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The airway microbiome has an important role in asthma pathophysiology. However, little is known on the relationships between the airway microbiome of asthmatic children, loss of asthma control, and severe exacerbations. Here we report that the microbiota’s dynamic patterns and compositions are related to asthma exacerbations. We collected nasal blow samples (n = 319) longitudinally during a clinical trial at 2 time-points within one year: randomization when asthma is under control, and at time of early loss of asthma control (yellow zone (YZ)). We report that participants whose microbiota was dominated by the commensal Corynebacterium   +   Dolosigranulum cluster at RD experience the lowest rates of YZs (p = 0.005) and have longer time to develop at least 2 episodes of YZ (p = 0.03). The airway microbiota have changed from randomization to YZ. A switch from the Corynebacterium   +   Dolosigranulum cluster at randomization to the Moraxella- cluster at YZ poses the highest risk of severe asthma exacerbation (p = 0.04). Corynebacterium’s relative abundance at YZ is inversely associated with severe exacerbation (p = 0.002). How the airway microbiome influences asthma pathophysiology remains unclear. Here, the authors analyse nasal samples of cohort of school-age children with persistent asthma and find that the microbiota’s patterns and composition at time of early loss of asthma control associate with severe asthma exacerbations.

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.

[Please see PDF for full article text] [Please see PDF for full article text] Objectives: To evaluate the effect of Z-spring appliance and clear aligners, used in anterior crossbite treatment, on dental plaque colonization in children. Methods: A 2-arm randomized controlled trial included 30 patients aged 7-12 years with anterior crossbite. Participants were divided into 2 groups: clear aligner (group A, n=15) and Z-spring appliance (group B, n=15). Dental plaque samples were collected at treatment initiation and completion, inoculated onto selective media, and analyzed for colony counts (cfu/ml) of Streptococcus mutans (S. mutans), Streptococcus mitis (S. mitis), Streptococcus salivarius (S. salivarius), and Candida albicans (C. albicans). Results: In group A, S. mutans counts significantly increased (p=0.006), while C. albicans counts decreased (p=0.039). In group B, S. mutans counts significantly decreased (p=0.002). No significant changes were observed in S. salivarius or S. mitis counts in either group. Conclusion: Short-term clear aligner use in children increases S. mutans colonization, suggesting a potential risk for dental caries with prolonged treatment. Early preventive measures are crucial to mitigate this risk and ensure better oral health outcomes. ClinicalTrials.gov Reg. No. ID: NCT06858033 Keywords: dental plaque, Streptococcus mutans, Candida albicans, orthodontic appliances, removable, oral microbiome

The numerous species that make up the oral microbiome are now understood to play a key role in establishment and maintenance of oral health. The ability to taxonomically identify community members at the species level is important to elucidating its diversity and association to health and disease. We report the overall ecological effects of using a toothpaste containing enzymes and proteins compared to a control toothpaste on the plaque microbiome. The results reported here demonstrate that a toothpaste containing enzymes and proteins can augment natural salivary defences to promote an overall community shift resulting in an increase in bacteria associated with gum health and a concomitant decrease in those associated with periodontal disease. Statistical analysis shows significant increases in 12 taxa associated with gum health including Neisseria spp. and a significant decrease in 10 taxa associated with periodontal disease including Treponema spp. The results demonstrate that a toothpaste containing enzymes and proteins can significantly shift the ecology of the oral microbiome (at species level) resulting in a community with a stronger association to health.

In this study, the essential oils of Orthosiphon stamineus Benth and Ficus deltoidea Jack were evaluated for their antibacterial activity against invasive oral pathogens, namely Enterococcus faecalis, Streptococcus mutans, Streptococcus mitis, Streptococcus salivarius, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis and Fusobacterium nucleatum. Chemical composition of the oils was analyzed using gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). The antibacterial activity of the oils and their major constituents were investigated using the broth microdilution method (minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)). Susceptibility test, anti-adhesion, anti-biofilm, checkerboard and time-kill assays were also carried out. Physiological changes of the bacterial cells after exposure to the oils were observed under the field emission scanning electron microscope (FESEM). O. stamineus and F. deltoidea oils mainly consisted of sesquiterpenoids (44.6% and 60.9%, respectively), and β-caryophyllene was the most abundant compound in both oils (26.3% and 36.3%, respectively). Other compounds present in O. stamineus were α-humulene (5.1%) and eugenol (8.1%), while α-humulene (5.5%) and germacrene D (7.7%) were dominant in F. deltoidea. The oils of both plants showed moderate to strong inhibition against all tested bacteria with MIC and MBC values ranging 0.63–2.5 mg/mL. However, none showed any inhibition on monospecies biofilms. The time-kill assay showed that combination of both oils with amoxicillin at concentrations of 1× and 2× MIC values demonstrated additive antibacterial effect. The FESEM study showed that both oils produced significant alterations on the cells of Gram-negative bacteria as they became pleomorphic and lysed. In conclusion, the study indicated that the oils of O. stamineus and F. deltoidea possessed moderate to strong antibacterial properties against the seven strains pathogenic oral bacteria and may have caused disturbances of membrane structure or cell wall of the bacteria.

The Streptococcus bovis/Streptococcus equinus complex (SBSEC) and possibly Streptococcus infantarius subsp. infantarius (Sii) are associated with human and animal diseases. Sii predominate in spontaneously fermented milk products with unknown public health effects. Sii/SBSEC prevalence data from West Africa in correlation with milk transformation practices are limited. Northern Co ˆte d'Ivoire served as study area due to its importance in milk production and consumption and to link a wider Sudano-Sahelian pastoral zone of cross-border trade. We aimed to describe the cow milk value chain and determine Sii/ SBSEC prevalence with a cross-sectional study. Dairy production practices were described as non-compliant with basic hygiene standards. The system is influenced by secular sociocultural practices and environmental conditions affecting product properties. Phenotypic and molecular analyses identified SBSEC in 27/43 (62.8%) fermented and 26/67 (38.8%) unfermented milk samples. Stratified by collection stage, fermented milk at producer and vendor levels featured highest SBSEC prevalence of 71.4% and 63.6%, respectively. Sii with 62.8% and 38.8% as well as Streptococcus gallolyticus subsp. macedonicus with 7.0% and 7.5% were the predominant SBSEC species identified among fermented and unfermented milk samples, respectively. The population structure of Sii/SBSEC isolates seems to reflect evolving novel dairy-adapted, non-adapted and potentially pathogenic lineages. Northern Co ˆte d'Ivoire was confirmed as area with high Sii presence in dairy products. The observed production practices and the high diversity of Sii/SBSEC supports in-depth

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.

To integrate a multiplex one-tube nested real-time polymerase chain reaction (mOTN-PCR) with a point-of-care testing (POCT) instrument for rapid, sensitive detection of (SPN), (GBS), and (GAS) in cerebrospinal fluid (CSF), achieving an automated \"sample-in, result-out\" workflow. The sensitivity of the mOTN-PCR was assessed using recombinant plasmids, and its specificity was evaluated using common pathogens associated with CSF infections. Simulated CSF samples were prepared using both manual nucleic acid extraction and fully automated nucleic acid extraction (POCT instrument), and then detected in parallel by mOTN-PCR and conventional real-time PCR. The clinical performance of the mOTN-PCR-POCT assay was further evaluated using 109 clinical samples (36 clinical CSF samples and 73 non-CSF clinical samples), and compared with the manual nucleic acid extraction followed by the conventional real-time PCR method. The mOTN-PCR achieved detection limits (LOD) of 5, 10, and 10 copies/μL for SPN, GBS, and GAS plasmids, respectively, with no cross-reactivity. In simulated CSF samples, the LODs were 20, 10, and 20 CFU/mL for SPN, GBS, and GAS, respectively, using manual extraction, within 4 hours. The mOTN-PCR-POCT method consistently detected all three pathogens at 20 CFU/mL, reducing total testing time to approximately 2 hours. Clinical sample testing revealed that the mOTN-PCR-POCT identified three additional positives in samples previously deemed negative by conventional real-time PCR, demonstrating superior detection capability and a more streamlined operational workflow of the mOTN-PCR-POCT. The mOTN-PCR-POCT was demonstrated to be simple to operate, requires no culture, and enables rapid, highly sensitive detection of SPN, GBS, and GAS in CSF.

The aim of our study was to investigate phenotypic and genotypic features of streptococci misidentified (misID) as Streptococcus pneumoniae, obtained over 20 years from hospital patients in Poland. Sixty-three isolates demonstrating microbiological features typical for pneumococci (optochin susceptibility and/or bile solubility) were investigated by phenotypic tests, lytA and 16S rRNA gene polymorphism and whole-genome sequencing (WGS). All isolates had a 6-bp deletion in the lytA 3′ terminus, characteristic for Mitis streptococc and all but two isolates lacked the pneumococcal signature cytosine at nucleotide position 203 in the 16S rRNA genes. The counterparts of psaA and ply were present in 100% and 81.0% of isolates, respectively; the spn9802 and spn9828 loci were characteristic for 49.2% and 38.1% of isolates, respectively. Phylogenetic trees and networks, based on the multilocus sequence analysis (MLSA) scheme, ribosomal multilocus sequence typing (rMLST) scheme and core-genome analysis, clearly separated investigated isolates from S. pneumoniae and demonstrated the polyclonal character of misID streptococci, associated with the Streptococcus pseudopneumoniae and Streptococcus mitis groups. While the S. pseudopneumoniae clade was relatively well defined in all three analyses, only the core-genome analysis revealed the presence of another cluster comprising a fraction of misID streptococci and a strain proposed elsewhere as a representative of a novel species in the Mitis group. Our findings point to complex phylogenetic and taxonomic relationships among S. mitis-like bacteria and support the notion that this group may in fact consist of several distinct species.

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