “inflammatory bacteriome” is enriched in OSSC.

Objective Increasing data supports the role of bacterial inflammation in adverse events of cardiovascular and cerebrovascular diseases. In our previous research, DNA of bacterial species found in coronary artery thrombus aspirates and ruptured cerebral aneurysms were mostly of endodontic and periodontal origin, where Streptococcus mitis group DNA was the most common. We hypothesized that the genomes of S mitis group could be identified in thrombus aspirates of patients with lower limb arterial and deep venous thrombosis. Methods Thrombus aspirates and control blood samples taken from 42 patients with acute or acute-on-chronic lower limb ischemia (Rutherford I-IIb) owing to arterial or graft thrombosis (n = 31) or lower limb deep venous thrombosis (n = 11) were examined using a quantitative real-time polymerase chain reaction to detect all possible bacterial DNA and DNA of S mitis group in particular. The samples were considered positive, if the amount of bacterial DNA in the thrombus aspirates was 2-fold or greater in comparison with control blood samples. Results In the positive samples the mean difference for the total bacterial DNA was 12.1-fold (median, 7.1), whereas the differences for S mitis group DNA were a mean of 29.1 and a median of 5.2-fold. Of the arterial thrombus aspirates, 57.9% were positive for bacterial DNA, whereas bacterial genomes were found in 75% of bypass graft thrombosis with 77.8% of the prosthetic grafts being positive. Of the deep vein thrombus aspirates, 45.5% contained bacterial genomes. Most (80%) of bacterial DNA-positive cases contained DNA from the S mitis group. Previous arterial interventions were significantly associated with the occurrence of S mitis group DNA ( P = .049, Fisher's exact test). Conclusions This is the first study to report the presence of bacterial DNA, predominantly of S mitis group origin, in the thrombus aspirates of surgical patients with lower limb arterial and deep venous thrombosis, suggesting their possible role in the pathogenesis of thrombotic events. Additional studies will, however, be needed to reach a final conclusion.

To improve our understanding about the severity of invasive pneumococcal disease (IPD), we investigated the association between the genotype of Streptococcus pneumoniae and disease outcomes for 349 bacteremic patients. A pneumococcal genome-wide association study (GWAS) demonstrated a strong correlation between 30-day mortality and the presence of the phage-derived gene pblB, encoding a platelet-binding protein whose effects on platelet activation were previously unknown. Platelets are increasingly recognized as key players of the innate immune system, and in sepsis, excessive platelet activation contributes to microvascular obstruction, tissue hypoperfusion, and finally multiorgan failure, leading to mortality. Our in vitro studies revealed that pblB expression was induced by fluoroquinolones but not by the beta-lactam antibiotic penicillin G. Subsequently, we determined pblB induction and platelet activation by incubating whole blood with the wild type or a pblB knockout mutant in the presence or absence of antibiotics commonly administered to our patient cohort. pblB-dependent enhancement of platelet activation, as measured by increased expression of the α-granule protein P-selectin, the binding of fibrinogen to the activated αIIbβ3 receptor, and the formation of platelet-monocyte complex occurred irrespective of antibiotic exposure. In conclusion, the presence of pblB on the pneumococcal chromosome potentially leads to increased mortality in patients with an invasive S. pneumoniae infection, which may be explained by enhanced platelet activation. This study highlights the clinical utility of a bacterial GWAS, followed by functional characterization, to identify bacterial factors involved in disease severity., IMPORTANCE The exact mechanisms causing mortality in invasive pneumococcal disease (IPD) patients are not completely understood. We examined 349 patients with IPD and found in a bacterial genome-wide association study (GWAS) that the presence of the phage-derived gene pblB was associated with mortality in the first 30 days after hospitalization. Although pblB has been extensively studied in Streptococcus mitis, its consequence for the interaction between platelets and Streptococcus pneumoniae is largely unknown. Platelets are important in immunity and inflammation, and excessive platelet activation contributes to microvascular obstruction and multiorgan failure, leading to mortality. We therefore developed this study to assess whether the expression of pblB might increase the risk of death for IPD patients through its effect on enhanced platelet activation. This study also shows the value of integrating extensive bacterial genomics and clinical data in predicting and understanding pathogen virulence, which in turn will help to improve prognosis and therapy.

Silicon-based thin films, such as silicon and silicon-based alloys, have been intensively studied, especially for their use in microelectronics and large area electronics. However, they also show interesting properties regarding their biological interaction, although these characteristics have been studied to a lower extent. Recently, promising antibacterial properties of some silicon-based materials have been investigated, showing their potential ability to reduce the adhesion of some types of bacteria on the surfaces onto which they are applied. Since these properties rely on the reduction of bacterial adhesion, instead of exploiting the release of antibacterial agents such as Ag ions or nanoparticles, they show the advantage of preventing potentially harmful long-time effects that could be exerted by these agents in the host tissues. Moreover, this approach avoids the risk that the release intensity of these agents is reduced over time due to their depletion inside the material. Due to the wide range of interesting properties of silicon-based materials, including biocompatibility increase, barrier action against the release of chemicals, and tuning of surface energy, the production of multifunctional coatings could also be possible. In this chapter, the most important properties of silicon-based thin films are reviewed, and some results concerning the potential antibacterial properties of silicon–oxygen amorphous thin-film alloys (a-SiO x ) are presented in detail.

口腔には種々のレンサ球菌が常在し、一部の口腔レンサ球菌は齲蝕、亜急性細菌性心内膜炎や誤嚥性肺炎を引き起こすことが知られている。従って、これらの疾患の発症リスクを評価するためには、簡便に採取できる唾液から各種口腔レンサ球菌を正確に検出することが重要な課題となる。本研究では、リアルタイム定量PCR を利用して唾液中の各種口腔レンサ球菌を定量的に検出することを試みた。15名の被験者から安静時の唾液を採取し、DNA を抽出した後、各種口腔レンサ球菌（Streptococcus mutans, S. sobrinus, S. salivarius,S. sanguinis あるいはS. mitis）を特異的に検出するPCR 用プライマーおよび全菌あるいは全口腔レンサ球菌を検出するユニバーサルプライマーを用いてリアルタイム定量PCR を行った。その結果、被験者15名の値から算出したS. mutans, S. sobrinus, S. salivarius, S. sanguinis あるいはS. mitis の全口腔レンサ球菌に占める割合は、それぞれ5％未満であった。また、全口腔レンサ球菌に占める割合は、S. sobrinus が5種類の口腔レンサ球菌の中で最も少ないことが明らかになった。本研究では、リアルタイム定量PCR を利用することによって、数種類の口腔レンサ球菌を同時に定量できることを示した。リアルタイム定量PCRを応用した唾液中の細菌数や割合の検査によって、将来的に、齲蝕や細菌性心内膜炎等の発症リスクを評価することが可能になるかもしれない。

Competence for genetic transformation in the genus Streptococcus depends on an alternative sigma factor, σ X , for coordinated synthesis of 23 proteins, which together establish the X state by permitting lysis of incompetent streptococci, uptake of DNA fragments, and integration of strands of that DNA into the resident genome. Initiation of transient accumulation of high levels of σ X is coordinated between cells by transcription factors linked to peptide pheromone signals. In Streptococcus pneumoniae , elevated σ X is insufficient for development of full competence without coexpression of a second competence-specific protein, ComW. ComW, shared by eight species in the Streptococcus mitis and Streptococcus anginosus groups, is regulated by the same pheromone circuit that controls σ X , but its role in expression of the σ X regulon is unknown. Using the strong, but not absolute, dependence of transformation on comW as a selective tool, we collected 27 independent comW bypass mutations and mapped them to 10 single-base transitions, all within rpoD , encoding the primary sigma factor subunit of RNA polymerase, σ A . Eight mapped to sites in rpoD region 4 that are implicated in interaction with the core β subunit, indicating that ComW may act to facilitate competition of the alternative sigma factor σ X for access to core polymerase.

We present the rare case of a 76-year-old female with infective endocarditis (IE) caused by Candida glabrata. Immediately before developing the present infection, she developed IE with vegetation on the mitral annular calcification, which was caused by Streptococcus mitis and successfully treated with penicillin-G and gentamicin. However, her fever recurred, and she developed disseminated intravascular coagulation. Blood culture revealed C. glabrata, and echocardiography revealed new vegetation on the mitral valve. After 4 weeks of treatment with micafungin, prosthetic valve replacement was performed, followed by additional administration of micafungin for 4 weeks (total of 8 weeks). No relapse at 9 months after surgery has been observed. C. glabrata endocarditis is extremely rare and difficult to manage. Our case and review of past reported cases suggest that early diagnosis and initiation of treatment contribute to good prognosis of C. glabrata endocarditis.

Aim To evaluate the effect of TEGDMA on human gingival fibroblasts (HGFs) in vitro co-cultured with Streptococcus mitis, focusing on the signalling pathways underlying cell tissue remodelling and inflammatory response processes. Methodology β1 integrin expression was evaluated by means of imaging flow cytometry. The Western blot technique was used to investigate the expression of protein kinase C (PKC), extracellular signal-regulated kinase (ERK), matrix metalloproteinase 9 (MMP9) and 3 (MMP3). RT-PCR was performed to quantify nuclear factor-kb subunits (Nf-kb1, ReLa), IkB kinase β (IkBkB), cyclooxygenase II (COX-2) and tumour necrosis factor-α (TNF-α) mRNA levels. Statistical analysis was performed using the analysis of variance (anova). Results When HGFs are co-cultured with S. mitis, β1 integrin intensity, phosphorylated PKC (p-PKC), activated ERK (p-ERK), IkBkB mRNA level and MMP9 expression increased (for all molecules P

The relevance of microorganisms in preterm birth is still under discussion. Using a diagnostic fluorescence in situ hybridization probe panel, we visualized Staphylococcus aureus and Streptococcus mitis group in two cases of acute chorioamnionitis. This technique provides spatial resolution and quantity of bacteria, clarifying the epidemiology and pathogenic pathways of acute chorioamnionitis.

The use of natural products as an alternative for treatment and prevention of diseases has been increasing daily. Lippia alba is a shrub widely distributed throughout Brazil and its aqueous extracts, as infusion and decoction made from its leaves, are currently being used in popular medicine. Thus, the objective of the study was to do a phytochemical screening of these solutions as well as evaluate in vitro antimicrobial activity of these solutions in the reduction of: Streptococcus mutans (ATCC 25175), Streptococcus mitis (ATCC 49456), Staphylococcus aureus (ATCC 12600) and Lactobacillus casei (ATCC 27216). The negative and positive controls were respectively, BHI and Chlorhexidine. Statistical analysis was performed using the Mann-Whitney and Kruskal-Wallis test with Bonferroni's penalization, showing significant difference between the products tested (p

Dental implant abutments that emerge through the mucosa are rapidly covered with a salivary protein pellicle to which bacteria bind, initiating biofilm formation. In this study, adherence of early colonizing streptococci, Streptococcus gordonii, Streptococcus oralis, Streptococcus mitis and Streptococcus sanguinis to two saliva-coated anodically oxidized surfaces was compared with that on commercially pure titanium (CpTi). Near edge X-ray absorption (NEXAFS) showed crystalline anatase was more pronounced on the anodically oxidized surfaces than on the CpTi. As revealed by fluorescence microscopy, a four-species mixture, as well as individual bacterial species, exhibited lower adherence after 2 h to the saliva-coated, anatase-rich surfaces than to CpTi. Since wettability did not differ between the saliva-coated surfaces, differences in the concentration and/or configuration of salivary proteins on the anatase-rich surfaces may explain the reduced bacterial binding effect. Anatase-rich surfaces could thus contribute to reduced overall biofilm formation on dental implant abutments through diminished adherence of early colonizers.

The genetically diverse viridans group streptococci (VGS) are increasingly recognized as the cause of a variety of human diseases. We used a recently developed multilocus sequence analysis scheme to define the species of 118 unique VGS strains causing bacteremia in patients with cancer; Streptococcus mitis (68 patients) and S. oralis (22 patients) were the most frequently identified strains. Compared with patients infected with non–S. mitis strains, patients infected with S. mitis strains were more likely to have moderate or severe clinical disease (e.g., VGS shock syndrome). Combined with the sequence data, whole-genome analyses showed that S. mitis strains may more precisely be considered as >2 species. Furthermore, we found that multiple S. mitis strains induced disease in neutropenic mice in a dose-dependent fashion. Our data define the prominent clinical effect of the group of organisms currently classified as S. mitis and lay the groundwork for increased understanding of this understudied pathogen.

Synthesis of cell-bound glucan from dietary sucrose by oral pathogenic bacteria may influence bacterial cell surface properties and colonization of surfaces. This study investigated the effects of the addition of 2 % sucrose to culture medium on cell surface properties (hydrophobicity, charge, and auto-aggregation) and colonization activities (attachment and biofilm formation) on three abiotic surfaces (hydroxyapatite, glass, and stainless steel) of two Streptococcus mutans strains, one Streptococcus salivarius strain, one Streptococcus mitis strain, and one Actinomyces naeslundii strain. The results showed that the additional sucrose reduced the hydrophobicity of three strains (44-62 %) and increased that of one strain (31 %). Cellular aggregation of one strain was decreased (13 %) and that of another increased (21 %). No change in the surface charge of strains was apparent. Additional sucrose also inhibited the attachment of three strains (0.6-1.3 log CFU cm−2) and enhanced that of one strain (0.5-1.3 log CFU cm−2) to glass and stainless steel. The attachment of two strains to hydroxyapatite was reduced (0.9-1.3 log CFU cm−2). Biofilm formation by four strains was enhanced on all surfaces (0.4-1 log CFU cm−2). No relationship between changes in cell surface properties and changes in colonization activities was apparent. Sucrose does not always enhance oral bacterial colonization of abiotic surfaces.

This study aimed to determine the phytochemical composition of a root extract of Solanum paniculatum Linn. (SPL), and to evaluate its in vitro antibacterial activity. The extract was characterized by phytochemical screening and thin-layer chromatography. The following oral bacteria were used to determine the minimum inhibitory concentration (MIC) and minimum inhibitory concentration of adherence (MICA) Streptococcus mitis (ATCC 903), Streptococcus mutans (ATCC 25175), Streptococcus sanguinis (ATCC 15300), Streptococcus oralis (ATCC 10557), Streptococcus salivarius (ATCC 7073), and Lactobacillus casei (ATCC 9595). Each assay was carried out in duplicate and the positive control (0.12% chlorhexidine digluconate) was submitted to the same procedure. S. mutans was used for the evaluation of bactericidal kinetics. The results were analyzed by determining the number of colony-forming units (CFU/mL). The SPL extract presented MIC and MICA values of 7.81 and 62.5 mg/mL, respectively. The extract was bactericidal at a concentration of 500 mg/mL within 2 h and at the MIC within 4 h. Phytochemical analysis revealed the presence of alkaloids, sulfur compounds, gums and lactones, as well as a high content of phenols. In conclusion, SPL presented in vitrobacteriostatic, anti-adhesion and bactericidal activity. The strong presence of phenols may explain the pharmacological activity of this extract. Key words: Solanum paniculatum, microbiology, phytotherapy, phenols.

The chemical composition of the essential oil isolated from the aerial parts of Melampodium divaricatum (Rich.) DC. (Asteraceae) was characterized by GC-FID and GC/MS analyses. (E)-Caryophyllene (56.0%), germacrene D (12.7%), and bicyclogermacrene (9.2%) were identified as the major oil components. The antimicrobial activity of the oil against seven standard strains of oral pathogens from the American Type Culture Collection (ATCC) was evaluated by determining minimum inhibitory concentrations (MICs) using the microdilution method. MIC Values below 100 μg/ml were obtained against Streptococcus sobrinus (90 μg/ml), Lactobacillus casei (30 μg/ml), S. mutans (20 μg/ml), and S. mitis (18 μg/ml). In contrast, the MIC values of the major oil compound (E)-caryophyllene were higher than 400 μg/ml against all pathogens, suggesting that the activity of the oil might depend on minor oil components and/or on synergistic effects. The M. divaricatum essential oil is a promising agent to include in anticariogenic oral rinse formulations for the control of oral pathogens.

Received : 2 December, 2013 Revised : 23 February, 2014 Accepted : 24 February, 2014 ABSTRACT Objectives : The purpose of the study is to investigate the activities of Nelumbo nucifera leaf extracts on Streptococcus mutans, Streptococcus mitis, Streptococcus sobrinus, Porphyromonas gingivalis, Prevotella intermedia, and Treponema denticola. Methods : The inhibitory effect of lotus leaf extracts on the growth of oral bacteria was assessed in experiments with extracts from freshly harvested and pulverized lotus leaves and bacterial cultures of dental caries. Corresponding Author Man Kyu Huh Department of Molecular Biology Dongeui University 176 Eomgwangno Busan jin gu, Busan 614-714, Korea. Tel : +82-51-890-1529 +82-10-9334-8592 Fax : +82-51-890-1521 E-mail : mkhuh@deu.ac.kr Results : The results showed that N. nucifera extracts possess antimicrobial activity on all bacterial strains. The minimal inhibitory concentration (MIC) values varied from 4 mg/ml to 10 mg/ml against antimicrobial activity. The relative growth ratio (RGR) against of N. nucifera extracts were determined as 50% in concentration of 4.0 mg/ml. The extract of N. nucifera was effective in reducing on the glucosyltransferase (GTase) activity of six strains in vitro. Conclusions : Methanol extracts of lotus leaves showed antimicrobial effects on three bacterial species causing dental caries and three bacterial species causing periodontitis, as well as inhibitory effects on GTase activity.

Small-colony variants (SCVs) of bacteria are associated with recurrent and persistent infections. We describe for the first time SCVs of Streptococcus tigurinus in a patient with a prosthetic joint infection. S. tigurinus is a novel pathogen of the Streptococcus mitis group and causes invasive infections. We sought to characterize S. tigurinus SCVs using experimental methods and find possible genetic explanations for their phenotypes. The S. tigurinus SCVs were compared with the wild-type (WT) isolate using phenotypic methods, including growth under different conditions, autolysis, and visualization of the cell ultrastructure by use of transmission electron microscopy (TEM). Furthermore, comparative genome analyses were performed. The S. tigurinus SCVs displayed reduced growth compared to the WT and showed either a very stable or a fluctuating SCV phenotype. TEM analyses revealed major alterations in cell separation and morphological abnormalities, which were partially explained by impaired autolytic behavior. Intriguingly, the SCVs were more resistant to induced autolysis. Whole-genome sequencing revealed mutations in the genes involved in general cell metabolism, cell division, stringent response, and virulence. Clinically, the patient recovered after a 2-stage exchange of the prosthesis. Comparative whole-genome sequencing in clinical strains is a useful tool for identifying novel genetic signatures leading to the most persistent bacterial forms. The detection of viridans streptococcal SCVs is challenging in a clinical laboratory due to the small colony size. Thus, it is of major clinical importance for microbiologists and clinicians to be aware of viridans streptococcal SCVs, such as those of S. tigurinus , which lead to difficult-to-treat infections.

Dental resin composites consist of organic polymers with inorganic fillers used as bonding resins and direct filling materials in dentine adhesives and as sealing agents for inlays, crowns and orthodontic brackets. Despite various modifications in the formulation, the chemical composition of composite resins includes inorganic filler particles and additives, which are incorporated into a mixture of an organic resin matrix. Among them, 2-hydroxyethylmethacrylate (HEMA) is one of the most frequently used. Several studies have attempted to clarify the mechanisms underlying HEMA cytotoxicity. Most of them support the hypothesis that this compound, once released in the oral environment, increases reactive oxygen species (ROS) production and oxidative DNA damage through double-strand breaks evidenced by in vitro presence of micronuclei. As a consequence, the glutathione detoxifying intracellular pool forms adducts with HEMA through its cysteine motif and inflammation begins to occur: transcription of early genes of inflammation such as tumour necrosis factor α or inducible cyclooxygenase up to the secretion of prostaglandins 2. These phenomena are counteracted by N-acetylcysteine (NAC), a nonenzymatic antioxidant, but not by vitamin E or other antioxidant. Consequently, NAC prevents HEMA-induced apoptosis acting as a direct ROS scavenger. This minireview collects the most significant papers on HEMA and tries to make an overview of its cytotoxicity on different cell types and experimental models.

The objective of the present investigation was to evaluate the antibacterial and antioxidant potential of n-hexane and methanolic extracts of Melia azedarach L. bark. The antibacterial potential of M. azedarach L. bark was tested against human pathogens causing diarrhoea and dysentery such as Shigella flexneri (MTCC- 9543), Salmonella enterica ser typhi (MTCC-733), Bacillus subtilis (MTCC-1305), Streptococcus mitis (MTCC- 2897), Klebsiella pneumoniae (MTCC-109) and Staphylococcus aureus (MTCC-1430) using agar well diffusion method. The results of the study revealed that n-hexane extract of bark sample was highly effective against Shigella flexneri whereas Streptococcus mitis, Staphylococcus aureus and Bacillus subtilis showed no response and other test pathogens under study responded moderately. It was observed that methanolic extract had high inhibition potential against Salmonella enterica ser typhi and Streptococcus mitis while moderate effect against other test bacteria. Studies on the antioxidant activity by DPPH scavenging method revealed significant antioxidant potential of n-hexane and methanolic extracts with IC50 value 84.37 and 66.79 respectively.

Genomic, taxonomic and biochemical studies were performed on two strains of α-haemolytic streptococci that showed them to be clustered with major members of the Streptococcus mitis group. These Gram-stain-positive strains were isolated from tooth surfaces of caries-free humans and showed the classical spherical shape of streptococcal species growing in chains. Sequence analysis from concatenated 16S and 23S rRNA gene and sodA genes showed that these strains belonged to the mitis group, but both of them clustered into a new phylogenetic branch. The genomes of these two isolates were sequenced, and whole-genome average nucleotide identity (ANI) demonstrated that these strains significantly differed from any streptococcal species, showing ANI values under 91 % even when compared with the phylogenetically closest species such as Streptococcus oralis and S. mitis . Biochemically, the two isolates also showed distinct metabolic features relative to closely related species, like α-galactosidase activity. From the results of the present study, the name Streptococcus dentisani sp. nov. is proposed to accommodate these novel strains, which have been deposited in open collections at the Spanish type Culture Collection (CECT) and Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures (DSMZ), being respectively identified as Streptococcus dentisani Str. 7746 ( = CECT 8313 = DSM 27089) and Streptococcus dentisani Str. 7747T ( = CECT 8312T = DSM 27088T).

Psidium guajava L. has extensive use in folk medicine. The aim of this study was to quantify the levels of phenolic, flavonoids, antioxidant activity, leathality assay and antibacterial and antitumoral activities of the extract of P. guajava. In the dry guava extract there were high levels of phenolics (766.08 ± 14.52 mg/g), flavonoids (118.90 ± 5.47 mg/g) and antioxidant activity (87.65%). The LD50 was 185.15 μg/ml. The MIC value was 250 μg/ml for Streptococcus mutans, Streptococcus mitis and Streptococcus oralis. IC50 of the extract tested in the HeLa, RKO and Wi cell lines was 15.6 ± 0.8 μg/ml, 21.2 ± 1.1 μg/ml and 68.9 ± 1.5 μg/ml, respectively. The results of all analyses allow us to conclude that the dry extract of guava leaves has promising activity to be applied topically in the oral cavity or in the development of antitumor formulation or even be used as a functional food.

This study proposes a bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. Sixty extracts were obtained from ten plants - (1) Jatropha weddelliana, (2) Attalea phalerata, (3) Buchenavia tomentosa, (4) Croton doctoris, (5) Mouriri elliptica, (6) Mascagnia benthamiana, (7) Senna aculeata, (8) Unonopsis guatterioides, (9) Allagoptera leucocalyx and (10) Bactris glaucescens - using different extraction methods - (A) 70° ethanol 72 h/25°C, (B) water 5 min/100°C, (C) water 1 h/55°C, (D) water 72 h/25°C, (E) hexane 72 h/25°C and (F) 90° ethanol 72 h/25°C. The plants were screened for antibacterial activity at 50 mg/ml using the agar well diffusion test against Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. The active extracts were tested to determine their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), cytotoxicity and chemical characterization. Forty-seven extracts (78%) were active against at least one microorganism. Extract 4A demonstrated the lowest MIC and MBC for all microorganisms except S. gordonii and the extract at MIC concentration was non-cytotoxic. The concentrated extracts were slightly cytotoxic. Electrospray ionization with tandem mass spectrometry analyses demonstrated that the extract constituents coincided with the mass of the terpenoids and phenolics. Overall, the best results were obtained for extraction methods A, B and C. The present work proved the antimicrobial activity of several plants. Particularly, extracts from C. doctoris were the most active against bacteria involved in dental caries disease.

Streptococcus mitis is a component of the normal oropharynx, skin, gastrointestinal system, and genital tract florae. It is generally considered as a relatively benign bacterium. We present a case of spontaneous Streptococcus mitis meningitis in a patient with liver cirrhosis and no known risk factors for invasive infectious diseases.

Methanethiol (methyl mercaptan) is an important contributor to oral malodour and periodontal tissue destruction. Porphyromonas gingivalis, Prevotella intermedia and Fusobacterium nucleatum are key oral microbial species that produce methanethiol via methionine gamma lyase (mgl) activity. The aim of this study was to compare an mgl knockout strain of P. gingivalis with its wild type using a 10-species biofilm co-culture model with oral keratinocytes and its effect on biofilm composition and inflammatory cytokine production. A P. gingivalis mgl knockout strain was constructed using insertion mutagenesis from wild type W50 with gas chromatographic head space analysis confirming lack of methanethiol production. 10-species biofilms consisting of Streptococcus mitis, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp polymorphum, Fusobacterium nucleatum ssp vincentii, Veillonella dispar, Actinomyces naeslundii, Prevotella intermedia and Aggregatibacter actinomycetemcomitans with either the wild type or mutant P. gingivalis were grown on Thermanox cover slips and used to stimulate oral keratinocytes (OKF6-TERT2), under anaerobic conditions for 4 and 24 hours. Biofilms were analysed by quantitative PCR with SYBR Green for changes in microbial ecology. Keratinocyte culture supernatants were analysed using a multiplex bead immunoassay for cytokines. Significant population differences were observed between mutant and wild type biofilms; V. dispar proportions increased (p

There is no clear relationship between the serum inhibition test and clinical outcome for Streptococcus mitis (S. mitis) endocarditis. We report an 84-year-old male with endocarditis caused by penicillin-tolerant S. mitis. The results for the serum inhibitory test (SIT) and serum bactericidal test (SBT) showed a trough level of SIT = 1:256 and SBT = 1:4 and a peak level of SIT ≥ 1:1024 and SBT = 1:16. In addition, the SIT/SBT ratio was 64 at peak level and more than 64 at trough level, which is compatible with penicillin-tolerant S. mitis. Following a 42-day high-dose penicillin treatment (24 M IU/day, via a continuous drip), the patient made a good recovery. In vitro inhibitory and bactericidal test results were not a valid predictor of medical treatment failure. Physicians need to continue to evaluate the surgical indications when treating patients with S. mitis endocarditis.

Submitted by ANA LUIZA DO ROSÁRIO PALMA null (ana.luiza.rp@hotmail.com) on 2017-01-11T17:41:58Z No. of bitstreams: 1 Ana Luiza do Rosário Palma, Biopatologia Bucal.pdf: 3299481 bytes, checksum: 9eb1d5e0b50827ce3d17e38baa851362 (MD5) Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-01-12T16:46:23Z (GMT) No. of bitstreams: 1 palma_alr_me_sjc.pdf: 3299481 bytes, checksum: 9eb1d5e0b50827ce3d17e38baa851362 (MD5) Made available in DSpace on 2017-01-12T16:46:23Z (GMT). No. of bitstreams: 1 palma_alr_me_sjc.pdf: 3299481 bytes, checksum: 9eb1d5e0b50827ce3d17e38baa851362 (MD5) Previous issue date: 2016-12-16 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) O objetivo foi avaliar as interações entre Candida albicans (ATCC 18804) com Streptococcus mitis (49456) e Streptococcus sanguinis (10556) in vitro e in vivo avaliando-se a possível influência destas associações, na expressão de genes, na filamentação e formação de biofilme de Candida albicans. A formação de biofilme, foi realizado mono e multiespécie em placa de 96 poços por 48 h à 37 ºC com 5% CO2. Os biofilmes foram desagregados e diluídos para semeadura em ágar e após incubação as UFC/mL foram contadas. A filamentação de C. albicans, in vitro foi realizada em placas de 24 poços e in vivo em Galleria mellonella, com análise histológica e contagem de UFC/mL. A avaliação da expressão gênica de ALS1, ALS3, BRC1, CPH1, EFG1 e HWP1, foi realizada por PCR em tempo real utilizando o gene normalizador ACT1. Os resultados da UFC/mL (p < 0.05), demonstrou que o biofilme de C. albicans monoespécie apresentou maior crescimento, quando comparado com o biofilme associado com S. mitis (p = 0,001) ou com S. sanguinis (p = 0,001). A filamentação in vitro demonstrou que a interação com S. mitis inibiu a filamentação de C. albicans (p = 0,0006), entretanto, a interação com S. sanguinis não inibiu (p = 0,1554). Os genes ALS1, ALS3 e HWP1 foram super expressos na interação com S. mitis. A interação com S. sanguinis, promoveu super expressão dos genes ALS3 e HWP1. Os genes BRC1, CPH1 e EFG1 foram super expressos na interação com S. mitis e sub expressos, na interação com S. sanguinis. Não houve diferença estatística nos estudos in vivo de filamentação e UFC/mL. Conclui-se que in vitro, S. mitis e S. sanguinis foram capazes de inibir a formação de biofilme de C. albicans. Assim como a interação com S. mitis inibiu a sua filamentação. A interação com S. mitis parece aumentar o fator de virulência de C. albicans, quanto a expressão dos genes de aderência ALS1, ALS3 e HWP1, bem como na associação com S. sanguinis (ALS3 e HWP1). Os genes de formação de biofilme, BRC1, CPH1 e EFG1, na interação com S. mitis promoveu aumento do fator de virulência. The objective was to evaluate the interactions between Candida albicans (ATCC 18804) with Streptococcus mitis (49456) and Streptococcus sanguinis (10556) in vitro and in vivo evaluating the possible influence of these associations, in the expression of genes, in the filamentation and biofilm formation of Candida albicans. Biofilm formation was performed mono- and multispecies in 96-well plate for 48 h at 37 ºC with 5% CO2. Biofilms sonicated and diluted for sowing on agar and after incubation the colonies counted to obtain the colony forming units (CFU/mL). The filamentation in vitro was performed in 24-well plate and in vivo Galleria mellonella, with histological and CFU/mL. The evaluation of gene expression ALS1, ALS3, BRC1, CPH1, EFG1 and HWP1 was performed by real-time PCR using the normalizing gene ACT1. The results of CFU/ml (p