In Vitro Impact of Fluconazole on Oral Microbial Communities, Bacterial Growth, and Biofilm Formation.

Antifungal agents are widely used to specifically eliminate infections by fungal pathogens. However, the specificity of antifungal agents has been challenged by a few studies demonstrating antibacterial inhibitory effects against Mycobacteria and Streptomyces species. Here, we evaluated for the first time the potential effect of fluconazole, the most clinically used antifungal agent, on a human oral microbiota biofilm model. The results showed that biofilm viability on blood and mitis salivarius agar media was increased over time in the presence of fluconazole at clinically relevant concentrations, despite a reduction in biomass. Targeted PCR revealed a higher abundance of Veillonella atypica, Veillonella dispar, and Lactobacillus spp. in the fluconazole-treated samples compared to the control, while Fusobacterium nucleatum was reduced and Streptococcus spp were not significantly affected. Further, we tested the potential impact of fluconazole using single-species models. Our results, using Streptococcus mutans and Streptococcus mitis luciferase reporters, showed that S. mutans planktonic growth was not significantly affected by fluconazole, whereas for S. mitis, planktonic growth, but not biofilm viability, was inhibited at the highest concentration. Fluconazole's effects on S. mitis biofilm biomass were concentration and time dependent. Exposure for 48 h to the highest concentration of fluconazole was associated with S. mitis biofilms with the most increased biomass. Potential growth inhibitory effects were further tested using four non-streptococcal species. Among these, the planktonic growth of both Escherichia coli and Granulicatella adiacens was inhibited by fluconazole. The data indicate bacterial responses to fluconazole that extend to a broader range of bacterial species than previously anticipated from the literature, with the potential to disturb biofilm communities.

Treatment of Mouse Infants with Amoxicillin, but Not the Human Milk-Derived Antimicrobial HAMLET, Impairs Lung Th17 Responses.

Emerging evidence suggests differential effects of therapeutic antibiotics on infant T cell responses to pathogens. In this study, we explored the impact of the treatment of mouse infants with amoxicillin and the human milk-derived antimicrobial HAMLET (human alpha-lactalbumin made lethal to tumor cells) on T cell responses to Streptococcus pneumoniae. Lung cells and splenocytes were isolated from the infant mice subjected to intranasal administration of amoxicillin, HAMLET, or a combination of HAMLET and amoxicillin, and cultured with S. pneumoniae to measure T cell responses. After in-vitro stimulation with S. pneumoniae, lung cells from amoxicillin- or amoxicillin plus HAMLET-treated mice produced lower levels of Th17 (IL-17A), but not Th1 (IFN-γ), cytokine than mice receiving HAMLET or PBS. IL-17A/IFN-γ cytokine levels produced by the stimulated splenocytes, on the other hand, revealed no significant difference among treatment groups. Further analysis of T cell cytokine profiles by flow cytometry showed that lung CD4+, but not CD8+, T cells from amoxicillin- or HAMLET plus amoxicillin-treated mice expressed decreased levels of IL-17A compared to those from HAMLET-exposed or control mice. Collectively, these results indicate that exposure of infant mice to amoxicillin, but not HAMLET, may suppress lung Th17 responses to S. pneumoniae.

Suppressive effect of therapeutic antibiotic regimen on antipneumococcal Th1/Th17 responses in neonatal mice.

BACKGROUND: Antibiotics are commonly used in human neonates, but their impact on neonatal T cell immunity remains poorly understood. The aim of this study was to investigate the impact of the antibiotic piperacillin with the beta-lactamase inhibitor tazobactam on neonatal CD4+ and CD8+ T cell responses to Streptococcus pneumoniae. METHODS: Splenic and lung cells were isolated from the neonatal mice receiving piperacillin and tazobactam or saline (sham) and cultured with S. pneumoniae to analyze T cell cytokine production by ELISA and flow cytometry. RESULTS: Antibiotic exposure to neonatal mice resulted in reduced numbers of CD4+/CD8+ T cells in the spleen and lungs compared to control mice. Upon in vitro stimulation with S. pneumoniae, splenocytes and lung cells from antibiotic-exposed mice produced lower levels of IFN-γ (Th1)/IL-17A (Th17) and IL-17A cytokines, respectively. Flow cytometric analysis revealed that S. pneumoniae-stimulated splenic CD4+ T cells from antibiotic-exposed mice expressed decreased levels of IFN-γ and IL-17A compared to control mice, whereas lung CD4+ T cells produced lower levels of IL-17A. However, no significant difference was observed for IL-4 (Th2) production. CONCLUSIONS: Neonatal mice exposure to piperacillin and tazobactam reduces the number of CD4+ and CD8+ T cells, and suppresses Th1 and Th17, but not Th2, responses to S. pneumoniae. IMPACT: Exposure of neonatal mice with a combination of piperacillin and tazobactam reduces CD4+/CD8+ T cells in the spleen and lungs. Antibiotic exposure suppresses neonatal Th1 and Th17, but not Th2, responses to Streptococcus pneumoniae. Our findings may have important implications for developing better therapeutic strategies in the neonatal intensive care unit.

Differential response to prolonged amoxicillin treatment: long-term resilience of the microbiome versus long-lasting perturbations in the gut resistome.

The collateral impact of antibiotics on the microbiome has attained increasing attention. However, the ecological consequences of long-term antibiotic exposure on the gut microbiome, including antibiotic resistance, are still limited. Here, we investigated long-term exposure effects to amoxicillin on the human gut microbiome and resistome. Fecal samples were collected from 20 patients receiving 3-months of amoxicillin or placebo treatment as part of a Norwegian multicenter clinical trial on chronic low back pain (AIM study). Samples were collected at baseline, last day of treatment, and 9 months after antibiotic cessation. The abundance and diversity of microbial and resistome composition were characterized using whole shotgun and functional metagenomic sequencing data. While the microbiome profiles of placebo subjects were stable over time, discernible changes in diversity and overall microbiome composition were observed after amoxicillin treatment. In particular, health-associated short-chain fatty acid producing species significantly decreased in proportion. However, these changes were short-lived as the microbiome showed overall recovery 9 months post-treatment. On the other hand, exposure to long-term amoxicillin was associated with an increase in total antimicrobial resistance gene load and diversity of antimicrobial resistance genes, with persistent changes even at 9 months post-treatment. Additionally, beta-lactam resistance was the most affected antibiotic class, suggesting a targeted response to amoxicillin, although changes at the gene level varied across individuals. Overall, our results suggest that the impact of prolonged amoxicillin exposure was more explicit and long-lasting in the fecal resistome than in microbiome composition. Such information is relevant for designing rational administration guidelines for antibiotic therapies.

Markerless Genome Editing in Competent Streptococci.

Selective markers employed in classical mutagenesis methods using natural genetic transformation can affect gene expression, risk phenotypic effects, and accumulate as unwanted genes during successive mutagenesis cycles. In this chapter, we present a protocol for markerless genome editing in Streptococcus mutans and Streptococcus pneumoniae achieved with an efficient method for natural transformation. High yields of transformants are obtained by combining the unimodal state of competence developed after treatment of S. mutans with sigX-inducing peptide pheromone (XIP) in a chemically defined medium (CDM) or of S. pneumoniae with the competence-stimulating peptide (CSP) together with use of a donor amplicon carrying extensive flanking homology. This combination ensures efficient and precise integration of a new allele by the recombination machinery present in competent cells.

Awareness regarding antimicrobial resistance and confidence to prescribe antibiotics in dentistry: a cross-continental student survey.

BACKGROUND: The antimicrobial resistance (AMR) crisis is a major global threat and one of its biggest drivers is the overuse of antibiotics in humans. Dentists are responsible for 5-10% antibiotic prescriptions worldwide and recent data suggest that knowledge and prescribing practices need improvement. METHODS: A cross-sectional web-survey was sent to dental students from six universities in Norway, Canada, and Brazil. Topics addressed covered awareness, confidence to prescribe antibiotics, and education needs. Data were presented descriptively and statistical testing was employed to compare group means when applicable. RESULTS: In total, 562 responses were collected across the three countries with a response rate of 28.6%. 'Antibiotic resistance' was among the highest priorities (scale 1-10) with an average of 8.86 (SEM ± 0.05), together with 'Gender inequality' (8.68 ± 0.07) and 'Climate change' (8.68 ± 0.07). Only 28.8% thought that Dentistry was engaged in national/international campaigns promoting awareness on the topic and 8.9% stated to have heard about the 'One Health' concept. Final year dental students showed an average confidence to prescribe antibiotics of 7.59 (± 0.14). Most students demonstrated interest in receiving additional education on all topics listed, with the three most pressing being 'antibiotic prescription for treatment of infections' (82.9%), 'drug interactions' (80.9%), and 'spread of antibiotic resistance' (79.6%). A trend was observed between higher awareness regarding the topic and higher confidence to prescribe. CONCLUSIONS: There is a need to revisit dental education on antibiotic resistance with a global perspective and to create more stewardship initiatives that promote awareness on the topic.

Effects of different amoxicillin treatment durations on microbiome diversity and composition in the gut.

Antibiotics seize an effect on bacterial composition and diversity and have been demonstrated to induce disruptions on gut microbiomes. This may have implications for human health and wellbeing, and an increasing number of studies suggest a link between the gut microbiome and several diseases. Hence, reducing antibiotic treatments may be beneficial for human health status. Further, antimicrobial resistance (AMR) is an increasing global problem that can be counteracted by limiting the usage of antibiotics. Longer antibiotic treatments have been demonstrated to increase the development of AMR. Therefore, shortening of antibiotic treatment durations, provided it is safe for patients, may be one measure to reduce AMR. In this study, the objective was to investigate effects of standard and reduced antibiotic treatment lengths on gut microbiomes using a murine model. Changes in the murine gut microbiome was assessed after using three different treatment durations of amoxicillin (3, 7 or 14 days) as well as a control group not receiving amoxicillin. Fecal samples were collected before and during the whole experiment, until three weeks past end of treatment. These were further subject for 16S rRNA Illumina MiSeq sequencing. Our results demonstrated significant changes in bacterial diversity, richness and evenness during amoxicillin treatment, followed by a reversion in terms of alpha-diversity and abundance of major phyla, after end of treatment. However, a longer restitution time was indicated for mice receiving amoxicillin for 14 days, and phylum Patescibacteria did not fully recover. In addition, an effect on the composition of Firmicutes was indicated to last for at least three weeks in mice treated with amoxicillin for 14 days. Despite an apparently reversion to a close to original state in overall bacterial diversity and richness, the results suggested more durable changes in lower taxonomical levels. We detected several families, genera and ASVs with significantly altered abundance three weeks after exposure to amoxicillin, as well as bacterial taxa that appeared significantly affected by amoxicillin treatment length. This may strengthen the argument for shorter antibiotic treatment regimens to both limit the emergence of antibiotic resistance and risk of gut microbiome disturbance.

Can polyphenolic surface modifications prevent fungal colonization of titanium dental implants?

Oral biofilms can be a major health problem causing infections and chronic inflammation of mucosal tissue. While much effort is put in the investigation of bacteria in biofilms, the role of fungi is often neglected, despite Candida albicans playing a key role in the formation of multispecies oral biofilms. With the rise of antibiotic resistance, new strategies to reduce microbial growth need to be found. Therefore, plant derived polyphenolic molecules have been suggested to reduce both adhesion and growth of pathogenic bacteria and fungi. In this study, we investigated the use of polyphenolic coatings to reduce adhesion and biofilm formation of C. albicans BWP17 on titanium implants. Tannic acid and pyrogallol coatings altered the hydrophobic and charge properties of titanium surfaces, and both compounds were gradually released as active molecules over time. Despite such effects, we found no significant inhibition on growth and biofilm formation of C. Albicans, indicating that the release of active molecules from the coatings did not reach relevant inhibitory concentrations. However, a potential antibiofilm effect was observed by the pH-dependent disassembly of the polyphenolic layer, which caused the biofilm to detach. Hence, further efforts are required to create tailored implant surfaces, which sustainably reduce microbial growth and adhesion.

Microbial DNA extraction of high-host content and low biomass samples: Optimized protocol for nasopharynx metagenomic studies.

Introduction: Low microbial biomass and high human DNA content in nasopharyngeal aspirate samples hinder comprehensive characterization of microbiota and resistome. We obtained samples from premature infants, a group with increased risk of developing respiratory disorders and infections, and consequently frequent exposure to antibiotics. Our aim was to devise an optimal protocol for handling nasopharyngeal aspirate samples from premature infants, focusing on host DNA depletion and microbiome and resistome characterization. Methods: Three depletion and three DNA extraction protocols were compared, using RT-PCR and whole metagenome sequencing to determine the efficiency of human DNA removal, taxonomic profiling and assignment of antibiotic resistance genes. Protocols were tested using mock communities, as well as pooled and individual patient samples. Results: The only extraction protocol to retrieve the expected DNA yield from mock community samples was based on a lytic method to improve Gram positive recovery (MasterPure™). Host DNA content in non-depleted aliquots from pooled patient samples was 99%. Only samples depleted with MolYsis™ showed satisfactory, but varied reduction in host DNA content, in both pooled and individual patient samples, allowing for microbiome and resistome characterisation (host DNA content from 15% to 98%). Other depletion protocols either retrieved too low total DNA yields, preventing further analysis, or failed to reduce host DNA content. By using Mol_MasterPure protocol on aliquots from pooled patient samples, we increased the number of bacterial reads by 7.6 to 1,725.8-fold compared to non-depleted reference samples. PCR results were indicative of achieved microbial enrichment. Individual patient samples processed with Mol_MasterPure protocol varied greatly in total DNA yield, host DNA content (from 40% to 98%), species and antibiotic resistance gene richness. Discussion: Despite high human DNA and low microbial biomass content in nasopharynx aspirates of preterm infants, we were able to reduce host DNA content to levels compatible with downstream shotgun metagenomic analysis, including bacterial species identification and coverage of antibiotic resistance genes. Whole metagenomic sequencing of microbes colonizing the nasopharynx may contribute to explaining the possible role of airway microbiota in respiratory conditions and reveal carriage of antibiotic resistance genes.

Effects of Expression of Streptococcus pneumoniae PspC on the Ability of Streptococcus mitis to Evade Complement-Mediated Immunity.

Streptococcus pneumoniae and Streptococcus mitis are genetically closely related and both frequently colonise the naso-oropharynx, yet S. pneumoniae is a common cause of invasive infections whereas S. mitis is only weakly pathogenic. We hypothesise that sensitivity to innate immunity may underlie these differences in virulence phenotype. We compared the sensitivity of S. pneumoniae and S. mitis strains to complement-mediated immunity, demonstrating S. mitis strains were susceptible to complement-mediated opsonophagocytosis. S. pneumoniae resistance to complement is partially dependent on binding of the complement regulator Factor H by the surface protein PspC. However, S. mitis was unable to bind factor H. The S. pneumoniae TIGR4 strain pspC was expressed in the S. mitis SK142 strain to create a S. mitis pspC+ strain. Immunoblots demonstrated the S. mitis pspC+ strain expressed PspC, and flow cytometry confirmed this resulted in Factor H binding to S. mitis, reduced susceptibility to complement and improved survival in whole human blood compared to the wild-type S. mitis strain. However, in mouse models the S. mitis pspC+ strain remained unable to establish persistent infection. Unlike S. pneumoniae strains, culture in serum or blood did not support increased CFU of the S. mitis strains. These results suggest S. mitis is highly sensitive to opsonisation with complement partially due to an inability to bind Factor H, but even when complement sensitivity was reduced by expression of pspC, poor growth in physiological fluid limited the virulence of S. mitis in mice.

Combined loss of expression of involucrin and cytokeratin 13 is associated with poor prognosis in squamous cell carcinoma of mobile tongue.

BACKGROUND: This study aimed to evaluate the prognostic significance of expression levels of involucrin (IVL), cytokeratin (CK)-10 and -13 at different intratumor sites (tumor center and invading area) of oral tongue squamous cell carcinoma (OTSCC). METHODS: IVL, CK13 and CK10 expression levels were examined in a multicenter cohort of 146 OTSCCs using immunohistochemistry. External mRNA datasets were used for expression analysis and/or to validate survival associations. RESULTS: External transcriptomic datasets showed downregulation of IVL and KRT13 in oral malignancies including OTSCC as compared to normal controls. The combined loss of IVL and CK13 expression at the invading core but not at the center core was significantly associated with poor differentiation and reduced 5-year overall survival. Multivariate Cox analysis confirmed the loss of CK13 and IVL expression to be an independent prognostic factor. Transcriptomic dataset corroborated immunohistochemistry results. CONCLUSIONS: Combined expression levlels of IVL and CK13 might be useful as prognostic biomarkers in OTSCC.

Vaccination With the Commensal Streptococcus mitis Expressing Pneumococcal Serotype 5 Capsule Elicits IgG/IgA and Th17 Responses Against Streptococcus pneumoniae.

Recent studies have identified a clinical isolate of the commensal Streptococcus mitis that expresses Streptococcus pneumoniae serotype 5 capsule (S. mitis serotype 5) and shows serospecificity toward pneumococcal serotype 5. However, it remains unknown whether S. mitis serotype 5 induces protective immunity against pneumococcal serotype 5. In this study, we evaluated the ability of S. mitis serotype 5 to generate protective immunity in a mouse model of lung infection with pneumococcal serotype 5. Upon challenge infection with S. pneumoniae serotype 5, mice intranasally immunized with S. mitis serotype 5 exhibited reduced pneumococcal loads in the lungs, nasal wash, and bronchoalveolar lavage fluid compared with those receiving PBS (control). The immunized mice displayed significantly higher levels of IgG and IgA antibodies reactive to S. mitis serotype 5, S. pneumoniae serotype 5 or S. pneumoniae serotype 4 than the antibody levels in control mice. In vaccinated mice, the IgG/IgA antibody levels reactive to S. mitis serotype 5 or S. pneumoniae serotype 5 were higher than the levels reactive to S. pneumoniae serotype 4. Furthermore, in-vitro restimulation of the lung-draining mediastinal lymph node cells and splenocytes from immunized mice with killed S. mitis serotype 5, S. pneumoniae serotype 5 or S. pneumoniae serotype 4 showed enhanced Th17, but not Th1 and Th2, responses. Overall, our findings show that mucosal immunization with S. mitis serotype 5 protects against S. pneumoniae serotype 5 infection and induces Th17 and predominant serotype-specific IgG/IgA antibody responses against pneumococcal infection.

ResistoXplorer: a web-based tool for visual, statistical and exploratory data analysis of resistome data.

The study of resistomes using whole metagenomic sequencing enables high-throughput identification of resistance genes in complex microbial communities, such as the human microbiome. Over recent years, sophisticated and diverse pipelines have been established to facilitate raw data processing and annotation. Despite the progress, there are no easy-to-use tools for comprehensive visual, statistical and functional analysis of resistome data. Thus, exploration of the resulting large complex datasets remains a key bottleneck requiring robust computational resources and technical expertise, which creates a significant hurdle for advancements in the field. Here, we introduce ResistoXplorer, a user-friendly tool that integrates recent advancements in statistics and visualization, coupled with extensive functional annotations and phenotype collection, to enable high-throughput analysis of common outputs generated from metagenomic resistome studies. ResistoXplorer contains three modules-the 'Antimicrobial Resistance Gene Table' module offers various options for composition profiling, functional profiling and comparative analysis of resistome data; the 'Integration' module supports integrative exploratory analysis of resistome and microbiome abundance profiles derived from metagenomic samples; finally, the 'Antimicrobial Resistance Gene List' module enables users to intuitively explore the associations between antimicrobial resistance genes and the microbial hosts using network visual analytics to gain biological insights. ResistoXplorer is publicly available at http://www.resistoxplorer.no.

Antibiotic Stewardship in Premature Infants: A Systematic Review.

INTRODUCTION: Antibiotic treatment in premature infants is often empirically prescribed, and practice varies widely among otherwise comparable neonatal intensive care units. Unnecessary and prolonged antibiotic treatment is documented in numerous studies. Recent research shows serious side effects and suggests long-term adverse health effects in prematurely born infants exposed to antibiotics in early life. One preventive measure to reduce unnecessary antibiotic exposure is implementation of antibiotic stewardship programs. Our objective was to review the literature on implemented antibiotic stewardship programs including premature infants with gestational age ≤34 weeks. METHODS: Six academic databases (PubMed [Medline], McMaster PLUS, Cochrane Database of Systematic Reviews, UpToDate, Cochrane Central Register of Controlled Trials, and National Institute for Health and Care Excellence) were systematically searched. PRISMA guidelines were applied. RESULTS: The search retrieved 1,212 titles of which 12 fitted inclusion criteria (11 observational studies and 1 randomized clinical trial). Included articles were critically appraised. We grouped the articles according to common area of implemented stewardship actions: (1) focus on reducing initiation of antibiotic therapy, (2) focus on shortening duration of antibiotic therapy, (3) various organizational stewardship implementations. The heterogeneity of cohort composition, of implemented actions and of outcome measures made meta-analysis inappropriate. We provide an overview of the reduction in antibiotic use achieved. CONCLUSION: Antibiotic stewardship programs can be effective for premature newborns especially when multifactorial and tailored to this population, focusing on reducing initiation or on shortening the duration of antibiotic therapy. Programs without specific measures were less effective.

The Dark Side of Antibiotics: Adverse Effects on the Infant Immune Defense Against Infection.

Although antibiotics confer significant health benefits in treating or preventing bacterial infections, an accumulating wealth of evidence illustrates their detrimental effect on host-microbiota homeostasis, posing a serious menace to the global public health. In recent years, it is becoming evident that infants, who are subjected to frequent antibiotic exposures due to their vulnerability to infection, reflect increased susceptibility to a wide spectrum of diseases, including infection, in later life. Antibiotics induce perturbations of the microbiota or dysbiosis, which in turn alters the host immune responses against pathogens. In comparison with adults, antibiotic treatments in infants have disproportionate consequences because the infant microbiota represents an evolving system that is unstable and immature until 2-3 years of age. However, relatively less knowledge is available on how antibiotics affect the infant microbiota and immunity. In this review article, we focus on how antibiotic treatment regimens influence the infant innate and adaptive immunity to pathogens in humans and animal models, and make the host susceptible to infections in later life. There is a critical need to better understand the effect of antibiotics on infant immune function, which may have implications for developing effective prophylactics and therapeutics against diseases in infants and adults.

Cyclic Di-adenosine Monophosphate Regulates Metabolism and Growth in the Oral Commensal Streptococcus mitis.

Cyclic di-adenosine monophosphate (c-di-AMP) has emerged as an important bacterial signaling molecule that functions both as an intracellular second messenger in bacterial cells and an extracellular ligand involved in bacteria-host cross-talk. In this study, we identify and characterize proteins involved in controlling the c-di-AMP concentration in the oral commensal and opportunistic pathogen Streptococcusmitis (S. mitis). We identified three known types of c-di-AMP turnover proteins in the genome of S. mitis CCUG31611: a CdaA-type diadenylate cyclase as well as GdpP-, and DhhP-type phosphodiesterases. Biochemical analyses of purified proteins demonstrated that CdaA synthesizes c-di-AMP from ATP whereas both phosphodiesterases can utilize c-di-AMP as well as the intermediary metabolite of c-di-AMP hydrolysis 5'-phosphadenylyl-adenosine (pApA) as substrate to generate AMP, albeit at different catalytic efficiency. Using deletion mutants of each of the genes encoding c-di-AMP turnover proteins, we show by high resolution MS/MS that the intracellular concentration of c-di-AMP is increased in deletion mutants of the phosphodiesterases and non-detectable in the cdaA-mutant. We also detected pApA in mutants of the DhhP-type phosphodiesterase. Low and high levels of c-di-AMP were associated with longer and shorter chains of S. mitis, respectively indicating a role in regulation of cell division. The deletion mutant of the DhhP-type phosphodiesterase displayed slow growth and reduced rate of glucose metabolism.

Helicobacter pylori was not detected in oral squamous cell carcinomas from cohorts of Norwegian and Nepalese patients.

Helicobacter pylori (HP) infection is an established causative agent for gastric cancer. Although the oral cavity is a part of the gastrointestinal system, the presence and possible causative role of HP in oral squamous cell carcinoma (OSCC) is a subject of controversy. Therefore, the current study aimed to investigate HP infection in two cohorts of OSCC patients with different demographic characteristics, lifestyles and habitual risk factors. A total of 242 formalin-fixed paraffin-embedded OSCC specimens from two different patient cohorts (Norway, n = 171 and Nepal, n = 71) were used to examine HP using immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR). Two different HP specific genes (23S rRNA and ureA) were used for TaqMan-based qPCR, and for subsequent verification using HP specific RIDAGENE HP kit and SYBR Green based qPCR. All of the OSCC specimens from both cohorts were found to be negative for HP infection with IHC and qPCR, although the positive control specimens tested positive. Our findings suggest that HP is absent in the examined OSCC cohorts, irrespective of race, lifestyle and habitual risk factors. This indicates that, in contrast to gastric cancer, HP is an unlikely contributing factor for OSCC pathogenesis.

Impact of narrow-spectrum penicillin V on the oral and faecal resistome in a young child treated for otitis media.

OBJECTIVES: Antibiotic overuse has led to the global emergence of antimicrobial-resistant bacteria, and children are among the most frequent users of antibiotics. Most studies with broad-spectrum antibiotics show a severe impact on resistome development in patients. Although narrow-spectrum antibiotics are believed to have fewer side effects, their impact on the microbiome and resistome is mostly unknown. The aim of this study was to investigate the impact of the narrow-spectrum antibiotic phenoxymethylpenicillin (penicillin V) on the microbiome and resistome of a child treated for acute otitis media. METHODS: Oral and faecal samples were collected from a 1-year-old child before (Day 0) and after (Days 5 and 30) receiving penicillin V for otitis media. Metagenomic sequencing data were analysed to determine taxonomic profiling using Kraken and Bracken software, and resistance profiling using KMA in combination with the ResFinder database. RESULTS: In the oral samples, antimicrobial resistance genes (ARGs) belonging to four classes were identified at baseline. At Day 5, the abundance of some ARGs was increased, whereas some remained unchanged and others could no longer be detected. At Day 30, most ARGs had returned to baseline levels or lower. In the faecal samples, seven ARGs were observed at baseline and five at Day 5. At Day 30, the number of ARGs had increased to 21. CONCLUSIONS: Following penicillin V, we observed a remarkable enrichment of the aecal resistome, indicating that even narrow-spectrum antibiotics may have important consequences in selecting for a more resistant microbiome.

In Vivo Relationship between the Nano-Biomechanical Properties of Streptococcal Polysaccharide Capsules and Virulence Phenotype.

In common with many bacterial pathogens, Streptococcus pneumoniae has a polysaccharide capsule which facilitates immune evasion and determines virulence. Recent data have shown that the closely related Streptococcus mitis also expresses polysaccharide capsules including those with an identical chemical structure to S. pneumoniae capsular serotypes. We utilized atomic force microscopy (AFM) techniques to investigate the biophysical properties of S. mitis and S. pneumoniae strains expressing the same capsular serotypes that might relate to differences in virulence potential. When comparing S. mitis and S. pneumoniae strains with identical capsule serotypes, S. mitis strains were susceptible to neutrophil killing, and electron microscopy and AFM demonstrated significant morphological differences. Force-volume mapping using AFM showed distinct force-curve profiles for the center and edge areas of encapsulated streptococcal strains. This "edge effect" was not observed in unencapsulated bacteria and therefore was a direct representation of the mechanical properties of the bacterial capsule. When two strains of S. mitis and S. pneumoniae expressed an identical capsular serotype, they presented similar biomechanical characteristics. This infers a potential relationship between capsule biochemistry and nanomechanics, independent of bacterial strain. Overall, this study demonstrates that it is possible to investigate reproducibly the mechanistic, structural, and mechanical properties of both the capsule and the body of individual living bacterial cells and relate the data to virulence phenotypes. We have demonstrated that using nanomechanics to investigate individual bacterial cells we can now begin to identify the surface properties bacterial pathogens require to avoid host-mediated immunity.