Prolonged hospitalization signature and early antibiotic effects on the nasopharyngeal resistome in preterm infants.

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. In this prospective cohort study, we investigate the nasopharynx resistome development in preterm infants, assess early antibiotic impact on its trajectory, and explore its association with clinical covariates using shotgun metagenomics. Our findings reveal 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 persist up to 8-10 months of age, representing a long-lasting hospitalization signature. The nasopharyngeal resistome strongly correlates 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.

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.

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.

Conserved Pheromone Production, Response and Degradation by Streptococcus mutans.

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. Interconnections 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 activity 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 able to degrade CSP, and that such activity was not significantly different in isogenic mutants with deletion of the protease gene htrA or the competence genes sigX, oppD, and comR. CSP cleavage was also detected in all the wild type strains, indicating that this is a conserved feature 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 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 the ability to degrade 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 favor diseases.