Francisella et antibio-resistance : aspects génétiques, phénotypiques et cliniques

Francisella tularensis is a gram-negative facultative intracellular bacterium, causing tularemia. This zoonosis is mainly related to two subspecies: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) in North America and throughout the Northern Hemisphere, respectively. Infections with this second subspecies, less virulent than the first one, predominantly induce glandular clinical forms of mild to moderate severity. Their treatment is based on antibiotherapy using a fluoroquinolone or a tetracycline. The use of aminoglycosides is reserved for severe clinical forms. The lymph nodes infection, however, often become chronic (20 to 40% of cases), despite administration of an appropriate antibiotic treatment.The aim of this study was to verify the hypothesis of the emergence of bacterial resistance in Francisella, which could explain treatment failures. It is based on the development and study of an in vitro evolutionary experiment of the bacterium in the presence of ciprofloxacin, a fluoroquinolone. Our work confirmed the bacterium's ability to evolve towards a high-level of resistance to fluoroquinolones, this evolution being correlated with the accumulation of mutations in the genes encoding for type II topoisomerases. In addition, we observed in all strains of F. tularensis subsp. holarctica resistant to fluoroquinolones at a clinically significant level, the presence of mutations altering the GyrA subunit of DNA gyrase at amino acids positions 83 and 87. The research of this marker in clinical samples from patients with treatment failure following appropriate antibiotic treatment was however unsuccessful.After checking the action of antibiotics on bacteria internalized in the intracellular compartment in fibroblast cells, we looked for other mutations induced during the evolution of Francisella to resistance to fluoroquinolones. This study unveiled the involvement of several transmembrane transport systems in antibiotic resistance. We also revealed the existence of a second major target involved in Francisella iron metabolism. The alteration of this target (FupA/B), in addition to being associated with an increase in fluoroquinolone resistance, is correlated with a sharp decrease in the ability of the bacteria to multiply in phagocytic cells.Francisella tularensis est une bactérie à Gram négatif intracellulaire facultative, agent causal de la tularémie. Cette zoonose est induite principalement par deux sous espèces : F. tularensis subsp. tularensis (type A) et F. tularensis subsp. holarctica (type B) retrouvées respectivement en Amérique du Nord et dans tout l’hémisphère Nord. Cette seconde sous espèce, moins virulente que la première induit majoritairement des formes cliniques de sévérité moyenne à modérée dites ganglionnaires. Leur traitement est basé sur l’utilisation des antibiotiques de la classe des fluoroquinolones ou des tetracyclines, l’utilisation des aminosides étant réservée aux formes graves. Les adénopathies évoluent cependant souvent vers la suppuration et la chronicité (20 à 40% des cas), malgré l’administration d’un traitement antibiotique adapté.Les travaux réalisés visent à étudier l’hypothèse de l’émergence de la résistance bactérienne chez Francisella, expliquant ces échecs thérapeutiques. Ils sont basés sur le développement et l’étude d’un modèle d’évolution in vitro de la bactérie en présence de ciprofloxacine, une fluoroquinolone. Nos travaux ont confirmé la capacité de la bactérie à évoluer vers un haut niveau de résistance à ces antibiotiques, corrélée à l’accumulation de mutations dans les gènes codant pour les topoïsomérases de type II. De plus, nous avons observé la présence sur l’ensemble des souches de F. tularensis subsp. holarctica d’un niveau de résistance cliniquement significatif induit par des mutations modifiant la sous-unité GyrA de l’ADN gyrase sur les acides aminés en position 83 et 87. La recherche de ce marqueur dans des prélèvements de patient en échec thérapeutique suite à divers traitements antibiotiques s’est avérée infructueuse.Après avoir vérifié l’action de l’antibiotique sur les bactéries dans le compartiment intracellulaire (fibroblates), nous avons recherché les autres mutations induites lors de l’évolution de Francisella en présence de fluoroquinolones. Cette étude a permis l’implication de plusieurs systèmes de transports transmembranaires dans la résistance antibiotique. Nous avons également révélé l’existence d’une seconde cible majeure impliquée dans le métabolisme du fer de la bactérie. L’altération de cette cible (FupA/B) en plus d’être associée à une augmentation de la résistance aux fluoroquinolones est corrélée à une forte diminution de la capacité de la bactérie à se multiplier dans les cellules phagocytaires

Francisella et antibio-resistance : aspects génétiques, phénotypiques et cliniques

Francisella tularensis is a gram-negative facultative intracellular bacterium, causing tularemia. This zoonosis is mainly related to two subspecies: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) in North America and throughout the Northern Hemisphere, respectively. Infections with this second subspecies, less virulent than the first one, predominantly induce glandular clinical forms of mild to moderate severity. Their treatment is based on antibiotherapy using a fluoroquinolone or a tetracycline. The use of aminoglycosides is reserved for severe clinical forms. The lymph nodes infection, however, often become chronic (20 to 40% of cases), despite administration of an appropriate antibiotic treatment.The aim of this study was to verify the hypothesis of the emergence of bacterial resistance in Francisella, which could explain treatment failures. It is based on the development and study of an in vitro evolutionary experiment of the bacterium in the presence of ciprofloxacin, a fluoroquinolone. Our work confirmed the bacterium's ability to evolve towards a high-level of resistance to fluoroquinolones, this evolution being correlated with the accumulation of mutations in the genes encoding for type II topoisomerases. In addition, we observed in all strains of F. tularensis subsp. holarctica resistant to fluoroquinolones at a clinically significant level, the presence of mutations altering the GyrA subunit of DNA gyrase at amino acids positions 83 and 87. The research of this marker in clinical samples from patients with treatment failure following appropriate antibiotic treatment was however unsuccessful.After checking the action of antibiotics on bacteria internalized in the intracellular compartment in fibroblast cells, we looked for other mutations induced during the evolution of Francisella to resistance to fluoroquinolones. This study unveiled the involvement of several transmembrane transport systems in antibiotic resistance. We also revealed the existence of a second major target involved in Francisella iron metabolism. The alteration of this target (FupA/B), in addition to being associated with an increase in fluoroquinolone resistance, is correlated with a sharp decrease in the ability of the bacteria to multiply in phagocytic cells.Francisella tularensis est une bactérie à Gram négatif intracellulaire facultative, agent causal de la tularémie. Cette zoonose est induite principalement par deux sous espèces : F. tularensis subsp. tularensis (type A) et F. tularensis subsp. holarctica (type B) retrouvées respectivement en Amérique du Nord et dans tout l’hémisphère Nord. Cette seconde sous espèce, moins virulente que la première induit majoritairement des formes cliniques de sévérité moyenne à modérée dites ganglionnaires. Leur traitement est basé sur l’utilisation des antibiotiques de la classe des fluoroquinolones ou des tetracyclines, l’utilisation des aminosides étant réservée aux formes graves. Les adénopathies évoluent cependant souvent vers la suppuration et la chronicité (20 à 40% des cas), malgré l’administration d’un traitement antibiotique adapté.Les travaux réalisés visent à étudier l’hypothèse de l’émergence de la résistance bactérienne chez Francisella, expliquant ces échecs thérapeutiques. Ils sont basés sur le développement et l’étude d’un modèle d’évolution in vitro de la bactérie en présence de ciprofloxacine, une fluoroquinolone. Nos travaux ont confirmé la capacité de la bactérie à évoluer vers un haut niveau de résistance à ces antibiotiques, corrélée à l’accumulation de mutations dans les gènes codant pour les topoïsomérases de type II. De plus, nous avons observé la présence sur l’ensemble des souches de F. tularensis subsp. holarctica d’un niveau de résistance cliniquement significatif induit par des mutations modifiant la sous-unité GyrA de l’ADN gyrase sur les acides aminés en position 83 et 87. La recherche de ce marqueur dans des prélèvements de patient en échec thérapeutique suite à divers traitements antibiotiques s’est avérée infructueuse.Après avoir vérifié l’action de l’antibiotique sur les bactéries dans le compartiment intracellulaire (fibroblates), nous avons recherché les autres mutations induites lors de l’évolution de Francisella en présence de fluoroquinolones. Cette étude a permis l’implication de plusieurs systèmes de transports transmembranaires dans la résistance antibiotique. Nous avons également révélé l’existence d’une seconde cible majeure impliquée dans le métabolisme du fer de la bactérie. L’altération de cette cible (FupA/B) en plus d’être associée à une augmentation de la résistance aux fluoroquinolones est corrélée à une forte diminution de la capacité de la bactérie à se multiplier dans les cellules phagocytaires

Francisella and antibiotic resistance : genetic, phenotypic and clinical aspects

Francisella tularensis est une bactérie à Gram négatif intracellulaire facultative, agent causal de la tularémie. Cette zoonose est induite principalement par deux sous espèces : F. tularensis subsp. tularensis (type A) et F. tularensis subsp. holarctica (type B) retrouvées respectivement en Amérique du Nord et dans tout l’hémisphère Nord. Cette seconde sous espèce, moins virulente que la première induit majoritairement des formes cliniques de sévérité moyenne à modérée dites ganglionnaires. Leur traitement est basé sur l’utilisation des antibiotiques de la classe des fluoroquinolones ou des tetracyclines, l’utilisation des aminosides étant réservée aux formes graves. Les adénopathies évoluent cependant souvent vers la suppuration et la chronicité (20 à 40% des cas), malgré l’administration d’un traitement antibiotique adapté.Les travaux réalisés visent à étudier l’hypothèse de l’émergence de la résistance bactérienne chez Francisella, expliquant ces échecs thérapeutiques. Ils sont basés sur le développement et l’étude d’un modèle d’évolution in vitro de la bactérie en présence de ciprofloxacine, une fluoroquinolone. Nos travaux ont confirmé la capacité de la bactérie à évoluer vers un haut niveau de résistance à ces antibiotiques, corrélée à l’accumulation de mutations dans les gènes codant pour les topoïsomérases de type II. De plus, nous avons observé la présence sur l’ensemble des souches de F. tularensis subsp. holarctica d’un niveau de résistance cliniquement significatif induit par des mutations modifiant la sous-unité GyrA de l’ADN gyrase sur les acides aminés en position 83 et 87. La recherche de ce marqueur dans des prélèvements de patient en échec thérapeutique suite à divers traitements antibiotiques s’est avérée infructueuse.Après avoir vérifié l’action de l’antibiotique sur les bactéries dans le compartiment intracellulaire (fibroblates), nous avons recherché les autres mutations induites lors de l’évolution de Francisella en présence de fluoroquinolones. Cette étude a permis l’implication de plusieurs systèmes de transports transmembranaires dans la résistance antibiotique. Nous avons également révélé l’existence d’une seconde cible majeure impliquée dans le métabolisme du fer de la bactérie. L’altération de cette cible (FupA/B) en plus d’être associée à une augmentation de la résistance aux fluoroquinolones est corrélée à une forte diminution de la capacité de la bactérie à se multiplier dans les cellules phagocytaires.Francisella tularensis is a gram-negative facultative intracellular bacterium, causing tularemia. This zoonosis is mainly related to two subspecies: F. tularensis subsp. tularensis (type A) and F. tularensis subsp. holarctica (type B) in North America and throughout the Northern Hemisphere, respectively. Infections with this second subspecies, less virulent than the first one, predominantly induce glandular clinical forms of mild to moderate severity. Their treatment is based on antibiotherapy using a fluoroquinolone or a tetracycline. The use of aminoglycosides is reserved for severe clinical forms. The lymph nodes infection, however, often become chronic (20 to 40% of cases), despite administration of an appropriate antibiotic treatment.The aim of this study was to verify the hypothesis of the emergence of bacterial resistance in Francisella, which could explain treatment failures. It is based on the development and study of an in vitro evolutionary experiment of the bacterium in the presence of ciprofloxacin, a fluoroquinolone. Our work confirmed the bacterium's ability to evolve towards a high-level of resistance to fluoroquinolones, this evolution being correlated with the accumulation of mutations in the genes encoding for type II topoisomerases. In addition, we observed in all strains of F. tularensis subsp. holarctica resistant to fluoroquinolones at a clinically significant level, the presence of mutations altering the GyrA subunit of DNA gyrase at amino acids positions 83 and 87. The research of this marker in clinical samples from patients with treatment failure following appropriate antibiotic treatment was however unsuccessful.After checking the action of antibiotics on bacteria internalized in the intracellular compartment in fibroblast cells, we looked for other mutations induced during the evolution of Francisella to resistance to fluoroquinolones. This study unveiled the involvement of several transmembrane transport systems in antibiotic resistance. We also revealed the existence of a second major target involved in Francisella iron metabolism. The alteration of this target (FupA/B), in addition to being associated with an increase in fluoroquinolone resistance, is correlated with a sharp decrease in the ability of the bacteria to multiply in phagocytic cells

A new dye uptake assay to test the activity of antibiotics against intracellular Francisella tularensis.

International audienceFrancisella tularensis, a facultative intracellular bacterium, is the aetiological agent of tularaemia. Antibiotic treatment of this zoonosis is based on the administration of a fluoroquinolone or a tetracycline for cases with mild to moderate severity, whereas an aminoglycoside (streptomycin or gentamicin) is advocated for severe cases. However, treatment failures and relapses remain frequent, especially in patients suffering from chronic lymph node suppuration. Therefore, new treatment alternatives are needed. We have developed a dye uptake assay for determination of minimal inhibitory extracellular concentrations (MIECs) of antibiotics against intracellular F. tularensis, and validated the method by comparing the results obtained using a CFU-enumerating method. We also compared MIECs with MICs of the same compounds determined using a CLSI broth microdilution method. We tested the activity of 11 antibiotics against two clinical strains of F. tularensis subsp. holarctica isolated in France. Both strains displayed low MICs (≤1 μg/mL) to fluoroquinolones (ciprofloxacin, levofloxacin and moxifloxacin), gentamicin, doxycycline and rifampicin. Higher MICs (≥8 μg/mL) were found for carbapenems (imipenem and meropenem), daptomycin and linezolid. Erythromycin MICs were 4.0 and 16.0 μg/mL, respectively, for the two clinical strains. MIECs were almost the same with the two methods used. They were concordant with MICs, except for erythromycin and linezolid (respectively, four and eight times more active against intracellular F. tularensis) and gentamicin (four to eight times less active against intracellular F. tularensis). This study validated the dye uptake assay as a new tool for determination of the activity of a large panel of antibiotics against intracellular F. tularensis. This test confirmed the intracellular activity of first-line antibiotics used for tularaemia treatment, but also revealed significant activity of linezolid against intracellular F. tularensis

Bis-indolic compounds as potential new therapeutic alternatives for tularaemia.

International audienceFrancisella tularensis is the etiological agent of tularaemia and a CDC class A biological threat agent. Few antibiotic classes are currently useful in treating tularaemia, including the aminoglycosides gentamicin and streptomycin, fluoroquinolones, and tetracyclines. However, treatment failures and relapses remain frequent and F. tularensis strains resistant to antibiotics have been easily selected in vitro. In this study, we evaluated the activity of new synthetic bis-indole derivatives against this pathogen. Minimum inhibitory concentrations (MICs) of four compounds (dcm01 to dcm04) were determined for the reference strains F. tularensis subsp. holarctica LVS NCTC10857, F. tularensis subsp. novicida CIP56.12 and F. philomiragia ATCC25015, and for 41 clinical strains of F. tularensis subsp. holarctica isolated in France. Minimal bactericidal concentrations (MBCs) were determined for the dcm02 and dcm04 compounds for the LVS and two clinical strains. Killing curves were also determined for the same three strains exposed to dcm04. All tested bis-indole compounds were bacteriostatic against F. tularensis subsp. holarctica strains, with a MIC90 of 8 μg/mL for dcm01, dcm02, and dcm03, and 2 μg/mL for dcm04. Only one strain was resistant to both dcm01 and dcm03, with MICs > 32 μg/mL. In contrast, F. tularensis subsp. novicida was resistant to all derivatives and F. philomiragia was only susceptible to dcm02 and dcm04, with MICs of 16 and 4 μg/mL, respectively. MBC and killing curve experiments revealed significant bactericidal activity (i.e., 3-log reduction of the bacterial inoculum) of the dcm02 and dcm04 compounds only for the LVS strain. In conclusion, we have identified novel synthetic bis-indole compounds that are active against F. tularensis subsp. holarctica. They may be drug candidates for the development of new therapeutic alternatives for tularaemia treatment. Their further characterization is needed, especially identification of their bacterial targets

Evolution toward high-level fluoroquinolone resistance in Francisella species.

International audienceOBJECTIVES: Francisella tularensis, a CDC class A potential bioterrorism agent, is a Gram-negative bacterium responsible for tularaemia. Understanding the mechanisms of resistance to antibiotics used as first-line treatment is of major security relevance. METHODS: We propagated the three parental reference strains Francisella tularensis subsp. holarctica live vaccine strain, Francisella novicida and Francisella philomiragia with increasing concentrations of ciprofloxacin, a fluoroquinolone used as curative and prophylactic treatment for tularaemia. This evolution procedure provided us with high-level ciprofloxacin-resistant mutants and all evolutionary intermediates towards high-level resistance. We determined the resistance levels to other fluoroquinolones (levofloxacin and moxifloxacin) and other antibiotic families (aminoglycosides, tetracyclines and macrolides) and characterized the genetic changes in the fluoroquinolone target genes encoding DNA gyrase and topoisomerase IV. RESULTS: All high-level resistant mutants shared cross-resistance to the tested fluoroquinolones, while some also revealed striking levels of cross-resistance to other clinically relevant antibiotic classes. High-level resistant mutants carried one to three mutations, including some not previously reported. We mapped all mutations onto known topoisomerase three-dimensional structures. Along the pathways towards high-level resistance, we identified complex evolutionary trajectories including polymorphic states and additional resistance mechanisms likely to be associated with efflux processes. CONCLUSIONS: Our data demonstrated the efficiency and speed of in vitro production of mutants highly resistant to fluoroquinolones in Francisella species. They emphasize the urgent need to identify all antibiotic resistance mechanisms in these species, develop molecular tools for their detection and design new therapeutic alternatives for tularaemia

Genomic trajectories to fluoroquinolone resistance in Francisella tularensis subsp. holarctica live vaccine strain

International audienceObjectives: Fluoroquinolone (FQ)-resistant mutants were previously selected from the live vaccine strain (LVS) of Francisella tularensis (F. tularensis) subsp. holarctica. This study further characterised all genetic changes that occurred in these mutants during the evolutionary trajectory toward high-level FQ resistance, and their potential impact on F. tularensis antibiotic resistance and intracellular fitness. Methods: The whole genomes of FQ-resistant mutants were determined and compared with those of their parental strain. All detected mutations were evaluated for their potential impact on FQ resistance and intracellular multiplication of F. tularensis. Results: As compared with the parental LVS genome, 28 mutations were found in the derived FQ-resistant mutants. These mutations involved all genes encoding type II topoisomerases (i.e. gyrA, gyrB, parC , and parE). Interestingly, some of them were not previously associated with FQ resistance, warranting further characterisation. Mutations associated with FQ resistance were also found in other genes, including three encoding proteins involved in transport processes. Most of the detected mutations did not alter multiplication of the corresponding mutants in J774 cells. In contrast, all mutations at locus FTL_0439 encoding FupA/B, a membrane protein involved in iron transport, were associated with FQ resistance and fitness loss. Conclusion: FQ resistance in F. tularensis is complex and may involve single or combined mutations in genes encoding type II topoisomerases, transport systems and FupA/B. In vivo studies are now required to assess the potential role of these mutations in FQ treatment failures

Functional Characterization of the DNA Gyrases in Fluoroquinolone-Resistant Mutants of Francisella novicida

International audienceFluoroquinolone (FQ) resistance is a major health concern in the treatment of tularemia. Because DNA gyrase has been described as the main target of these compounds, our aim was to clarify the contributions of both GyrA and GyrB mutations found in Francisella novicida clones highly resistant to FQs. Wild-type and mutated GyrA and GyrB subunits were overexpressed so that the in vitro FQ sensitivity of functional reconstituted complexes could be evaluated. The data obtained were compared to the MICs of FQs against bacterial clones harboring the same mutations and were further validated through complementation experiments and structural modeling. Whole-genome sequencing of highly FQ-resistant lineages was also done. Supercoiling and DNA cleavage assays demonstrated that GyrA D87 is a hot spot FQ resistance target in F. novicida and pointed out the role of the GyrA P43H substitution in resistance acquisition. An unusual feature of FQ resistance acquisition in F. novicida is that the first-step mutation occurs in GyrB, with direct or indirect consequences for FQ sensitivity. Insertion of P466 into GyrB leads to a 50% inhibitory concentration (IC50) comparable to that observed for a mutant gyrase carrying the GyrA D87Y substitution, while the D487E-ΔK488 mutation, while not active on its own, contributes to the high level of resistance that occurs following acquisition of the GyrA D87G substitution in double GyrA/GyrB mutants. The involvement of other putative targets is discussed, including that of a ParE mutation that was found to arise in the very late stage of antibiotic exposure. This study provides the first characterization of the molecular mechanisms responsible for FQ resistance in Francisella