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Summary Mitis‐group streptococci are ubiquitous oral commensals that can promote polybacterial biofilm virulence. Using a novel murine oral mucosal co‐infection model we sought to determine for the first time whether these organisms promote the virulence of C. albicans mucosal biofilms in oropharyngeal infection and explored mechanisms of pathogenic synergy. We found that Streptococcus oralis colonization of the oral and gastrointestinal tract was augmented in the presence of C. albicans. S. oralis and C. albicans co‐infection significantly augmented the frequency and size of oral thrush lesions. Importantly, S. oralis promoted deep organ dissemination of C. albicans. Whole mouse genome tongue microarray analysis showed that when compared with animals infected with one organism, the doubly infected animals had genes in the major categories of neutrophilic response/chemotaxis/inflammation significantly upregulated, indicative of an exaggerated inflammatory response. This response was dependent on TLR2 signalling since oral lesions, transcription of pro‐inflammatory genes and neutrophil infiltration, were attenuated in TLR2−/− animals. Furthermore, S. oralis activated neutrophils in a TLR2‐dependent manner in vitro. In summary, this study identifies a previously unrecognized pathogenic synergy between oral commensal bacteriaand C. albicans. This is the first report of the ability of mucosal commensal bacteria to modify the virulence of an opportunistic fungal pathogen.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS ( ) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner \"What's in My Pot?\" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes ( = 514), clinical isolates ( = 31), and reference strains ( = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates ( = 8, = 17 and = 6) and 4 reference strains ( = 1, = 1, = 1, and = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except , and . The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

BackgroundDifferentiation of Streptococcus pneumoniae from other viridans group streptococci is well known to be challenging in clinical laboratories. Matrix assisted laser desorption ionisation–time of flight mass spectrometry (MALDI-TOF MS) had been reported to be a good alternative for Streptococcus species level identification. However, differentiation of S. pneumoniae from other Streptococcus mitis group organisms was found to be problematic using the Bruker MALDI Biotyper system.MethodsThis study used the Bruker MALDI Biotyper system in addition to a mass spectra model analysis generated by 10 reference strains of S. pneumoniae, 8 strains of S. mitis and 2 strains of S. oralis in the ClinProTools to identify 28 clinical isolates of S. pneumoniae and 47 isolates of S. mitis/oralis. The results were compared with those generated by the MALDI Biotyper system alone.ResultsThe percentages of correct species level identification using the MALDI Biotyper system alone and the direct transfer and extraction method were 66.7% (50/75) and 70.7% (53/75), respectively. With the additional ClinProTools mass spectra analysis, the percentages of correct identification by the direct transfer and extraction method increased to 85.3% (64/75) and 100% (75/75), respectively. This new workflow significantly improved the accuracy of S. pneumoniae and S. mitis/oralis identification.ConclusionsThe additional ClinProTools mass spectra analysis with extraction method after MALDI Biotyper identification significantly improved the accuracy of identification among S. pneumoniae, S. oralis and S. mitis. The extra 15 min processing time of spectra analysis should be affordable in most clinical laboratories. We suggest that the same approach could be further explored in handling other bacterial species with high similarities.

Background: Mitis group non-pneumococcal streptococci (MGNPS), specifically Streptococcus mitis, Streptococcus infantis and Streptococcus oralis, have recently been shown to cause pneumonia and/or bacteremia. These organisms often have capsular (cps) operons resembling those in pneumococci, and some express cps-generated polysaccharides that antigenically cross-react with pneumococcal capsular serotypes. But, to date, a series of MGNPS isolates has not been studied by electron microscopy (EM) for the presence of a capsule. Methods: We studied 21 MGNPS; 11 were isolated from sputum and determined to have caused pneumonia, 3 were isolated from blood, and 7 were commensal isolates cultured from the oral cavity of healthy adults. Two reacted with a pneumococcal anticapsular antibody. Isolates were fixed with two different protocols and examined by transmission EM. Results: EM of MGNPS after standard fixation and staining with uranyl acetate did not show capsules. In contrast, the 21 MGNPS isolates that we studied after fixation with ruthenium red and lysine acetate were all shown to be encapsulated. The thickness and density of capsules was related to their species: Streptococcus pneumoniae had the most prominent encapsulation and Streptococcus oralis had the least. However, within a species, there was no apparent difference in capsules between disease-causing and colonizing strains. Conclusions: EM with ruthenium red staining demonstrated capsules on 21 MGNPS, but within a species, there was no apparent difference between disease-causing and commensal isolates. It seems reasonable to conclude that the capsule, together with inoculum size, host’s ability to clear aspirated organisms, and other as yet unidentified virulence factors, all contribute to the pathogenesis of MGNPS pneumonia.

subspecies is explored as an anti-cariogenic probiotic. Here, subjecting freshly stimulated saliva samples of 35 healthy volunteers, six epidemiologically unrelated and two related strains were isolated (prevalence around 20%) applying a newly developed three-step procedure. Furthermore, the probiotic strain 7746 (AB-Dentisanium®) was tested under a variety of environmental conditions for its inhibitory effect on six , two , 15 other oral or intestinal streptococci, 15 strains, and six representatives of other species including periodontopathogens. All except one of the strains were inhibited by 7746 colonies or culture supernatant concentrate but only if either the test cell number was low or the producer or its bacteriocin concentration, respectively, was high. OMI 332, OMI 315, OMI 335, OMI 238, and the intestinal OMI 339 were not inhibited, while the other 10 streptococcal strains (especially OMI 334 and intestinal OMI 326) showed a certain degree of inhibition. From the panel of other bacterial species only was slightly inhibited. With the exception of OMI 285 and OMI 291 that possessed a 7746 bacteriocin-like gene cluster, all strains and especially type strain 7747 were strongly inhibited by 7746. In conclusion, probiotic strain 7746 might antagonize the initiation and progression of dental caries by reducing if not too abundant. strains inhibit each other, but strains with similar bacteriocin-related gene clusters, including immunity genes, are able to co-exist due to cross-resistance. In addition, development of resistance and adaptation to 7746-bacteriocins was observed during our study and needs attention. Hence, mechanisms underlying such processes need to be further investigated using omics-approaches. On the manufacturing level, probiotic strains should be continuously tested for function. Further clinical studies investigating inhibition of by AB-Dentisanium® are required that should also monitor the impact on the oral microbiome composition including resident strains.

Background The incidence of neonatal sepsis in the United States of America is 1–2 cases per 1,000 live births. The majority are bacterial infections due to Escherichia coli and Group B Streptococcus. Only a few reported cases due to Streptococcus mitis oralis exist in the literature with limited information regarding management and treatment. Case presentation We report a case of Streptococcus mitis oralis bacteremia and meningitis in a full-term 38 week and 6-day-old female neonate born to a 37-year-old G2P2 mother initially treated with ampicillin and gentamicin for presumed Group B Streptococcus (GBS) sepsis and meningitis, then completing a 14-day treatment course with cefepime. Conclusion Clinicians should be aware of S. mitis oralis as it can cause significant illness in both pre-term and term neonates, a population more susceptible to infection. Due to the low incidence of cases, there are no specific guidelines for management of Streptococcus mitis sepsis or meningitis in the newborn. We highlight our clinical decision-making regarding antibiotic selection and treatment duration while focusing on enhancing treatment efficacy based on antimicrobial susceptibilities.

Polycystic ovary syndrome (PCOS) is a hormonal disorder of women that not only is the leading cause of infertility but also shows a reciprocal link with oral health. This study aimed to investigate the hypothesis that the levels of putative periodontal pathogens in saliva and their antibody response in serum are elevated in PCOS, compared to systemic health. A total of 125 women were included in four groups; 45 women with PCOS and healthy periodontium, 35 women with PCOS and gingivitis, 25 systemically and periodontally healthy women, 20 systemically healthy women with gingivitis. Salivary levels of seven putative periodontal pathogens were analyzed by quantitative real-time polymerase chain reaction and serum antibody levels were analyzed by ELISA. In women with PCOS, salivary Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus oralis and Tannerella forsythia levels were higher than matched systemically healthy women, particularly in the case of gingivitis. Aggregatibacter actinomycetemcomitans and Treponema denticola levels were similar among study groups. The presence of PCOS also enhanced P. gingivalis, Prevotella intermedia and S. oralis serum antibody levels, when gingivitis was also present. Gingival inflammation correlated positively with levels of the studied taxa in saliva, particularly in PCOS. The presence of P. gingivalis and F. nucleatum in saliva also exhibited a strong positive correlation with the corresponding serum antibody levels. In conclusion, as an underlying systemic endocrine condition, PCOS may quantitatively affect the composition of oral microbiota and the raised systemic response to selective members of this microbial community, exerting a confounding role in resultant gingival inflammation and periodontal health. The most consistent effect appeared to be exerted on P. gingivalis.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. Our aim was to identify conserved targets in pneumococci that showed positive prediction for lipoprotein and extracellular subcellular location using bioinformatics programs and verify the distribution and the degree of conservation of these targets in pneumococci. These targets can be considered potential vaccine candidate to be evaluated in the future. A set of 13 targets were analyzed and confirmed the presence in all pneumococci tested. These 13 genes were highly conserved showing around >96 % of amino acid and nucleotide identity, but they were also present and show high identity in the closely related species Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. S. oralis clusters away from S. pneumoniae, while S. pseudopneumoniae and S. mitis cluster closer. The divergence between the selected targets was too small to be observed consistently in phylogenetic groups between the analyzed genomes of S. pneumoniae. The proteins analyzed fulfill two of the initial criteria of a vaccine candidate: targets are present in a variety of different pneumococci strains including different serotypes and are conserved among the samples evaluated.

Background Streptococcus oralis belongs to the Streptococcus mitis group and is part of the normal flora of the nasal and oropharynx (Koneman et al., The Gram-positive cocci part II: streptococci, enterococci and the ‘Streptococcus-like’ bacteria. Color atlas and textbook of diagnostic microbiology, 1997). Streptococcus oralis is implicated in meningitis in patients with decreased immune function or from surgical manipulation of the central nervous system. We report a unique case of meningitis by S treptococcus oralis in a 58-year-old patient with cerebral spinal fluid leak due to right sphenoid meningoencephalocele. Case presentation A 58-year-old female presented in the emergency department due to altered mental status, fevers, and nuchal rigidity. Blood cultures were positive for S treptococcus oralis . Magnetic resonance stereotactic imaging of head with intravenous gadolinium showed debris in lateral ventricle occipital horn and dural thickening/enhancement consistent with meningitis. There was also a right sphenoidal roof defect, and meningoencephalocele with cerebrospinal fluid leak as a result. The patient was treated with ceftriaxone and had endoscopic endonasal repair of defect. She had complete neurologic recovery 3 months later. Conclusions Cerebrospinal fluid leak puts patients at increased risk for meningitis. Our case is unique in highlighting S treptococcus oralis as the organism implicated in meningitis due to cerebrospinal fluid leak.

Chewing of gum contributes to the maintenance of oral health. Many oral diseases, including caries and periodontal disease, are caused by bacteria. However, it is unknown whether chewing of gum can remove bacteria from the oral cavity. Here, we hypothesize that chewing of gum can trap bacteria and remove them from the oral cavity. To test this hypothesis, we developed two methods to quantify numbers of bacteria trapped in chewed gum. In the first method, known numbers of bacteria were finger-chewed into gum and chewed gums were molded to standard dimensions, sonicated and plated to determine numbers of colony-forming-units incorporated, yielding calibration curves of colony-forming-units retrieved versus finger-chewed in. In a second method, calibration curves were created by finger-chewing known numbers of bacteria into gum and subsequently dissolving the gum in a mixture of chloroform and tris-ethylenediaminetetraacetic-acid (TE)-buffer. The TE-buffer was analyzed using quantitative Polymerase-Chain-Reaction (qPCR), yielding calibration curves of total numbers of bacteria versus finger-chewed in. Next, five volunteers were requested to chew gum up to 10 min after which numbers of colony-forming-units and total numbers of bacteria trapped in chewed gum were determined using the above methods. The qPCR method, involving both dead and live bacteria yielded higher numbers of retrieved bacteria than plating, involving only viable bacteria. Numbers of trapped bacteria were maximal during initial chewing after which a slow decrease over time up to 10 min was observed. Around 10(8) bacteria were detected per gum piece depending on the method and gum considered. The number of species trapped in chewed gum increased with chewing time. Trapped bacteria were clearly visualized in chewed gum using scanning-electron-microscopy. Summarizing, using novel methods to quantify and qualify oral bacteria trapped in chewed gum, the hypothesis is confirmed that chewing of gum can trap and remove bacteria from the oral cavity.