Draft Genome Sequence of the Lignocellulolytic and Thermophilic Bacterium Thermobacillus xylanilyticus XE.

Thermobacillus xylanilyticus is a thermophilic and hemicellulolytic bacterium able to use several lignocelluloses as its main carbon source. This draft genome sequence gives insight into the genomic potential of this bacterium and provides new resources to understand the enzymatic mechanisms used by the bacterium during lignocellulose degradation and will allow the identification of robust lignocellulolytic enzymes.

Contribution of Fluorescence Techniques in Determining the Efficiency of the Non-thermal Plasma Treatment.

We have recently developed a non-thermal plasma (NTP) equipment intended to sterilize fragile medical devices and maintain the sterile state of items downstream the treatment. With traditional counts on agar plate a six log reduction of Staphylococcus aureus viability was obtained within 120 min of O2, Ar, or N2 NTP treatments. However to determine the best NTP process, we studied the different physiological states of S. aureus by flow cytometry (FC) and confocal laser scanning microscopy (CLSM) focusing on the esterasic activity and membrane integrity of the bacteria. Two fluorochromes, 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate and propidium iodide were used in order to distinguish three sub-populations: metabolically active, permeabilized, and damaged bacteria that can be in the viable but nonculturable state. FC and CLSM highlight that O2 and Ar NTP treatments were the most attractive processes. Indeed, a 5 min of Ar NTP generated a high destruction of the structure of bacteria and a 120 min of O2 NTP treatment led to the higher decrease of the total damaged bacteria population. SEM observations showed that in presence of clusters, bacteria of upper layers are easily altered compared to bacteria in the deeper layers. In conclusion, the plate counting method is not sufficient by itself to determine the best NTP treatment. FC and CLSM represent attractive indicator techniques to select the most efficient gas NTP treatment generating the lowest proportion of viable bacteria and the most debris.

Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters.

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-R(SDZ) and ME-49-R(SDZ), by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied.

Identification of differentially expressed proteins in sulfadiazine resistant and sensitive strains of Toxoplasma gondii using difference-gel electrophoresis (DIGE).

Treatment options for toxoplasmosis in humans are generally limited to the use of sulfonamide and/or pyrimethamine-based compounds. However, there is increasing evidence for clinical therapy failures in patients suggesting the existence of drug resistance in these classes of drug. In vitro resistance to sulfadiazine has been detected in three strains of Toxoplasma gondii isolated from clinical cases. In order to begin to understand the mechanisms of resistance, we undertook a difference-gel electrophoresis (DIGE) approach combined with mass spectrometry to identify proteins that are differentially expressed in sulfadiazine-resistance strains of the parasite. Naturally resistant strains TgA 103001 (Type I), TgH 32006 (Type II) and TgH 32045 (Type II variant) were compared to sensitive strains RH (Type I) and ME-49 (Type II) using DIGE and the modulated proteins analyzed using LC-MS/MS. In total, 68 differentially expressed protein spots were analyzed by mass spectrometer and 31 unique proteins, including four hypothetical proteins, were identified. Among the differentially expressed proteins, 44% were over-expressed in resistant strains and 56% were over-expressed in sensitive strains. The virulence-associated rhoptry protein, ROP2A, was found in greater abundance in both naturally resistant Type II strains TgH 32006 and TgH 32045 compared to the sensitive strain ME-49. Enolase 2 and IMC1 were found to be in greater abundance in sensitive strains RH and ME-49, and MIC2 was found to be more abundant in the sensitive strain ME-49. Proteins regulation of ROP2, MIC2, ENO2, IMC1 and GRA7 were confirmed by Western blot analysis. In addition, gene expression patterns of ROP2, MIC2, ENO2 and IMC1 were analyzed with qRT-PCR. This study provides the first proteomics insights into sulfadiazine resistance in T. gondii resistant strains isolated from clinical cases.