[Melioidosis: an emerging tropical disease]. / La mélioïdose: une maladie tropicale en quête de reconnaissance.

Melioidosis is an infection affecting both human and animal health. The causative agent is Burkholderia pseudomallei, a Gram-negative soil bacterium. Melioidosis is endemic in tropical areas of Southeast Asia and Northern Australia, and sporadic in many other countries. Clinical presentation is variable ranging from acute septicemia, isolated pulmonary infection, or chronic granulomatous lesions to asymptomatic forms with positive serology. There is no vaccine and treatment is difficult because B. pseudomallei is resistant to a wide range of antibiotics. Relapses are common. B. pseudomallei is listed as a biological risk class 3 and considered as a potential bioterrorism agent due to its high virulence by inhalation, to the difficulty of treatment, and to the lack of vaccine.

Identification and discrimination of Burkholderia pseudomallei, B. mallei, and B. thailandensis by real-time PCR targeting type III secretion system genes.

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.

Antibiotic susceptibility of 65 isolates of Burkholderia pseudomallei and Burkholderia mallei to 35 antimicrobial agents.

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.

The PmlI-PmlR quorum-sensing system in Burkholderia pseudomallei plays a key role in virulence and modulates production of the MprA protease.

Burkholderia pseudomallei is the causative agent of melioidosis, an often fatal infection of humans and animals. The virulence of this pathogen is thought to depend on a number of secreted proteins, including the MprA metalloprotease. We observed that MprA is produced upon entry into the stationary phase, when the cell density is high, and this prompted us to study cell density-dependent regulation in B. pseudomallei. A search of the B. pseudomallei genome led to identification of a quorum-sensing system involving the LuxI-LuxR homologs PmlI-PmlR. PmlI directed the synthesis of an N-acylhomoserine lactone identified as N-decanoylhomoserine lactone. A B. pseudomallei pmlI mutant was significantly less virulent than the parental strain in a murine model of infection by the intraperitoneal, subcutaneous, and intranasal routes. Inactivation of pmlI resulted in overproduction of MprA at the onset of the stationary phase. A wild-type phenotype was restored following complementation with pmlI or addition of cell-free culture supernatant. In contrast, there was no significant difference between the virulence of a B. pseudomallei mprA mutant and the virulence of the wild-type strain. These results suggest that the PmlI-PmlR quorum-sensing system of B. pseudomallei is essential for full virulence in a mouse model and downregulates the production of MprA at a high cell density.

Strategies for PCR based detection of Burkholderia pseudomallei DNA in paraffin wax embedded tissues.

Recently, several cases of melioidosis imported to Europe have been reported. The diagnosis of the acute or chronic infection remains challenging. This report describes an optimised protocol for fast and reliable DNA preparation for use in two different polymerase chain reaction (PCR) assays, namely: (1) a seminested PCR assay targeting a genus specific sequence of the ribosomal protein subunit 21 (rpsU) gene and (2) a nested PCR assay targeting the gene encoding the filament forming flagellin (fliC). Various strains of Burkholderia spp, strains of closely related genera, and spleen tissue samples of experimentally infected mice were investigated. The combination of PCR and sequencing of the amplicons resulted in high sensitivity and specificity. These procedures may allow rapid, sensitive, and reliable detection of B pseudomallei DNA in routinely formalin fixed and paraffin wax embedded samples, thus providing a safe diagnostic tool and avoiding the cultivation of a risk group 3 agent. In addition, this method could be useful for retrospective histopathological investigations.

[The Rel/NF-kappa-B transcription factors: complex role in cell regulation]. / Les facteurs de transcription Rel/NF-kappa B: rôle complexe dans les régulations cellulaires.

The transcription factor NF-kappa B has attracted widespread attention among researchers. NF-kappa B displays some original characteristics including rapid regulation, the wide range of genes that it controls and its probable involvement in several diseases. In resting cells, NF-kappa B is kept in an inactive form in the cytoplasm where it is bound to a member of the I kappa B family of inhibitory proteins. NF-kappa B can be activated by exposure of cells to physiological as well as non physiological stimuli. Upon cell activation, the inhibitors are modified through site specific phosphorylations which target them for subsequent ubiquitination and proteolytic degradation by the proteasome. Removal of the inhibitor unmasks the nuclear localization signals on subunits of NF-kappa B. Free NF-kappa B moves to the nucleus where it binds to target DNA elements and activate transcription of genes encoding proteins involved in immune responses, inflammation or cell proliferation. NF-kappa B could be considered as a co-ordinating element in the body's responses to situations of stress, infection or inflammation. A tight regulation of NF-kappa B seems to be crucial since a dysfunction could promote pathogenic processes including AIDS (acquired immunodeficiency syndrome), rheumatoid arthritis and cancer. Additionally, it will be important to understand the exact roles for NF-kappa B in regulating apoptosis. NF-kappa B is now regarded as a good therapeutic target and the development of specific inhibitors should lead in the next future to novel therapeutics.

[Inactivation of bacterial spores by high hydrostatic pressure]. / Inactivation des spores bactériennes par les hautes pressions hydrostatiques.

High pressure biotechnology was developed in Japan in the 90's. This new technology has been used in several domains, including chemical synthesis, food industry, physical chemistry of proteins. Moreover, it could be used instead of heat or chemical treatment for inactivation of pathogenic micro-organisms. Hydrostatic pressures ranging from 100 to 300 MPa cause the inactivation of viruses, parasites, yeast and bacteria. However, bacterial spores are particularly resistant and their inactivation by high hydrostatic pressure can be achieved in combination with synergistic treatments (heat, chemicals and ultrasound).

Study on the pathophysiology of experimental Burkholderia pseudomallei infection in mice.

Burkholderia pseudomallei is the etiological agent of melioidosis, a potentially fatal disease occurring in man and animals. The aim of this study was to investigate the pathophysiological course of experimental melioidosis, and to identify the target organs, in an animal model. For this purpose SWISS mice were infected intraperitoneally with the virulent strain B. pseudomallei 6068. The bacterial load of various organs was quantified daily by bacteriological analysis and by an enzyme-linked immunosorbent assay (ELISA) based on a monoclonal antibody specific to B. pseudomallei exopolysaccharide (EPS). Electron microscopic investigation of the spleen was performed to locate the bacteria at the cellular level. In this model of acute melioidosis, B. pseudomallei had a marked organ tropism for liver and spleen, and showed evidence of in vivo growth with a bacterial burden of 1.6x10(9) colony forming units (CFU) per gram of spleen 5 days after infection with 200 CFU. The highest bacterial loads were detected in the spleen at all time points, in a range from 2x10(6) to 2x10(9) CFU g(-1). They were still 50-80 times greater than the load of the liver at the time of peak burden. Other investigated organs such as lungs, kidneys, and bone marrow were 10(2)-10(4)-fold less infected than the spleen, with loads ranging from 3x10(2) to 3x10(6) CFU g(-1). The heart and the brain were sites of a delayed infection, with counts in a range from 10(3) to 10(7) times lower than bacterial counts in the spleen. The EPS-specific ELISA proved to be highly sensitive, particularly at the level of those tissues in which colony counting on agar revealed low contamination. In the blood, EPS was detected at concentrations corresponding to bacterial loads ranging from 8x10(3) to 6x10(4) CFU ml(-1). Electron microscopic examination of the spleen revealed figures of phagocytosis, and the presence of large numbers of intact bacteria, which occurred either as single cells or densely packed into vacuoles. Sparse figures suggesting bacterial replication were also observed. In addition, some bacteria could be seen in vacuoles that seemed to have lost their membrane. These observations provide a basis for further investigations on the pathogenesis of the disease.

Protease production by Burkholderia pseudomallei and virulence in mice.

The aim of this study was to assess protease production and virulence of various Burkholderia pseudomallei strains. Protease activity was evaluated in filtrates from cultures grown for 50 h in TSB Dialysate by azocasein hydrolysis, and expressed as absorbancy at 405 nm. Virulence was assessed in 8 weeks old SWISS mice, by intraperitoneal injection of 6-6 x 10(5) CFU, and the LD50 was calculated after 30 days by the method of Reed and Muench. The lethal activity was studied for five strains of B. pseudomallei and the type strains of Burkholderia pseudomallei, Burkholderia mallei, and Burkholderia cepacia. The three type strains appeared to be low protease producers (A405 = 0.11, 0.09 and 0.00, respectively) and avirulent. The two more virulent B. pseudomallei strains exhibited significantly different LD50, 3.5 x 10(2) (IPP 6068 VIR) versus 2.1 x 10(5) CFU/mouse (40/97), and protease activities (A405 = 0.046 and 0.79, respectively). Moreover, the avirulent parent of IPP 6068 (AG), was a better protease producer than the 6068 VIR strain, A405 = 0.26 versus 0.046. These results suggest that there is no correlation between virulence and level of exoproteolytic activity, when B. pseudomallei is injected to mice via the intraperitoneal route.

A parametric study of the destruction efficiency of Bacillus spores in low pressure oxygen-based plasmas.

The destruction of Bacillus spores in oxygen-based plasmas sustained in the millitorr pressure range has been studied as functions of various biological and plasma parameters, namely Bacillus species, surface concentration of spores, treatment temperature, and gas composition. In an oxygen plasma, Bacillus stearothermophilus appears less plasma-resistant than the other spores tested. Oxygen, H2O2 and chiefly CO2 plasmas are clearly shown to be much more efficient in destroying Bacillus subtilis spores than pure argon plasma. The bacterial surface concentration on the spore carriers and the treatment temperature also lead to significant variations in the destruction efficiency of spores when using CO2 plasma.

[Bacterial biofilms and resistance to disinfectants]. / Biofilms bactériens et résistance aux désinfectants.

When bacteria grow in close association with solid surface, they constitute a microbial community tight included in the exopolymer glycocalyx. Many laboratory studies have shown that these bacteria are 10 to 100 folds more resistant to disinfectants than the bacteria of the same strain in suspension. Several factors are responsible for this resistance: the glycocalyx which limits the diffusion and reacts with the disinfectant, the more or less dense repartition of the bacteria inside the biofilm, their physiologic state with reduced metabolism, and the surface on which is the biofilm. The activity assessment of disinfectant agents is achieved with standardized methods. They must take into account not only the conditions in which the disinfectants are employed, but also the micro-organism state. Experimental results showing the resistance of biofilm bacteria must lead to elaborate methods allowing the assessment of bactericide activity of disinfectants against biofilm bacteria.

Immunosuppressive effects of Pseudomonas aeruginosa exotoxin A on human B-lymphocytes.

In this study we investigated the effects of exotoxin A on proliferation and differentiation of human B-cells in vitro. Exotoxin A at a concentration of 1 microgram/ml inhibited the proliferation of B-cells preactivated by insolubilized anti-IgM antibody or by formalinized Staphylococcus aureus particles, plus IL-2 or IL-4. B-cell blasts obtained after preactivation of tonsillar B-cells produce IgG and IgM in culture supernatants, and this Ig production is enhanced by IL-2 or IL-4. Exotoxin A inhibited the production of IgG and IgM by the B-blasts at the concentration of 1 microgram/ml.

[Biological safety in the laboratory. Biological risk, standardization and practice]. / Biosécurité au laboratoire. Risque biologique, normalisation et pratique.

Working with pathogens or genetically engineered micro-organisms is a potential hazard for scientists, health care workers, employees of pharmaceutical industry, and also for the environment. Carelessness, poor technique in the handling of infectious materials, needle sting or infectious aerosol exposure are the cause of laboratory acquired infection. Biosafety, corollary of biocontamination, is based on the combination of good microbiological techniques, facility design of the laboratory and safety equipment. So, four biosafety levels are appropriate for the operations performed and the hazard posed by the infectious agents.

Genetics of lactic acid bacteria with special reference to lactococci.

Lactic acid bacteria play an important role in the manufacture of fermented foods. Genetic studies have made these microorganisms, particularly lactococci, accessible to genetic manipulation. The instability of key metabolic traits of lactococci has been explained by the presence of plasmid DNA species. Most genetic interest has been focused to solve the quoted instability and the sensitivity to bacteriophage infection. At the same time, gene transfer systems have been developed and specific genes with commercial significance have been identified and cloned. Lactic acid bacteria can be also used as production organisms of heterologous proteins, e.g. chymosin, lysozyme.

[Paradoxic effect of anti-T-2 toxin antibodies in the curative treatment of acute poisoning in mice]. / Effet paradoxal des anticorps anti-toxine T-2 dans le traitement curatif de l'intoxication aiguë de la Souris.

We have obtained polyclonal anti-T-2 toxin antibodies with high affinity to T-2 toxin (K = 0.34 x 10(9) l/M at 4 degrees C), crossreacting with HT-2 toxin, one of the major metabolites. We have assessed these antibodies in prevention and therapy of the acute intoxication. When given 24 h before 1 or 2 LD50 of T-2 toxin (5 or 10 mg/kg), the antibodies decreased the mortality from 67% to 37%, or from 100% to 83% respectively with an optimal antibody dosage of 100 mg/kg. In contrast when given 30 mn after the toxin, anti-T-2 toxin antibodies increased the mortality; in the same way non specific antibodies increased the mortality of mice intoxicated with T-2 toxin.