Antimicrobial Resistance, Virulence Genes, and Genetic Lineages of Staphylococcus pseudintermedius in Healthy Dogs in Tunisia

Nasal swabs of 100 healthy dogs were obtained in 2011 in Tunisia and tested for Staphylococcus pseudintermedius recovery. Antimicrobial resistance profile and virulence gene content were determined. Multilocus-sequence-typing (MLST) and SmaI-pulsed-field gel electrophoresis (PFGE) were investigated. S. pseudintermedius was recovered in 55 of the 100 tested samples (55 %), and one isolate per sample was further studied. All 55 S. pseudintermedius isolates were susceptible to methicillin (MSSP) but showed resistance to the following antimicrobials (% resistant isolates/resistance gene): penicillin (56.4/blaZ), tetracycline (40/tetM), trimethoprim-sulfamethoxazole (23.7), fusidic acid (9), kanamycin (3.7/aph(3´)-Ia), erythromycin-clindamycin (1.8/erm(B)), streptomycin (1.8/ant(6)-Ia), chloramphenicol (1.8) and ciprofloxacin (1.8). The following toxin genes were identified (% of isolates): lukS/F-I (98.2), expA (5.5), se-int (98.2), sec _canine (1.8), siet (100), sea (5.5), seb (3.6), sec (10.9), sed (54.5), sei (5.5), sej (29.1), sek (3.6), ser (9.1), and hlg _v (38.2). Ten different sequence-types were detected among 11 representative MSSP isolates: ST20, ST44, ST69, ST70, ST78, ST100, ST108, ST160, ST161, and ST162, the last three ones revealing novel alleles or allele combinations. Eleven different PFGE-patterns were identified in these isolates. The nares of healthy dogs could be a reservoir of antimicrobial resistant and virulent MSSP, highlighting the presence of the recently described exfoliating gene expA and several enterotoxin genes.     

Biodiversity within Medicago truncatula genotypes toward response to iron deficiency: Investigation of main tolerance mechanisms

Iron (Fe) deficiency is one of the major environmental stresses affecting plant production in the world. The selection of tolerant genotypes is considered an effective remediation strategy for this stress. The present study was carried out in order to investigate the biodiversity within Medicago truncatula plants in response to Fe deficiency, to identify tolerant genotypes and to assess the main tolerance mechanisms. To do this, a screening test was performed on 20 M. truncatula genotypes cultivated in minimal medium. Biometric and physiological markers were analyzed, including plant biomass, chlorophyll and root architecture. Results showed a biodiversity among the 20 genotypes. Interestingly, Fe deficiency tolerance was highest in TN8.20 and A17 genotypes. However, the lowest tolerance behavior was observed in TN1.11 and TN6.18. In order to investigate the main tolerance mechanisms, an experiment was conducted in the hydroponic system on already selected genotypes. Assessment of Fe deficiency tolerance was performed mainly on plant growth parameters, Fe (III)-chelate-reductase activity, rhizosphere acidification and antioxidant system defense. The relative better tolerance of A17 and TN8.20 to Fe deficiency was positively correlated with their capacity to maintain higher Fe-acquisition efficiency in roots via rhizosphere acidification and the stimulation of Fe (III)-chelate-reductase activity. Moreover, tolerant genotypes showed the lowest decreases in chlorophyll content and photosynthetic activity (CO₂ assimilation) compared to the sensitive ones. The efficiency of antioxidant capacity of the tolerant genotypes was revealed in stimulation of catalase (CAT) and peroxidase (POX) activities as well as accumulation of polyphenols, leading to the maintenance of cell integrity under Fe deficiency.     

Sequence Polymorphism of Cytochrome b Gene in Theileria annulata Tunisian Isolates and Its Association with Buparvaquone Treatment Failure

Background

Buparvaquone (BW 720C) is the major hydroxynaphtoquinone active against tropical theileriosis (Theileria annulata infection). Previous studies showed that buparvaquone, similarly to others hydroxynaphtoquinone, probably acts by binding to cytochrome b (cyt b) inhibiting the electron transport chain in the parasite. Several observations suggested that T. annulata is becoming resistant to buparvaquone in many endemic regions (Tunisia, Turkey and Iran), which may hinder the development of bovine livestock in these areas.

Methodology/Principal Findings

In the present study we sought to determine whether point mutations in T. annulata cytochrome b gene could be associated to buparvaquone resistance. A total of 28 clones were studied in this work, 19 of which were obtained from 3 resistant isolates (ST2/12, ST2/13 and ST2/19) collected at different time after treatment, from a field treatment failure and nine clones isolated from 4 sensitive stocks of T. annulata (Beja, Battan, Jed4 and Sousse). The cytochrome b gene was amplified and sequenced. We identified five point mutations at the protein sequences (114, 129, 253, 262 and 347) specific for the clones isolated from resistant stocks. Two of them affecting 68% (13/19) of resistant clones, are present in the drug-binding site Q₀₂ region at the position 253 in three resistant clones and at the position 262 in 11 out of 19 resistant clones. These two mutations substitute a neutral and hydrophobic amino acids by polar and hydrophilic ones which could interfere with the drug binding capabilities. When we compared our sequences to the Iranian ones, the phylogenetic tree analyses show the presence of a geographical sub-structuring in the population of T. annulata.

Conclusions/Significance

Taken together, our results suggest that the cytochrome b gene may be used as a tool to discriminate between different T. annulata genotypes and also as a genetic marker to characterize resistant isolates of T. annulata.     

The alternative oxidase pathway is involved in optimizing photosynthesis in Medicago truncatula infected by Fusarium oxysporum and Rhizoctonia solani

Phytopathogen infection alters primary metabolism status and plant development. The alternative oxidase (AOX) has been hypothesized to increase under pathogen attack preventing reductions, thus optimizing photosynthesis and growth. In this study, two genotypes of Medicago truncatula, one relatively resistant (Jemalong A17) and one susceptible (TN1.11), were infected with Fusarium oxysporum and Rhizoctonia solani. The in vivo foliar respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) were measured using the oxygen isotope fractionation. Gas exchange and photosynthesis-related parameters were measured and calculated together with antioxidant enzymes activities and organic acids contents. Our results show that the in vivo activity of AOX (v_alt) plays a role under fungal infection. When infected with R. solani, the increase of v_alt in A17 was concomitant to an increase in net assimilation, in mesophyll conductance, to an improvement in the maximum velocity of Rubisco carboxylation and to unchanged malate content. However, under F. oxysporum infection, the induced v_alt was accompanied by an enhancement in the antioxidant enzymes, superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6) and guaiacol peroxidase (GPX; EC1.11.1.7), activities and to an unchanged tricarboxylic acid cycle intermediates. These results provide new insight into the role of the in vivo activity of AOX in coordinating primary metabolism interactions that, partly, modulate the relative resistance of M. truncatula to diseases caused by soil-borne pathogenic fungi.     

Involvement of Hydrogen Peroxide, Peroxidase and Superoxide Dismutase in Response of Medicago truncatula Lines Differing in Susceptibility to Phoma medicaginis Infection

This study was undertaken to assess the involvement of hydrogen peroxide (H₂O₂), peroxidase (POX; EC 1.11.1.7) and superoxide dismutase (SOD; EC 1.15.1.1) in Medicago truncatula in relation with susceptibility to Phoma medicaginis infection. Several M. truncatula lines were studied in terms of their response to P. medicaginis infection. Fifteen days after inoculation (dai), differences in susceptibility were found. DZA45.5 was the least susceptible line and F83005.5 was the most susceptible line. Microscopic analysis of fungal development was performed in inoculated detached leaves of the DZA45.5 and F83005.5 lines. No significant difference was observed in events from conidia germination to penetration. Differences became apparent during the colonization process as the pathogen was able to sporulate rapidly increasing its concentration on the tissue of F83005.5 in comparison with DZA45.5. To characterize the susceptibility of the two lines, histochemical detection of H₂O₂ was made in detached leaves. H₂O₂ detection showed an early accumulation of this component in cells of DZA45.5 at 1 dai. However, H₂O₂ was detected in few, if any, cells in the tissues of the most susceptible line, F83005.5. The activity of POX and SOD were determined spectrophotometrically in leaves of intact inoculated plants of both lines. Phoma medicaginis inoculation of DZA45.5 and F83005.5 did not affect POX activity level in leaves when compared with control uninoculated plants. SOD activity showed a significant decrease in F83005.5 and DZA45.5 leaves at 4 dai and 9 dai, respectively, in comparison with control plants. In control plants POX activity was significantly higher in the least susceptible line DZA45.5 in comparison with F83005.5. Early and higher production of H₂O₂ and elevated basal POX activity in cells of the least susceptible line, DZA45.5 could explain its ability to be less favourable to the colonization and reproduction of P. medicaginis in comparison with the most susceptible line, F83005.5.     

Rhizobial strain involvement in plant growth, nodule protein composition and antioxidant enzyme activities of chickpea-rhizobia symbioses: modulation by salt stress.

Mesorhizobium ciceri, Mesorhizobium mediterraneum and Sinorhizobium medicae strains showed different symbiotic performances when inoculated to chickpea (Cicer arietinum L., cv. chetoui) at unstressed conditions and under salt stress. The analysis of nodular proteic composition and antioxidant enzyme activities revealed a polymorphism of patterns on SDS and native PAGE suggesting a potential dependence on the bacterial partner. Salt effect was analysed on plant growth, nitrogen fixation and antioxidant enzymes. M. ciceri, the most efficient strain, seemed to allow a best tolerance to chickpea plants under salt stress. This constraint did not affect the nodular superoxide dismutase (SOD, E.C. 1.15.1.1) activity of the symbiosis implicating the latter strain. This symbiosis showed the least decrease for the nodule protein level and the catalase (CAT, E.C. 1.11.1.6) activity, and the highest increase of peroxidase (POX, E.C. 1.11.1.7) activity that seemed to be related with the tolerance to salt.     

Salinity tolerance is related to cyanide‐resistant alternative respiration in Medicago truncatula under sudden severe stress

Salt respiration is defined as the increase of respiration under early salt stress. However, the response of respiration varies depending on the degree of salt tolerance and salt stress. It has been hypothesized that the activity of the alternative pathway may increase preventing over‐reduction of the ubiquinone pool in response to salinity, which in turn can increase respiration. Three genotypes of Medicago truncatula are reputed as differently responsive to salinity: TN1.11, A17 and TN6.18. We used the oxygen‐isotope fractionation technique to study the in vivo respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) in leaves and roots of these genotypes treated with severe salt stress (300 mM) during 1 and 3 days. In parallel, AOX capacity, gas exchange measurements, relative water content and metabolomics were determined in control and treated plants. Our study shows for first time that salt respiration is induced by the triggered AOP in response to salinity. Moreover, this phenomenon coincides with increased levels of metabolites such as amino and organic acids, and is shown to be related with higher photosynthetic rate and water content in TN6.18.     

Rapid protein production using CHO stable transfection pools

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post‐transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence‐activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three‐fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin‐opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Adaptive Packet-size Control for Improved Throughput in Dynamic Access Networks

Cluster Computing - Cognitive radio (CR) is a new intelligent wireless technology that aims at improving spectrum utilization by allowing opportunistic access to the underutilized licensed...