Vancomycin-resistant Enterococcus faecium is a leading cause of hospital-acquired infections and outbreaks. Regulatory RNAs (sRNAs) are major players in adaptive responses, including antibiotic resistance. They were extensively studied in gram-negative bacteria, but less information is available for gram-positive pathogens. No sRNAs are described in E. faecium. We sought to identify a set of sRNAs expressed in vancomycin-resistant E. faecium Aus0004 strain to assess their roles in daptomycin response and resistance. Genomic and transcriptomic analyses revealed a set of 61 sRNA candidates, including 10 that were further tested and validated by Northern and qPCR. RNA-seq was performed with and without subinhibitory concentrations (SICs) of daptomycin, an antibiotic used to treat enterococcal infections. After daptomycin SIC exposure, the expression of 260 coding and srna genes was altered, with 80 upregulated and 180 downregulated, including 51% involved in carbohydrate and transport metabolisms. Daptomycin SIC exposure significantly affected the expression of seven sRNAs, including one experimentally confirmed, sRNA_0160. We studied sRNA expression in isogenic mutants with increasing levels of daptomycin resistance and observed that expression of several sRNAs, including sRNA_0160, was modified in the stepwise mutants. This first genome-wide sRNA identification in E. faecium suggests that some sRNAs are linked to antibiotic stress response and resistance.

Periodontitis are bacterial inflammatory diseases, where the bacterial biofilms present on the tooth-supporting tissues switch from a healthy state towards a pathogenic state. Among bacterial species involved in the disease, Porphyromonas gingivalis has been shown to induce dysbiosis, and to induce virulence of otherwise healthy bacteria like Streptococcus gordonii. During biofilm development, primary colonizers such as S. gordonii first attach to the surface and allow the subsequent adhesion of periodontal pathogens such as P. gingivalis. Interactions between those two bacteria have been extensively studied during the adhesion step of the biofilm. The aim of the study was to understand interactions of both species during the growing phase of the biofilm, for which little knowledge is available, using a mathematical model. This two-species biofilm model was based on a substrate-dependent growth, implemented with damage parameters, and validated thanks to data obtained on experimental biofilms. Three different hypothesis of interactions were proposed and assayed using this model: independence, competition between both bacteria species, or induction of toxicity by one species for the other species. Adequacy between experimental and simulated biofilms were found with the last hypothetic mathematical model. This new mathematical model of two species bacteria biofilms, dependent on different substrates for growing, can be applied to any bacteria species, environmental conditions, or steps of biofilm development. It will be of great interest for exploring bacterial interactions in biofilm conditions.

The human pathogen Staphylococcus aureus expresses a set of transcriptional factors and small RNAs (sRNAs) to adapt to environmental variations. Recent harmonization of staphylococcal sRNA data allowed us to search for novel sRNAs using DETR’PROK, a computational pipeline for identifying sRNA in prokaryotes. We performed RNA-Seq on Newman strain and identified a set of 48 sRNA candidates. To avoid bioinformatic artefacts, we applied a series of cut-offs and tested experimentally each selected intergenic region. This narrowed the field to 24 expressed sRNAs, of which 21 were new and designated with Srn identifiers. Further examination of these loci revealed that one exhibited an unusual condensed sRNA cluster of about 650 nucleotides. We determined the transcriptional start sites within this region and demonstrated the presence of three contiguous sRNA genes (srn_9342, srn_9344 and srn_9345) expressed from the positive strand, and two others (srn_9343 and srn_9346) transcribed from the opposite one. Using comparative genomics, we showed that genetic organization of the srn_9342-9346 locus is specific to Newman and that its expression is growth-phase dependent and subjected to nutrient deprivation and oxidative stress. Finally, we demonstrated that srn_9343 encodes a secreted peptide that could belong to a novel S. aureus toxin-antitoxin system.