François M. Thibault

Involvement of the efflux pumps in chloramphenicol selected strains of Burkholderia thailandensis: proteomic and mechanistic evidence.

Burkholderia is a bacterial genus comprising several pathogenic species, including two species highly pathogenic for humans, B. pseudomallei and B. mallei. B. thailandensis is a weakly pathogenic species closely related to both B. pseudomallei and B. mallei. It is used as a study model. These bacteria are able to exhibit multiple resistance mechanisms towards various families of antibiotics. By sequentially plating B. thailandensis wild type strains on chloramphenicol we obtained several resistant variants. This chloramphenicol-induced resistance was associated with resistance against structurally unrelated antibiotics including quinolones and tetracyclines. We functionally and proteomically demonstrate that this multidrug resistance phenotype, identified in chloramphenicol-resistant variants, is associated with the overexpression of two different efflux pumps. These efflux pumps are able to expel antibiotics from several families, including chloramphenicol, quinolones, tetracyclines, trimethoprim and some β-lactams, and present a partial susceptibility to efflux pump inhibitors. It is thus possible that Burkholderia species can develop such adaptive resistance mechanisms in response to antibiotic pressure resulting in emergence of multidrug resistant strains. Antibiotics known to easily induce overexpression of these efflux pumps should be used with discernment in the treatment of Burkholderia infections.

Phenotypic and genetic characterization of macrolide resistance in Francisella tularensis subsp. holarctica biovar I

OBJECTIVES Francisella tularensis subsp. holarctica strains are classified as biovars I and II, which are susceptible and naturally resistant to the macrolide erythromycin, respectively. The present study was aimed at both selecting biovar I strains with increased levels of erythromycin resistance and characterizing the underlying genetic mechanisms. METHODS Serial cultures in the presence of increasingly high erythromycin concentrations were performed to select independent high- and intermediate-level erythromycin-resistant mutants from each of three different biovar I strains. The mutants were characterized for cross-resistance to several antibiotics, presence of mutations in the genes encoding the 23S rRNA and the L4 and L22 ribosomal proteins, and overexpression of efflux pumps. RESULTS Mutants displayed cross-resistance to all macrolide compounds tested but not to other classes of antibiotics. We found mutations in domain V of the 23S rRNA gene (G2057A, A2058G, A2058T and C2611T) and in the gene encoding L22, leading to either the G91D substitution or the M82K83R84 deletion. Analysis of mutants with intermediate resistance levels obtained over the course of the selection process revealed both a positive correlation between the number of mutated ribosomal operons and the resistance level, and an additional resistance mechanism in the early steps of selection. CONCLUSIONS We showed that high-level resistance to macrolides can be easily obtained in vitro in F. tularensis subsp. holarctica biovar I strains, thereby suggesting that in vivo selection for resistance may explain reported failures of antibiotic treatment. Ketolides were the most effective macrolides tested, which may limit the risk of selection for resistance.

Susceptibility of 71 French isolates of Francisella tularensis subsp. holarctica to eight antibiotics and accuracy of the Etest® method

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Interplay between three RND efflux pumps in doxycycline-selected strains of Burkholderia thailandensis.

Background Efflux systems are involved in multidrug resistance in most Gram-negative non-fermentative bacteria. We have chosen Burkholderia thailandensis to dissect the development of multidrug resistance phenotypes under antibiotic pressure. Methodology/Principal Findings We used doxycycline selection to obtain several resistant B. thailandensis variants. The minimal inhibitory concentrations of a large panel of structurally unrelated antibiotics were determined ± the efflux pump inhibitor phenylalanine-arginine ß-naphthylamide (PAßN). Membrane proteins were identified by proteomic method and the expressions of major efflux pumps in the doxycycline selected variants were compared to those of the parental strains by a quantitative RT-PCR analysis. Doxycycline selected variants showed a multidrug resistance in two major levels corresponding to the overproduction of two efflux pumps depending on its concentration: AmrAB-OprA and BpeEF-OprC. The study of two mutants, each lacking one of these pumps, indicated that a third pump, BpeAB-OprB, could substitute for the defective pump. Surprisingly, we observed antagonistic effects between PAßN and aminoglycosides or some ß-lactams. PAßN induced the overexpression of AmrAB-OprA and BpeAB-OprB pump genes, generating this unexpected effect. Conclusions/Significance These results may account for the weak activity of PAßN in some Gram-negative species. We clearly demonstrated two antagonistic effects of this molecule on bacterial cells: the blocking of antibiotic efflux and an increase in efflux pump gene expression. Thus, doxycycline is a very efficient RND efflux pump inducer and PAßN may promote the production of some efflux pumps. These results should be taken into account when considering antibiotic treatments and in future studies on efflux pump inhibitors.

Melioidosis in Travelers Returning from Vietnam to France.

To the Editor: Melioidosis, a potentially fatal infectious disease, occurs predominantly across much of Asia and in northern Australia because of the soil and water bacterium Burkholderia pseudomallei (1). We report 2 related cases of suppurative cervical lymphadenitis, an unusual adult presentation of melioidosis, in 2 men who returned to France from Vietnam on the same trip (2).

Decontamination of a field laboratory dedicated to Ebola virus- infected patients

In 2015, the French Armed Forces deployed a biosafety level 3 (BSL3) field laboratory as a part of an Ebola treatment center in Guinea. When closing the center, laboratory decontamination operations were necessary. We present the decontamination protocols applied for the BSL3 field laboratory, making the entire module ready for a future use.

Réponse à la menace biologique : le réseau des laboratoires Biotox-Piratox

Resume Le reseau national des laboratoires Biotox-Piratox est une des composantes des plans nationaux de sauvegarde des populations. Il tire profit des lecons de la crise « charbon » de 2001, afin d’assurer l’utilisation optimale des capacites analytiques des laboratoires nationaux. L’organisation generale du reseau est articulee en trois niveaux de competence, les laboratoires sentinelles, les laboratoires de zones de defense et les laboratoires a competence nationale. Il met ainsi a la disposition des autorites locales et nationales une capacite de reponse flexible et adaptee aux actes malveillants ou alertes mettant en cause des agents biologiques ou des toxiques de guerre ou industriels.

Tularémie, guerre bactériologique et bioterrorisme

Resume Il importe d’obtenir que les professionnels de sante se donnent les moyens de diagnostiquer la tularemie, cette infection largement meconnue, et trop souvent consideree comme une maladie rurale exclusivement infeodee aux lievres et aux chasseurs. Pour repondre a une menace terroriste, les acteurs de sante, cliniciens, biologistes, anatomo-pathologistes et epidemiologistes doivent se familiariser avec cette affection en dehors de toute crise epidemique, ameliorer leurs outils diagnostics, declarer sans delai les cas diagnostiques, etre capables d’isoler les souches afin de tester rapidement leurs sensibilites aux antibiotiques et de pouvoir les comparer sans delai. Cette veille sur la sante humaine doit etre completee et coordonnee avec le maintien de la veille sur la sante de la faune sauvage et sur l’epidemiologie de la tularemie animale en France, en Europe et au-dela.

Burkholderia pseudomallei : une bactérie à ne pas méconnaître

Resume Burkholderia pseudomallei est l’agent de la melioidose. Il s’agit d’une bacterie de l’environnement hydrotellurique, hautement pathogene pour l’homme. En France et en Europe, les cas de melioidose sont des cas d’importation, alors qu’en Asie du Sud-est et en Australie, cette maladie sevit sous forme d’endemie. La repartition geographique de cette maladie s’etend et touche maintenant le Bresil, Madagascar et l’Ile de la Reunion. Le diagnostic phenotypique de cette bacterie est aise, mais il faut savoir penser a ce micro-organisme, qui n’est pas frequent sous nos contrees. Son antibiogramme specifique constitue une aide precieuse au diagnostic. Cette maladie est d’autant plus grave que les possibilites de traitement sont tres restreintes et reposent sur une lourde antibiotherapie.

Le diagnostic biologique au laboratoire de la maladie du charbon

Resume La maladie du charbon est une zoo-anthroponose touchant les troupeaux et parfois l’homme en contact avec les produits animaux dans un contexte de maladies professionnelles, industrielles ou de toxi-infections alimentaires. Elle se presente sous formes cutanee, digestive et respiratoire. Bacillus anthracis est l’agent responsable de la maladie du charbon. C’est un bacille a Gram positif, immobile, capsule, non hemolytique formant des spores. Alors qu’il est facile de traiter le charbon cutane par les antibiotiques, le charbon digestif, le charbon meninge et le charbon pulmonaire (ou d’inhalation) sont redoutables. Le cycle du charbon met en jeu une forme sporulee tellurique et une forme vegetative capsulee produisant les toxines charbonneuses chez l’hote. Toujours presente dans l’environnement hydrotellurique des zones d’enzootie, la maladie reemerge periodiquement en fonction des evolutions climatiques et ecologiques ou des activites humaines. La grande resistance des spores dans l’environnement et la virulence du bacille du charbon en font un agent potentiel de guerre bacteriologique et de bioterrorisme de premier plan. Il est donc important de savoir faire le diagnostic biologique de cette maladie qui s’appuie sur des techniques de bacteriologie classique, de biologie moleculaire et d’immunologie.