Biology subjects
Publications and source records attributed to Petersen, F. C..
We present a novel protocol for intranasal colonization of germ-free mice with human nasal microbiota, coupled with an optimized DNA extraction method for murine nasal samples compatible with 16S rRNA sequencing. This protocol may facilitate investigation of the functional roles and causal effects of nasal microbial communities in chronic rhinosinusitis and antimicrobial resistance.
Natural products are ubiquitously produced by many species that we encounter during our daily lives. One genus, Streptococcus, can produce a wide array of quorum sensing linked natural products known as RaS-RiPPs (ribosomally synthesized and post-translationally modified peptides). Their production is triggered by the induction of an Rgg-SHP quorum sensing system, which senses the presence of SHPs (short hydrophobic peptides) and induces the gene expression of these operons. Previous work has found that streptococcal RaS-RiPPs modulate the growth of different streptococci and might play a role in antibiotic tolerance. This is of particular importance to the oral microbiome, where streptococci are a predominant genus. This study examines the impact of the RaS-RiPP Tryglysin A on ex-vivo oral systems and provides the groundwork for determining how producer Streptococci might impact the composition and function of oral microbiome communities. We additionally explore important factors to consider when culturing ex-vivo oral systems.
Challenges from infections caused by biofilms and antimicrobial resistance highlight the need for novel antimicrobials that work synergistically with antibiotics and minimize resistance risk. In this study we investigated the potential synergistic effect of HAMLET (human alpha-lactalbumin made lethal to tumor cells), a human milk protein-lipid complex and amoxicillin on microbial ecology using an ex-vivo oral biofilm model. HAMLET was chosen due to its multi-targeted antimicrobial mechanism, together with its synergistic effect with antibiotics on single species pathogens, and low risk of resistance development. The combination of HAMLET and amoxicillin significantly reduced biofilm viability, while each of them alone had little or no impact. Using a whole metagenomics approach, we found that the combination group promoted a most remarkable shift in overall microbial composition compared to the untreated samples. Up to 90% of the bacterial species in the combined treatment were Lactobacillus crispatus, a species with probiotic effects, whereas it was detected in minor fraction in untreated samples. Resistome analysis indicated no major shifts on alpha-diversity, while beta-diversity revealed distinct clustering patterns for each treatment group, signifying that each treatment group harbors a unique resistome. TEM beta-lactamase genes were detected in low proportions in all treated samples but absent in untreated samples. Our study highlights the potential of HAMLET to synergize with amoxicillin in an ex-vivo model of the oral microbiome and modulate the proportion of probiotic bacteria. The findings extend the knowledge on the synergistic effects of HAMLET and antibiotics from single-species studies to polymicrobial biofilms of human origin. ImportancePolymicrobial infections are challenging to treat and prevent, requiring the use of antibiotics that exhibit reduced efficacy due to biofilm formation. HAMLET has recently emerged as an antimicrobial agent that can synergize with antibiotics while limiting microbial resistance. We investigated the effects of HAMLET, alone and combined with low concentrations of amoxicillin, on ex vivo oral biofilms to simulate complex microbial interactions observed in the oral cavity. The combination of HAMLET and amoxicillin effectively targeted polymicrobial biofilms and led to an increase in Lactobacillus crispatus. The potency of this combination appears to be due to the synergistic effect of HAMLET and amoxicillin. These findings underscore the potential of combining antimicrobials with different modes of action for the development of more effective strategies for preventing and treating polymicrobial infections.
Respiratory pathogens, commonly colonizing nasopharynx, are among the leading causes of death due to antimicrobial resistance. Yet, antibiotic resistance determinants within nasopharyngeal microbial communities remain poorly understood. Utilizing shotgun metagenomics, we investigated the nasopharynx resistome development in preterm infants, assessed early antibiotic impact on its trajectory, and explored its association with clinical covariates. Our findings revealed widespread nasopharyngeal carriage of antibiotic resistance genes (ARGs) with resistomes undergoing transient changes, including increased ARG diversity, abundance, and composition alterations due to early antibiotic exposure. ARGs associated with the critical nosocomial pathogen Serratia marcescens persisted up to 8-10 months of age, representing a long-lasting hospitalization signature. The nasopharyngeal resistome strongly correlated with microbiome composition, with inter-individual differences and postnatal age explaining most of the variation. Our report on the collateral effects of antibiotics and prolonged hospitalization underscores the urgency of further studies focused on this relatively unexplored reservoir of pathogens and ARGs.
Anti-fungal agents are widely used to specifically eliminate infections by fungal pathogens. However, the specificity of anti-fungal agents has been challenged by a few studies demonstrating anti-bacterial inhibitory effects against Mycobateria 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 progressively increased in the presence of fluconazole at clinically relevant concentrations, despite of a reduction in biomass. Target PCR revealed higher proportions 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 was 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, while for S, mitis planktonic growth, but not biofilm viability, was inhibited at the highest concentration. Fluconazole effects on S. mitis biofilm biomass were concentration and time-dependent. Exposure for 48h 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, planktonic growth of both Escherichia coli and Granulicatella adiacens were inhibited by fluconazole. Conclusions: 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 microbial biofilm communities. Future studies are warranted to further identify the breath of species for which growth may be impacted by anti-fungal agents, and possible mechanisms involved.
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.
BackgroundAntibiotic overuse has led to a global emergence of resistant bacteria, and children are among the frequent users. Most studies with broad-spectrum antibiotics show severe impact on the resistome development of patients. Although narrow-spectrum antibiotics are believed to have less 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 (OM).\n\nMethodsOral and fecal samples were collected from a one-year child before (day 0) and after (day 5 and 30) receiving Penicillin V against OM. Metagenomic sequencing data was analysed to determine taxonomic profiling, using Kraken and Bracken software, and resistance profiling, using KMA in combination with the ResFinder database.\n\nResultsIn the oral samples, 11 antimicrobial resistance genes (ARGs), belonging to four classes, were identified at baseline. At day 5, the abundance of some ARGs was increased, some remained unchanged, while others disappeared. At day 30, most ARGs had returned to baseline levels, or lower. In the fecal samples we observed seven ARGs at baseline and five at day 5, with only one gene observed at day 5 being present at baseline. At day 30 the number of ARGs increased to 21 ARGs from 7 different classes.\n\nConclusionsPenicillin V had a remarkable impact on the fecal resistome indicating that even narrow-pectrum antibiotics may have important consequences in selecting for a more resistant microbiome.
Streptococcus mutans, a bacterium with high cariogenic potential, coordinates competence for natural transformation and bacteriocin production via the XIP and CSP pheromones. CSP is effective in inducing bacteriocin responses, but not competence in chemically defined media (CDM). This is in contrast to XIP, which is a strong inducer of competence in CDM, but can also stimulate bacteriocin genes as a late response. Inter-connections between the pathways activated by the two pheromones have been characterized in certain detail in S. mutans UA159, but it is mostly unknown whether such findings are representative for the species. In this study, we used bioassays based on luciferase reporters for the bacteriocin gene cipB and the alternative sigma factor sigX to investigate various S. mutans isolates for production and response to CSP and XIP pheromones in CDM. Similar to S. mutans UA159, endogenous CSP was undetectable in the culture supernatants of all tested strains. During optimization of the bioassay using the cipB reporter, we discovered that the acivity of exogenous CSP used as a standard was reduced over time during S. mutans growth. Using a FRET-CSP reporter peptide, we found that S. mutans UA159 was indeed able to degrade CSP, and that such proteolytic activity was not significantly different in isogenic mutants with deletion of the protease gene htrA, or the competence genes sigX, oppD, and comR. CSP proteolysis was also detected in all the wild type strains, indicating that such activity is conserved in S. mutans. For the XIP pheromone, endogenous production was observed in the supernatants of all 34 tested strains at peak concentrations in culture supernatants that varied between 200 nM and 26000 nM. Transformation in the presence of exogenous XIP was detected in all, but one, of the isolates. The efficiency of transformation varied, however, among the different strains, and for those with the highest transformation rates, endogenous XIP peak concentrations in the supernatants were above 2000 nM XIP. We conclude that XIP production and inducing effect on transformation, as well as proteolytic activity leading to the inactivation of CSP are conserved functions among different S. mutans isolates. Understanding the functionality and conservation of pheromone systems in S. mutans may lead to novel strategies to prevent or treat unbalances in oral microbiomes that may favour diseases.
Here we show that mouse IgG2a and IgG1 antibodies specific for the commensal Streptococcus mitis cross-react with the pathogen Streptococcus pneumoniae, although the cross-reactivity conferred by IgG2a is stronger than IgG1 antibodies. These findings may have implications for designing S. mitis-based vaccines against pneumococcal infections.