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(1) Background: Bacillus anthracis is a spore-forming, Gram-positive bacterium causing anthrax, a zoonosis affecting mainly livestock. When occasionally infecting humans, B. anthracis provokes three different clinical forms: cutaneous, digestive and inhalational anthrax. More recently, an injectional anthrax form has been described in intravenous drug users. (2) Case presentation: We report here the clinical and microbiological features, as well as the strain phylogenetic analysis, of the only injectional anthrax case observed in France so far. A 27-year-old patient presented a massive dermohypodermatitis with an extensive edema of the right arm, and the development of drug-resistant shocks. After three weeks in an intensive care unit, the patient recovered, but the microbiological identification of B. anthracis was achieved after a long delay. (3) Conclusions: Anthrax diagnostic may be difficult clinically and microbiologically. The phylogenetic analysis of the Bacillus anthracis strain PF1 confirmed its relatedness to the injectional anthrax European outbreak group-II.

Mycoplasma (M.) bovis has recently emerged as a major, worldwide etiological agent of bovine respiratory diseases leading to huge economic losses mainly due to high morbidity and mortality as well as poor growth rates. The spread of M. bovis infections between different animals, herds, regions or countries has been often reported to be connected to the movement of animals. However, despite recent considerable efforts, no universal subtyping method is yet available to trace M. bovis isolates circulation at an international scale. Moreover in France, the overall population diversity of M. bovis isolates has not been assessed since the early 1990s. This study was conducted to fill in these gaps. The genotypic diversity between sixty isolates collected in France over the last 35 years was assessed using two molecular subtyping methods that addressed either the long-term epidemiological relationships (Multi Locus Sequence Typing, MLST) or the genetic microvariations (Multiple Locus VNTR Analysis, MLVA) between isolates. Phenotypic diversity was also analyzed by using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) to compare the main protein patterns of isolates. All proposed subtyping approaches were optimized and led to the same pattern in the French M. bovis population that consisted of two clusters, the first one comprising isolates collected before 2000 and the second, those collected after 2000. Recent strains were further shown to be more homogeneous than older ones, which is consistent with the spread of a single clone throughout the country. Because this spread was concomitant with the emergence of multiresistant M. bovis isolates, several hypotheses are discussed to explain the homogeneity of M. bovis isolates in France, even though the M. bovis species is fully equipped to generate diversity.

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).

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.

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.

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.

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.

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.

International audience; Multiplelocus variablenumber of tandem repeat (VNTR) analysis (MLVA) has been shown to be very promising for the typing of Burkholderia pseudomallei and mallei. The currently available set of loci requires high resolution allele size measurement due to short repeat units. The present work was aimed at expanding the available set of VNTR loci, and generating data from a collection of 102 B. pseudomallei strains isolated in Singapore between 1988 and 2004 including few additional strains of various origins as references. Ten new VNTRs with a longer array size have been identified compatible with standard agarose gel separation, and a reference database of 72 genotypes was created which can be queried on the Internet.

Melioidosis is a severe gram-negative infection caused by the facultative intracellular bacterium Burkholderia pseudomallei, which is responsible for a broad spectrum of symptoms in both humans and animals. No licensed vaccine currently exists. This study evaluated the protective effect of a monoclonal antibody (Mab Ps6F6) specific to B. pseudomallei exopolysaccharide in an outbred murine model of sub-acute melioidosis. When administered before the infectious challenge, Ps6F6 significantly increased resistance to infection and restrained bacterial burden in the spleen over a 30-days period. Patterns of IFN-gamma production were similar in the treated and non treated groups of mice. However, Ps6F6 lowered IFN-gamma levels over the duration of the assay period, except on day 1, suggesting a transient and rapid production of IFN-gamma under Ps6F6 control. Minor but persisting increases occurred in IL-12 levels while TNF-alpha was detected only in the controls at the later stages of infection. No IL-10 secretion was detected in both groups of mice. These data suggest that passive prophylaxis with Mab Ps6F6 provide a moderate and transient induction of inflammatory responses in infected mice but failed to trigger a sterilizing protective immunity.

Burkholderia pseudomallei and B. mallei are two highly pathogenic bacteria, responsible for melioidosis and glanders, respectively. The two are closely related and can also be mistaken for B. thailandensis , a nonpathogenic species. To improve their differential identification, we describe a hydrolysis probe-based real-time PCR method using the uneven distribution of type III secretion system genes among these three species.

Burkholderia pseudomallei, a gram-negative bacterium, is the causative agent of melioidosis in humans and animals (9, 13, 45). Melioidosis is endemic in tropical areas of Southeast Asia and northern Australia, but it is also sporadically found in many other countries. This highly pathogenic microorganism is deemed a potential agent of bioterrorism (listed as category B by the Centers for Disease Control and Prevention). The clinical manifestations of melioidosis vary greatly, ranging from an asymptomatic state to acute septicemia, pulmonary forms, and chronic granulomatous lesions (34). The latency period of the disease is between 2 days and 26 years (21, 34). Successful treatment is difficult because B. pseudomallei is inherently resistant to a wide range of antibiotics and relapse is common (43). Although melioidosis was first described in 1912 (44), the virulence determinants of B. pseudomallei have not been well characterized. Studies of B. pseudomallei pathogenicity have mostly concentrated on exoproducts, which include a protease, a lipase, a phospholipase C, and a hemolysin (3, 34, 45). The regulatory circuits governing the production of exoproducts in B. pseudomallei remain unknown. However, many gram-negative pathogens regulate the production of extracellular virulence factors by quorum sensing, a cell-density-dependent mechanism. Quorum sensing is the process of producing and responding to high intracellular concentrations of N-acylhomoserine lactone (AHL) autoinducers and relies on two proteins: (i) an AHL synthase belonging to the LuxI family, which directs the synthesis of AHL; and (ii) a transcriptional regulator belonging to the LuxR family, which, after binding of AHL, is thought to activate or repress transcription of targeted genes (11, 22, 29). In Burkholderia cepacia, which is phylogenetically related to B. pseudomallei (13), the CepI-CepR quorum-sensing system positively regulates protease production and represses synthesis of the siderophore ornibactin (19). There has been much interest in the contribution of the B. pseudomallei protease to virulence. This enzyme was purified from culture supernatants as a 36-kDa metalloenzyme and appeared to be necessary for full virulence in a rat model of lung infection (32). In contrast, we found no correlation between virulence and the level of protease activity after intraperitoneal infection of mice with B. pseudomallei (12). These results suggested that the role played by the protease in pathogenesis could be dependent on the route of infection. Recently, the mprA gene of B. pseudomallei was cloned and was found to encode a 50-kDa serine metalloprotease, the only protease produced by B. pseudomallei (17). However, the discrepancy with the size determined by Sexton et al. (32) has not been explained yet; perhaps the difference is due to proteolysis or the use of different techniques (17). Our previous investigations on the B. pseudomallei protease (12, 25) led us to examine whether enzyme production is regulated by quorum sensing. The role of quorum-sensing systems in the pathogenicity of different gram-negative bacteria has been demonstrated previously (28, 35). The existence of such a system in B. pseudomallei could provide new data on the virulence determinants of this organism. Here we describe identification of the PmlI-PmlR quorum-sensing system of B. pseudomallei. This system is crucial for full virulence of B. pseudomallei in a murine model of infection and modulates the production of the MprA protease.

We report here the draft sequence of strain CEB14_0017, alias HIAD_DUP, recovered from a human patient and initially identified as Yersinia pestis by mass spectrometry analysis. Genotyping based on tandem repeat polymorphism assigned the strain to Yersinia pseudotuberculosis sequence type 42 (ST42). The total assembly length is 4,894,739 bp.

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.

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.

International audience; 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.

Objectives Fifty isolates of Burkholderia pseudomallei and 15 isolates of Burkholderia mallei were tested for their susceptibilities to 35 antimicrobial agents, including agents not previously tested against these bacteria. Methods MICs were determined by agar dilution in Mueller-Hinton medium. Results Among the antibiotics tested, lower MICs were obtained with imipenem, ceftazidime, piperacillin, piperacillin/tazobactam, doxycycline and minocycline. Fluoroquinolones and aminoglycosides had poor activities. A single clinical isolate of B. pseudomallei was resistant to ceftazidime, co-amoxiclav and doxycycline but remained susceptible to imipenem. Conclusions Although B. mallei MICs are often lower, the overall results underline the importance of resistance in both species. The susceptibilities measured are consistent with the current recommendations for the treatment of B. pseudomallei and B. mallei infections.