Rôle des sARN dans la pathogénie d'Enterococcus faecium

Enterococcus faecium is a normal inhabitant of the human gut intestinal tract (GIT) and an opportunistic pathogen multi-resistant to antibiotics and responsible for hospital outbreaks. Since the existence of regulatory RNAs in E. faecium (Ern) has been recently discovered, the aim of this study was to evaluate their involvement in vivo. A murine GIT colonization model was developed, based on several published models thatwere compared, and a new one combining ceftriaxone and amoxicillin was developed. Then, the regulatory RNA Ern0160 was studied. Four E. faecium strains were used, Aus0004 (WT), an ern0160-deleted Aus0004 mutant (Δ0160), a trans-complemented Δ0160 strain overexpressing ern0160 (Δ0160_0160), and a strain Δ0160 with an empty pAT29 vector (Δ0160_pAT29). In in vitro experiments, no difference was observed between WT and Δ0160 strains cultured individually while Δ0160_0160 strain grew more slowly than Δ0160_pAT29. In competitive assays, the WT strain was predominant compared to the deleted strain Δ0160 at the end of the experiment. 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 + Δ0160 suspension, a significant advantage for the WT strain was observed, suggesting the involvement of ern0160 in GIT colonization. With Δ0160_0160 + Δ0160_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. Infection models have also been developed during this work but remain to be finalized.Enterococcus faecium est une bactérie commensale du tube digestif de l’homme, pathogène opportuniste multi-résistant aux antibiotiques et responsable d’épidémies hospitalières. L’existence d’ARN régulateurs chez E. faecium (Ern) ayant récemment été découverte, ce travail de thèse avait pour objectif d’évaluer leur implication in vivo. Un modèle de colonisation a été mis au point chez la souris, à partir de plusieurs modèles décrits dans la littérature. Ils ont été comparés, et un nouveau modèle associant la ceftriaxone et l’amoxicilline a été développé. L’ARN régulateur Ern0160 a ensuite été étudié. Quatre souches d’E. faecium ont été utilisées, Aus0004 (WT), la souche mutante WT délétée d‘ern0160 (Δ0160), la souche Δ0160 trans-complémentée surexprimant ern0160 (Δ0160_0160), et la souche Δ0160 contenant le vecteur pAT29 vide (Δ0160_pAT29). Dans les expériences in vitro, aucune différence n'a été observée entre les souches WT et Δ0160 cultivées seules, tandis que la souche Δ0160_0160 s'est développée plus lentement que Δ0160_pAT29. In vivo, aucune différence significative de colonisation n’a été observée avec les souches inoculées seules. Dans les essais de compétition in vitro et in vivo, la souche WT était prédominante par rapport à la souche délétée Δ0160. Avec la suspension « Δ0160_0160 + Δ0160_pAT29 », la souche Δ0160_pAT29 était prédominante, suggérant un effet délétère de la surexpression d’ern016. Ces résultats suggèrent l'implication d'Ern0160 dans la colonisation intestinale par E. faecium. Des modèles d’infection (endocardite infectieuse et bactériémie secondaire à une translocation intestinale) ont été développés au cours de ce travail mais restent à finaliser

sRNA and Enterococcus faecium pathogenesis

Enterococcus faecium est une bactérie commensale du tube digestif de l’homme, pathogène opportuniste multi-résistant aux antibiotiques et responsable d’épidémies hospitalières. L’existence d’ARN régulateurs chez E. faecium (Ern) ayant récemment été découverte, ce travail de thèse avait pour objectif d’évaluer leur implication in vivo. Un modèle de colonisation a été mis au point chez la souris, à partir de plusieurs modèles décrits dans la littérature. Ils ont été comparés, et un nouveau modèle associant la ceftriaxone et l’amoxicilline a été développé. L’ARN régulateur Ern0160 a ensuite été étudié. Quatre souches d’E. faecium ont été utilisées, Aus0004 (WT), la souche mutante WT délétée d‘ern0160 (Δ0160), la souche Δ0160 trans-complémentée surexprimant ern0160 (Δ0160_0160), et la souche Δ0160 contenant le vecteur pAT29 vide (Δ0160_pAT29). Dans les expériences in vitro, aucune différence n'a été observée entre les souches WT et Δ0160 cultivées seules, tandis que la souche Δ0160_0160 s'est développée plus lentement que Δ0160_pAT29. In vivo, aucune différence significative de colonisation n’a été observée avec les souches inoculées seules. Dans les essais de compétition in vitro et in vivo, la souche WT était prédominante par rapport à la souche délétée Δ0160. Avec la suspension « Δ0160_0160 + Δ0160_pAT29 », la souche Δ0160_pAT29 était prédominante, suggérant un effet délétère de la surexpression d’ern016. Ces résultats suggèrent l'implication d'Ern0160 dans la colonisation intestinale par E. faecium. Des modèles d’infection (endocardite infectieuse et bactériémie secondaire à une translocation intestinale) ont été développés au cours de ce travail mais restent à finaliser.Enterococcus faecium is a normal inhabitant of the human gut intestinal tract (GIT) and an opportunistic pathogen multi-resistant to antibiotics and responsible for hospital outbreaks. Since the existence of regulatory RNAs in E. faecium (Ern) has been recently discovered, the aim of this study was to evaluate their involvement in vivo. A murine GIT colonization model was developed, based on several published models thatwere compared, and a new one combining ceftriaxone and amoxicillin was developed. Then, the regulatory RNA Ern0160 was studied. Four E. faecium strains were used, Aus0004 (WT), an ern0160-deleted Aus0004 mutant (Δ0160), a trans-complemented Δ0160 strain overexpressing ern0160 (Δ0160_0160), and a strain Δ0160 with an empty pAT29 vector (Δ0160_pAT29). In in vitro experiments, no difference was observed between WT and Δ0160 strains cultured individually while Δ0160_0160 strain grew more slowly than Δ0160_pAT29. In competitive assays, the WT strain was predominant compared to the deleted strain Δ0160 at the end of the experiment. 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 + Δ0160 suspension, a significant advantage for the WT strain was observed, suggesting the involvement of ern0160 in GIT colonization. With Δ0160_0160 + Δ0160_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. Infection models have also been developed during this work but remain to be finalized

Rôle des sARN dans la pathogénie d'Enterococcus faecium

Enterococcus faecium is a normal inhabitant of the human gut intestinal tract (GIT) and an opportunistic pathogen multi-resistant to antibiotics and responsible for hospital outbreaks. Since the existence of regulatory RNAs in E. faecium (Ern) has been recently discovered, the aim of this study was to evaluate their involvement in vivo. A murine GIT colonization model was developed, based on several published models thatwere compared, and a new one combining ceftriaxone and amoxicillin was developed. Then, the regulatory RNA Ern0160 was studied. Four E. faecium strains were used, Aus0004 (WT), an ern0160-deleted Aus0004 mutant (Δ0160), a trans-complemented Δ0160 strain overexpressing ern0160 (Δ0160_0160), and a strain Δ0160 with an empty pAT29 vector (Δ0160_pAT29). In in vitro experiments, no difference was observed between WT and Δ0160 strains cultured individually while Δ0160_0160 strain grew more slowly than Δ0160_pAT29. In competitive assays, the WT strain was predominant compared to the deleted strain Δ0160 at the end of the experiment. 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 + Δ0160 suspension, a significant advantage for the WT strain was observed, suggesting the involvement of ern0160 in GIT colonization. With Δ0160_0160 + Δ0160_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. Infection models have also been developed during this work but remain to be finalized.Enterococcus faecium est une bactérie commensale du tube digestif de l’homme, pathogène opportuniste multi-résistant aux antibiotiques et responsable d’épidémies hospitalières. L’existence d’ARN régulateurs chez E. faecium (Ern) ayant récemment été découverte, ce travail de thèse avait pour objectif d’évaluer leur implication in vivo. Un modèle de colonisation a été mis au point chez la souris, à partir de plusieurs modèles décrits dans la littérature. Ils ont été comparés, et un nouveau modèle associant la ceftriaxone et l’amoxicilline a été développé. L’ARN régulateur Ern0160 a ensuite été étudié. Quatre souches d’E. faecium ont été utilisées, Aus0004 (WT), la souche mutante WT délétée d‘ern0160 (Δ0160), la souche Δ0160 trans-complémentée surexprimant ern0160 (Δ0160_0160), et la souche Δ0160 contenant le vecteur pAT29 vide (Δ0160_pAT29). Dans les expériences in vitro, aucune différence n'a été observée entre les souches WT et Δ0160 cultivées seules, tandis que la souche Δ0160_0160 s'est développée plus lentement que Δ0160_pAT29. In vivo, aucune différence significative de colonisation n’a été observée avec les souches inoculées seules. Dans les essais de compétition in vitro et in vivo, la souche WT était prédominante par rapport à la souche délétée Δ0160. Avec la suspension « Δ0160_0160 + Δ0160_pAT29 », la souche Δ0160_pAT29 était prédominante, suggérant un effet délétère de la surexpression d’ern016. Ces résultats suggèrent l'implication d'Ern0160 dans la colonisation intestinale par E. faecium. Des modèles d’infection (endocardite infectieuse et bactériémie secondaire à une translocation intestinale) ont été développés au cours de ce travail mais restent à finaliser

Rôle des sARN dans la pathogénie d'Enterococcus faecium

Enterococcus faecium is a normal inhabitant of the human gut intestinal tract (GIT) and an opportunistic pathogen multi-resistant to antibiotics and responsible for hospital outbreaks. Since the existence of regulatory RNAs in E. faecium (Ern) has been recently discovered, the aim of this study was to evaluate their involvement in vivo. A murine GIT colonization model was developed, based on several published models thatwere compared, and a new one combining ceftriaxone and amoxicillin was developed. Then, the regulatory RNA Ern0160 was studied. Four E. faecium strains were used, Aus0004 (WT), an ern0160-deleted Aus0004 mutant (Δ0160), a trans-complemented Δ0160 strain overexpressing ern0160 (Δ0160_0160), and a strain Δ0160 with an empty pAT29 vector (Δ0160_pAT29). In in vitro experiments, no difference was observed between WT and Δ0160 strains cultured individually while Δ0160_0160 strain grew more slowly than Δ0160_pAT29. In competitive assays, the WT strain was predominant compared to the deleted strain Δ0160 at the end of the experiment. 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 + Δ0160 suspension, a significant advantage for the WT strain was observed, suggesting the involvement of ern0160 in GIT colonization. With Δ0160_0160 + Δ0160_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. Infection models have also been developed during this work but remain to be finalized.Enterococcus faecium est une bactérie commensale du tube digestif de l’homme, pathogène opportuniste multi-résistant aux antibiotiques et responsable d’épidémies hospitalières. L’existence d’ARN régulateurs chez E. faecium (Ern) ayant récemment été découverte, ce travail de thèse avait pour objectif d’évaluer leur implication in vivo. Un modèle de colonisation a été mis au point chez la souris, à partir de plusieurs modèles décrits dans la littérature. Ils ont été comparés, et un nouveau modèle associant la ceftriaxone et l’amoxicilline a été développé. L’ARN régulateur Ern0160 a ensuite été étudié. Quatre souches d’E. faecium ont été utilisées, Aus0004 (WT), la souche mutante WT délétée d‘ern0160 (Δ0160), la souche Δ0160 trans-complémentée surexprimant ern0160 (Δ0160_0160), et la souche Δ0160 contenant le vecteur pAT29 vide (Δ0160_pAT29). Dans les expériences in vitro, aucune différence n'a été observée entre les souches WT et Δ0160 cultivées seules, tandis que la souche Δ0160_0160 s'est développée plus lentement que Δ0160_pAT29. In vivo, aucune différence significative de colonisation n’a été observée avec les souches inoculées seules. Dans les essais de compétition in vitro et in vivo, la souche WT était prédominante par rapport à la souche délétée Δ0160. Avec la suspension « Δ0160_0160 + Δ0160_pAT29 », la souche Δ0160_pAT29 était prédominante, suggérant un effet délétère de la surexpression d’ern016. Ces résultats suggèrent l'implication d'Ern0160 dans la colonisation intestinale par E. faecium. Des modèles d’infection (endocardite infectieuse et bactériémie secondaire à une translocation intestinale) ont été développés au cours de ce travail mais restent à finaliser

Streptogramins for the treatment of infections caused by Gram-positive pathogens.

International audienceINTRODUCTION: Streptogramins (pristinamycin and quinupristin-dalfopristin) can be interesting options for the treatment of infections due to Gram-positive cocci, especially multidrug-resistant isolates. AREAS COVERED: This review provides an updated overview of structural and activity characteristics, mechanisms of action and resistance, pharmacokinetic/pharmacodynamic, and clinical use of streptogramins. EXPERT OPINION: The streptogramin antibiotics act by inhibition of the bacterial protein synthesis. They are composed of two chemically distinct compounds, namely type A and type B streptogramins, which exert a rapid bactericidal activity against a wide range of Gram-positive bacteria (including methicillin-resistant staphylococci and vancomycin-resistant enterococci). Several mechanisms of resistance have been identified in staphylococci and enterococci but the prevalence of streptogramin resistance among clinical isolates remains very low. Even if only a few randomized clinical trials have been conducted, the efficacy of pristinamycin has been largely demonstrated with an extensive use for 50 years in France and some African countries. Despite its effectiveness in the treatment of severe Gram-positive bacterial infections demonstrated in several studies and the low rate of reported resistance, the clinical use of quinupristin-dalfopristin has remained limited, mainly due to its poor tolerance. Altogether, streptogramins (especially pristinamycin) can be considered as potential alternatives for the treatment of Gram-positive infections

Recent Trends in Antimicrobial Resistance among Anaerobic Clinical Isolates

International audienceAnaerobic bacteria are normal inhabitants of the human commensal microbiota and play an important role in various human infections. Tedious and time-consuming, antibiotic susceptibility testing is not routinely performed in all clinical microbiology laboratories, despite the increase in antibiotic resistance among clinically relevant anaerobes since the 1990s. β-lactam and metronidazole are the key molecules in the management of anaerobic infections, to the detriment of clindamycin. β-lactam resistance is usually mediated by the production of β-lactamases. Metronidazole resistance remains uncommon, complex, and not fully elucidated, while metronidazole inactivation appears to be a key mechanism. The use of clindamycin, a broad-spectrum anti-anaerobic agent, is becoming problematic due to the increase in resistance rate in all anaerobic bacteria, mainly mediated by Erm-type rRNA methylases. Second-line anti-anaerobes are fluoroquinolones, tetracyclines, chloramphenicol, and linezolid. This review aims to describe the up-to-date evolution of antibiotic resistance, give an overview, and understand the main mechanisms of resistance in a wide range of anaerobes. © 2023 by the authors

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

Temocillin Resistance in the Enterobacter cloacae Complex Is Conferred by a Single Point Mutation in BaeS, Leading to Overexpression of the AcrD Efflux Pump

International audienceThe Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward beta-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections. Here, we aimed at deciphering the never-investigated mechanisms of temocillin resistance acquisition in Enterobacterales. By comparative genomic analysis of two clonally related ECC clinical isolates, one susceptible (Temo_S [MIC of 4 mg/L]) and the other resistant (Temo_R [MIC of 32 mg/L]), we found that they differed by only 14 single-nucleotide polymorphisms, including one nonsynonymous mutation (Thr175Pro) in the two-component system (TCS) sensor histidine kinase BaeS. By site-directed mutagenesis in Escherichia coli CFT073, we demonstrated that this unique change in BaeS was responsible for a significant (16-fold) increase in temocillin MIC. Since the BaeSR TCS regulates the expression of two resistance-nodulation-cell division (RND)-type efflux pumps (namely, AcrD and MdtABCD) in E. coli and Salmonella, we demonstrated by quantitative reverse transcription-PCR that mdtB, baeS, and acrD genes were significantly overexpressed (15-, 11-, and 3-fold, respectively) in Temo_R. To confirm the role of each efflux pump in this mechanism, multicopy plasmids harboring mdtABCD or acrD were introduced into either Temo_S or the reference strain E. cloacae subsp. cloacae ATCC 13047. Interestingly, only the overexpression of acrD conferred a significant increase (from 8- to 16-fold) of the temocillin MIC. Altogether, we have shown that temocillin resistance in the ECC can result from a single BaeS alteration, likely resulting in the permanent phosphorylation of BaeR and leading to AcrD overexpression and temocillin resistance through enhanced active efflux. The Enterobacter cloacae complex (ECC) has become a major opportunistic pathogen with antimicrobial resistance issues. Temocillin, an "old" carboxypenicillin that is remarkably stable toward beta-lactamases, has been used as an alternative for the treatment of multidrug-resistant ECC infections