Regulatory RNA-mediated regulations for adaptation of Enterococcus faecium and Staphylococcus aureus to biocide stress

Enterococcus faecium et Staphylococcus aureus sont des bactéries pathogènes opportunistes de l’homme responsables d’épidémies hospitalières. Les antiseptiques sont très utilisés pour prévenir la survenue de ces infections et leur présence dans l’environnement hospitalier, même à faibles concentrations, dites subinhibitrices, constitue un stress auquel ces bactéries doivent d’adapter. Les ARN régulateurs (sARN) sont des acteurs importants pour l’adaptation aux variations environnementales des bactéries. Les preuves de leur existence sont anciennes chez S. aureus mais récentes chez E. faecium, entrainant un état des connaissances très hétérogène. Dans un premier temps, nous avons souhaité caractériser les sARN dont l’expression était altérée en cas de stress antiseptique exercé par de la chlorhexidine, du chlorure de benzalkonium, de la povidone iodée et du triclosan. Nous avons montré que ce stress entrainait une répression globale des sARN étudiés chez E. faecium mais pas chez S. aureus. Dans un second temps, l’étude approfondie d’un sARN d’E. faecium sélectionné, l’Ern0160, a permis de mettre en évidence la toute première cible directe d’un sARN de E. faecium. En effet, l’Ern0160 se lie spécifiquement à un ARN messager dont la protéine correspondante serait impliquée dans la virulence d’après un modèle expérimental murin. L’Ern0160 régule négativement l’expression de sa cible in vitro. L’impact de l’Ern0160 sur l’atténuation de la virulence de E. faecium reste à évaluer in vivo afin d’avoir une vision globale de son rôle dans la pathogénicité de ce dernier.Enterococcus faecium and Staphylococcus aureus are opportunistic pathogens in human and responsible for hospital outbreaks. Antiseptics are widely used in the hospital to prevent these infections. Even at low concentrations, called subinhibitory concentrations, antiseptics likely provide a stress that bacteria must cope with. Regulatory RNAs (sRNAs) are key players in adaptive networks of bacteria to environmental changes. They have been well-studied in S. aureus by contrast with E. faecium, leading to uneven knowledges. We first aimed to characterize the sRNAs whose expression was altered under antiseptic stress of chlorhexidine, benzalkonium chloride, PVP iodine and triclosan. We showed that it led to a global repression of the studied sRNAs in E. faecium but not in S. aureus. Then, we focused on a selected sRNA of E. faecium, Ern0160 and highlighted the very first direct target of a sRNA of E. faecium. Indeed, Ern0160 specifically binds to a messenger RNA that encode a protein that has been previously described as involved in virulence in mouse model. Ern0160 downregulates the expression of its target in vitro. The impact of Ern0160 on the attenuation of virulence of E. faecium remains to be investigated in vivo to further characterize its role in E. faecium pathogenicity

Rôles d'ARN régulateurs exprimés par Enterococcus faecium et Staphylococcus aureus en réponse au stress antiseptique

Enterococcus faecium and Staphylococcus aureus are opportunistic pathogens in human and responsible for hospital outbreaks. Antiseptics are widely used in the hospital to prevent these infections. Even at low concentrations, called subinhibitory concentrations, antiseptics likely provide a stress that bacteria must cope with. Regulatory RNAs (sRNAs) are key players in adaptive networks of bacteria to environmental changes. They have been well-studied in S. aureus by contrast with E. faecium, leading to uneven knowledges. We first aimed to characterize the sRNAs whose expression was altered under antiseptic stress of chlorhexidine, benzalkonium chloride, PVP iodine and triclosan. We showed that it led to a global repression of the studied sRNAs in E. faecium but not in S. aureus. Then, we focused on a selected sRNA of E. faecium, Ern0160 and highlighted the very first direct target of a sRNA of E. faecium. Indeed, Ern0160 specifically binds to a messenger RNA that encode a protein that has been previously described as involved in virulence in mouse model. Ern0160 downregulates the expression of its target in vitro. The impact of Ern0160 on the attenuation of virulence of E. faecium remains to be investigated in vivo to further characterize its role in E. faecium pathogenicity.Enterococcus faecium et Staphylococcus aureus sont des bactéries pathogènes opportunistes de l’homme responsables d’épidémies hospitalières. Les antiseptiques sont très utilisés pour prévenir la survenue de ces infections et leur présence dans l’environnement hospitalier, même à faibles concentrations, dites subinhibitrices, constitue un stress auquel ces bactéries doivent d’adapter. Les ARN régulateurs (sARN) sont des acteurs importants pour l’adaptation aux variations environnementales des bactéries. Les preuves de leur existence sont anciennes chez S. aureus mais récentes chez E. faecium, entrainant un état des connaissances très hétérogène. Dans un premier temps, nous avons souhaité caractériser les sARN dont l’expression était altérée en cas de stress antiseptique exercé par de la chlorhexidine, du chlorure de benzalkonium, de la povidone iodée et du triclosan. Nous avons montré que ce stress entrainait une répression globale des sARN étudiés chez E. faecium mais pas chez S. aureus. Dans un second temps, l’étude approfondie d’un sARN d’E. faecium sélectionné, l’Ern0160, a permis de mettre en évidence la toute première cible directe d’un sARN de E. faecium. En effet, l’Ern0160 se lie spécifiquement à un ARN messager dont la protéine correspondante serait impliquée dans la virulence d’après un modèle expérimental murin. L’Ern0160 régule négativement l’expression de sa cible in vitro. L’impact de l’Ern0160 sur l’atténuation de la virulence de E. faecium reste à évaluer in vivo afin d’avoir une vision globale de son rôle dans la pathogénicité de ce dernier

Distinct expression profiles of regulatory RNAs in the response to biocides in Staphylococcus aureus and Enterococcus faecium

International audienceThe aim of the study was to characterize the antimicrobial activity of clinically-relevant biocides (chlorhexidine digluconate, benzalkonium chloride, PVP-iodine and triclosan) and to determine the sRNA expression profiles under biocide exposure in two major bacterial opportunistic pathogens, Enterococcus faecium and Staphylococcus aureus. In vitro activities were evaluated against S. aureus HG003 and E. faecium Aus0004. We determined MIC, MBC, sub-inhibitory concentrations (SIC) and growth curves under SIC conditions. sRNA expression study under SIC exposure of biocides was performed by RT-qPCR on 3 sRNAs expressed in S. aureus (RNAIII, SprD and SprX) and the first 9 sRNAs identified as expressed in E. faecium. MICs were higher against E. faecium than for S. aureus. Growth curves under increasing biocide concentrations highlighted two types of bactericidal activity: "on/off" effect for chlorhexidine, benzalkonium chloride, PVP-iodine and a "concentration-dependent" activity for triclosan. Exposure to biocide SICs led to an alteration of several sRNA expression profiles, mostly repressed. The distinct biocide activity profiles must be evaluated with other compounds and bacterial species to enrich the prediction of resistance risks associated with biocide usage. Biocide exposure induces various sRNA-mediated responses in both S. aureus and E. faecium, and further investigations are needed to decipher sRNA-driven regulatory networks

Small RNA-mediated regulation of the tet(M) resistance gene expression in Enterococcus faecium

International audienceWe investigated the role of a novel small RNA expressed in Enterococcus faecium (named Ern0030). We revealed that ern0030 was encoded within the 5’untranslated region of tet(M), a gene conferring tetracycline resistance through ribosomal protection. By RACE mapping, we accurately determined the boundaries of ern0030, which corresponded to Ptet. This upstream sequence of tet(M), Ptet, was previously described within transcriptional attenuation mechanism. Here, Northern blot analyses revealed three transcripts of different lengths (ca. 230, 150 and 100 nucleotides) expressed from Ptet. Phenotypically, the total deletion of ern0030 conferred a decrease in tetracycline MICs that was consistent with gene expression data showing no significant tet(M) induction under tetracycline SIC in ern0030-deleted mutant as opposed to a 10-fold increase of tet(M) expression in the wild-type strain. We investigated the transcriptional attenuation mechanism by toeprint assay. Whereas the expected tet(M) RBS was detected, the RBS of the putative leader peptide was not highlighted by toeprint assay, suggesting the transcriptional attenuation was unlikely. Here, we demonstrate that Ern0030 has a role in regulation of tet(M) expression and propose a novel model of tet(M) regulation alternative or complementary to transcriptional attenuation

The Role of Next-Generation Sequencing (NGS) in the Management of Tuberculosis: Practical Review for Implementation in Routine

International audienceNext-generation sequencing (NGS) has modernized the field of tuberculosis (TB) research by enabling high-throughput sequencing of the entire genome of Mycobacterium tuberculosis (MTB), which is the causative agent of TB. NGS has provided insights into the genetic diversity of MTB, which are crucial for understanding the evolution and transmission of the disease, and it has facilitated the identification of drug-resistant strains, enabling rapid and accurate tailoring of treatment. However, the high cost and the technical complexities of NGS currently limit its widespread use in clinical settings. International recommendations are thus necessary to facilitate the interpretation of polymorphisms, and an experimental approach is still necessary to correlate them to phenotypic data. This review aims to present a comparative, step-by-step, and up-to-date review of the techniques available for the implementation of this approach in routine laboratory workflow. Ongoing research on NGS for TB holds promise for improving our understanding of the disease and for developing more efficacious treatments

In vitro activity of eravacycline and mechanisms of resistance in enterococci

International audienceEravacycline (ERC), the first fluorocycline, is a new tetracycline with superior activity to tigecycline (TGC) against many bacterial species. This work aimed to determine the in vitro activity of ERC compared with other tetracyclines against enterococcal clinical isolates and to analyse corresponding resistance mechanisms. A collection of 60 enterococcal strains was studied: 54 epidemiologically unrelated clinical isolates (46 Enterococcus faecium and 8 Enterococcus faecalis) including 42 vancomycin-resistant enterococci (VRE) (33 vanA and 9 vanB), 3 in vitro TGC-resistant mutants (E. faecium AusTig, HMtig1 and HMtig2) and 3 reference wild-type strains (E. faecium Aus0004 and HM1070, E. faecalis ATCC 29212). In vitro susceptibility was determined using Etest strips (for ERC) or by broth microdilution (for TGC, doxycycline, minocycline and tetracycline). Resistance genes [tet(M), tet(L), tet(O) and tet(S)] were screened by PCR for TGC- and/or ERC-resistant strains as well as sequencing of the rpsJ gene (encoding ribosomal protein S10). MIC values were 0.016/0.08, ≤0.03/0.5, 4/32, 8/16 and 32/>32 mg/L for ERC, TGC, doxycycline, minocycline and tetracycline, respectively. According to EUCAST guidelines, nine strains were categorised as resistant to TGC (MIC, 0.5-8 mg/L), including four E. faecium vanA(+) strains also resistant to ERC (MIC, 0.19-1.5 mg/L). These four strains all possessed at least one mutation in rpsJ and two tet determinants: tet(M) + tet(L) (n = 2); and tet(M) + tet(S) (n = 2). Although ERC has excellent in vitro activity against enterococci (including VRE), emergence of resistance is possible due to combined mechanisms (rpsJ mutations + tet genes)

Ventriculitis: A Severe Complication of Central Nervous System Infections

International audienceBACKGROUND: Ventriculitis, a dreaded complication of brain abscess, meningitis, and various neurosurgical procedures, has attracted limited attention in the medical literature. METHODS: This is a retrospective, single-center study. We screened the medical files of all patients who had a brain imaging report that included the word "ventriculitis" during years 2005-2019. Only patients with clinical, microbiological, and imaging features of ventriculitis were included. Data were collected through a standardized questionnaire. RESULTS: Ninety-eight patients fulfilled inclusion criteria: 42 women and 56 men, and the median age was 60 years (interquartile range, 48-68). The primary mechanism for ventriculitis was classified as follows: brain abscess (n = 29, 29.6%), meningitis (n = 27, 27.6%), intraventricular catheter-related (n = 17, 17.3%), post-neurosurgery (n = 13, 13.3%), and hematogenous (n = 12, 12.2%). The main neuroimaging features were intraventricular pus (n = 81, 82.7%), ependymal enhancement (n = 70, 71.4%), and intraventricular loculations (n = 15, 15.3%). The main pathogens were streptococci (n = 44, 44.9%), Gram-negative bacilli (n = 27, 27.6%), and staphylococci (n = 15, 15.3%). In-hospital and 1-year mortality rates were 30.6% (n = 30) and 38.8% (n = 38), respectively. Neurological sequelae were reported in 34 of 55 (61.8%) survivors, including cognitive impairment (n = 11), gait disturbances (n = 9), paresis (n = 7), behavior disorder (n = 6), and epilepsy (n = 5). On multivariate analysis, age >65 years, Glasgow Coma Scale score <13 at initial presentation, status epilepticus, hydrocephalus, and positive cerebrospinal fluid culture were associated with 1-year mortality. We built a scoring system to stratify patients with ventriculitis into low risk (12.5%), intermediate risk (36.5%), and high risk (71.4%) of death. CONCLUSIONS: Ventriculitis is a severe complication of brain abscess, meningitis, or neurosurgery, with an in-hospital mortality rate of 30% and neurological sequelae in 60% of survivors

The Regulatory RNA ern0160 Confers a Potential Selective Advantage to Enterococcus faecium for Intestinal Colonization

International audienceThe aim of this study was to evaluate the role of the regulatory small RNA (sRNA) Ern0160 in gastrointestinal tract (GIT) colonization by Enterococcus faecium. For this purpose, four strains of E. faecium were used, Aus0004 (WT), an ern0160-deleted Aus0004 mutant (Delta 0160), a trans-complemented Delta 0160 strain overexpressing ern0160 (Delta 0160_0160), and a strain Delta 0160 with an empty pAT29 vector (Delta 0160_pAT29). Strains were studied both in vitro and in vivo, alone and in competitive assays. In in vitro experiments, no difference was observed between WT and Delta 0160 strains cultured single while Delta 0160_0160 strain grew more slowly than Delta 0160_pAT29. In competitive assays, the WT strain was predominant compared to the deleted strain Delta 0160 at the end of the experiment. Then, in vivo experiments were performed using a GIT colonization mouse model. Several existing models of GIT colonization were compared while a novel one, combining ceftriaxone and amoxicillin, was developed. A GIT colonization was performed with each strain alone, and no significant difference was noticed. By contrast, significant results were obtained with co-colonization experiments. With WT + Delta 0160 suspension, a significant advantage for the WT strain was observed from day 5 to the end of the protocol, suggesting the involvement of ern0160 in GIT colonization. With Delta 0160_0160 + Delta 0160_pAT29 suspension, the strain with the empty vector took the advantage from day 3 to the end of the protocol, suggesting a deleterious effect of ern0160 overexpression. Altogether, these findings demonstrate the potential implication of Ern0160 in GIT colonization of E. faecium. Further investigations are needed for the identification of sRNA target(s) in order to decipher underlying molecular mechanisms

Genetic features of the poxtA linezolid resistance gene in human enterococci from France

International audienceOBJECTIVES: To describe the prevalence of poxtA among clinical linezolid-resistant enterococci (LRE) collected in France from 2016 to 2020 and to extensively characterize its genetic supports and environments. METHODS: All LRE clinical isolates received at the National Reference Centre for Enterococci from French hospitals between 2016 and 2020 were included. LRE isolates were screened for linezolid resistance genes (cfr-like, optrA and poxtA) by real-time PCR and phenotypically characterized. A collection of 11 representative poxtA-positive isolates (10 Enterococcus faecium and 1 Enterococcus faecalis) underwent WGS by hybrid assembly combining short-read (Illumina MiSeq) and long-read (MinION) approaches. Transferability of poxtA was attempted by filter-mating experiments. RESULTS: Out of 466 LRE received at the National Reference Centre for Enterococci over the period, 47 (10.1%) were poxtA-positive, including 42 E. faecium. The 11 isolates characterized by WGS were confirmed to be epidemiologically unrelated by core genome analysis and eight different STs were assigned to E. faecium isolates. The poxtA gene was found to be plasmid carried and flanked by IS1216E transposase genes in all isolates and frequently linked with fexB, tet(M) and tet(L). A total of seven distinct poxtA-harbouring plasmids were obtained after hybrid assembly and plasmid transfer of poxtA was successful in three cases. For the two poxtA/optrA-positive isolates, those genes were carried by different plasmids. CONCLUSIONS: The poxtA gene has been circulating among clinical enterococci in France since at least 2016, mostly in E. faecium and independently from optrA. The poxtA-carrying plasmids often co-carried resistance genes to phenicols and tetracyclines, and could have been co-selected through their veterinary use

Molecular and functional analysis of the novel cfr(D) linezolid resistance gene identified in Enterococcus faecium

International audienceObjectives - To characterize the novel cfr(D) gene identified in an Enterococcus faecium clinical isolate (15-307.1) collected from France. Methods - The genome of 15-307.1 was entirely sequenced using a hybrid approach combining short-read (MiSeq, Illumina) and long-read (GridION, Oxford Nanopore Technologies) technologies in order to analyse in detail the genetic support and environment of cfr(D). Transfer of linezolid resistance from 15-307.1 to E. faecium BM4107 was attempted by filter-mating experiments. The recombinant plasmid pAT29Ωcfr(D), containing cfr(D) and its own promoter, was transferred to E. faecium HM1070, Enterococcus faecalis JH2-2 and Escherichia coli AG100A. Results - As previously reported, 15-307.1 belonged to ST17 and was phenotypically resistant to linezolid (MIC, 16 mg/L), vancomycin and teicoplanin. A hybrid sequencing approach confirmed the presence of several resistance genes including vanA, optrA and cfr(D). Located on a 103 kb plasmid, cfr(D) encoded a 357 amino acid protein, which shared 64%, 64%, 48% and 51% amino acid identity with Cfr, Cfr(B), Cfr(C) and Cfr(E), respectively. Both optrA and cfr(D) were successfully co-transferred to E. faecium BM4107. When expressed in E. faecium HM1070 and E. faecalis JH2-2, pAT29Ωcfr(D) did not confer any resistance, whereas it was responsible for an expected PhLOPSA resistance phenotype in E. coli AG100A. Analysis of the genetic environment of cfr(D) showed multiple IS1216 elements, putatively involved in its mobilization. Conclusions - Cfr(D) is a novel member of the family of 23S rRNA methyltransferases. While only conferring a PhLOPSA resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D)