Biology subjects

Junges, R.

Publications and source records attributed to Junges, R..

6 recordsLinked to original sources

bioRxiv2026

Genetic and functional characterization of the natural transformation system in Streptococcus constellatus

Streptococcus constellatus is an opportunistic pathogen frequently associated with abscess formation in various body sites. While the species has been shown to acquire exogenous DNA through natural transformation, functional analyses of its underlying mechanisms and optimized genetic editing protocols remain limited. Thus, our aim was to characterize the natural transformation system in S. constellatus and investigate environmental factors regulating its natural transformation system. In addition, we sought to develop an optimized protocol for genome editing. Genomic analysis revealed that 73% of analyzed strains possess orthologs for essential competence regulon genes, with 55% harboring both a complete ComCDE-based operon and the necessary transformation machinery. While all complete genomes harbored three copies of the master regulator sigX, the accessory regulator comW was seemingly absent. Lacking the peptide exporter comAB, we demonstrated that S. constellatus utilizes the bacteriocin transporter silED for competence-stimulating peptide export. Gene expression assays indicated system activation at peptide concentrations as low as 4 nM, with peak sigX expression obtained over 60 nM. With the goal of optimizing gene editing strategies, we developed a protocol utilizing rich media supplemented with bovine serum albumin and calcium chloride, substantially increasing transformation frequencies. Furthermore, we observed that environmental stressors can upregulate the system, including hydrogen peroxide and subinhibitory concentrations of the antibiotics erythromycin, chloramphenicol, and ampicillin. Given the increasing clinical relevance of the anginosus group, elucidating horizontal gene transfer mechanisms can provide critical insights into the evolutionary success and pathogenic potential of these species.

microbiology↗

bioRxiv2026

Mapping antibiotic resistance determinants in oral streptococci

BackgroundAntibiotic resistance is a global priority in healthcare. Leveraging thousands of whole-genome sequences, here we reveal the core resistance determinants of oral streptococci, focusing on assessing pattern variability and gene exchange across commensals and pathogens. MethodsGenomic information was obtained from the National Center for Biotechnology Information. Determinants of antibiotic resistance were identified using AMRFinderPlus and CARD. ICEscreen was employed for calling of integrative and conjugative elements. Variability and recombination in penicillin-binding protein sequences were assessed with MMseqs2 and fastGEAR. ResultsA total of 2,087 genomes from 15 species were included with members from the mitis, mutans, anginosus, salivarius, and bovis groups. We observed 3,576 hits from 55 unique resistance genes conferring resistance to 11 antibiotic classes. The species with most resistance determinants per genome were identified as Streptococcus mitis (2.7), Streptococcus oralis (2.5), Streptococcus parasanguinis (1.9), Streptococcus gallolyticus (1.3), and Streptococcus anginosus (1.2). The two latter species also presented the most diverse composition of determinants. Over 1,800 integrative and conjugative elements were shown across all genomes, with nearly 18% of them carrying at least one antibiotic resistance gene. Penicillin-binding protein variation analyses showed a high diversity in the mitis group. Even though S. mitis and S. oralis composed less than 4% of the genomes included in the analyses, they were recognized as sources of DNA for over a third of recombination events in pbp1a and nearly half for both pbp2b and pbp2x in resistant isolates of Streptococcus pneumoniae. ConclusionsWe show that tetracycline and macrolide resistance were highly abundant and tightly connected to integrative and conjugative elements. Further, recent recombination data show frequent genetic exchange from oral streptococci to beta-lactam-resistant S. pneumoniae. Finally, assessing the dynamics of genetic exchange across species is central for the development of strategies to mitigate the impact of antibiotic resistance.

microbiology↗

bioRxiv2025

Large-scale global molecular epidemiology of antibiotic resistance determinants in Streptococcus pneumoniae

BackgroundStreptococcus pneumoniae is a leading pathogen in terms of deaths attributable to or associated with antimicrobial resistance globally. Thus, monitoring antibiotic resistance determinants constitutes a key aspect of surveillance efforts. AimLeveraging on publicly available whole-genome sequencing data, we aimed to investigate the presence and distribution patterns of antibiotic resistance in S. pneumoniae with a focus on multi-drug resistance and serotype distribution. MethodsMetadata and genomes were obtained from the National Center for Biotechnology Information Pathogen Detection database. Curation and harmonization were performed in R and SPSS. Data on resistance patterns was defined according to AMRFinderPlus and a combination of prediction tools were employed for in silico serotyping. ResultsAnalyses involved 75,161 genomes totaling 122,673 gene/allele counts from 14 antibiotic classes. Multi-drug resistance was observed in 16.7% of isolates with the highest increasing rates in Asia and South America. Within antibiotic classes, increase in macrolide resistance genes was highlighted, particularly in the proportion of genomes presenting mefA/msrD. Over a third of isolates with serotypes 19F, 23F, 15A, 6B, and 19A showed multi-drug resistance. We further observed the highest significant increases in the presence of resistance in 33F, 22F, 10A, and 23A. Serotype 13, not included in any vaccine formulation, presented high multi-drug resistance rates with a strong increasing trend. ConclusionsThe findings of this study highlight variations in resistance determinants globally and across serotypes over time. Collectively these data underscore the added value of utilizing public whole-genome sequencing data to investigate the effectiveness and repercussions of treatment and vaccination strategies on managing antibiotic resistance.

microbiology↗

bioRxiv2023

HAMLET, a human milk protein-lipid complex, modulates amoxicillin-induced changes in an ex vivo biofilm model of the oral microbiome

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.

immunology↗

bioRxiv2023

In vitro impact of fluconazole on oral microbial communities, bacterial growth and biofilm formation

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.

microbiology↗

bioRxiv2020

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.

microbiology↗