Semi-automated repetitive sequence-based PCR amplification for species of the Scedosporium apiospermum complex

International audiencePurpose: The Scedosporium apiospermum species complex usually ranks second among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF), but little is known about the molecular epidemiology of the airway colonization.Methods: Polymerase chain reaction (PCR) amplification of repetitive sequences (rep-PCR) was applied to the retrospective analysis of a panel of isolates already studied by random amplification of polymorphic DNA (RAPD) and comprising 63 isolates recovered from sputa from 9 CF patients. Results were compared to those obtained previously by RAPD, and herein by beta-tubulin (TUB) gene sequencing and Multilocus Sequence Typing (MLST).Results: Within the panel of isolates studied, S. apiospermum sensu stricto and Sce-dosporium boydii, as expected, were the predominant species with 21 and 36 isolates, respectively. Four isolates from one patient were identified as Scedosporium auranti-acum, whereas two isolates belonged to the Pseudallescheria ellipsoidea subgroup of S. boydii. rep-PCR analysis of these isolates clearly differentiated the three species and P. ellipsoidea isolates, whatever the rep-PCR kit used, and also permitted strain differentiation. When using the mold primer kit, results from rep-PCR were in close agreement with those obtained by MLST. For both S. apiospermum and S. boydii, 8 genotypes were differentiated by rep-PCR and MLST compared to 10 by RAPD. All S. aurantiacum isolates shared the same RAPD genotype and exhibited the same rep-PCR profile and sequence type.Conclusions: These results illustrate the efficacy of rep-PCR for both species identification within the S. apiospermum complex and genotyping for the two major species of this comple

Repurposing of auranofin and honokiol as antifungals against Scedosporium species and the related fungus Lomentospora prolificans.

peer reviewedThe slowing-down de novo drug-discovery emphasized the importance of repurposing old drugs. This is particularly true when combating infections caused by therapy-refractory microorganisms, such as Scedosporium species and Lomentospora prolificans. Recent studies on Scedosporium responses to oxidative stress underscored the importance of targeting the underlying mechanisms. Auranofin, ebselen, PX-12, honokiol, and to a lesser extent, conoidin A are known to disturb redox-homeostasis systems in many organisms. Their antifungal activity was assessed against 27 isolates belonging to the major Scedosporium species: S. apiospermum, S. aurantiacum, S. boydii, S. dehoogii, S. minutisporum, and Lomentospora prolificans. Auranofin and honokiol were the most active against all Scedosporium species (mean MIC50 values of 2.875 and 6.143 μg/ml, respectively) and against L. prolificans isolates (mean MIC50 values of 4.0 and 3.563μg/ml respectively). Combinations of auranofin with voriconazole or honokiol revealed additive effects against 9/27 and 18/27 isolates, respectively. Synergistic interaction between auranofin and honokiol was only found against one isolate of L. prolificans. The effects of auranofin upon exposure to oxidative stress were also investigated. For all species except S. dehoogii, the maximal growth in the presence of auranofin significantly decreased when adding a sublethal dose of menadione. The analysis of the expression of genes encoding oxidoreductase enzymes upon exposure of S. apiospermum to honokiol unveiled the upregulation of many genes, especially those coding peroxiredoxins, thioredoxin reductases, and glutaredoxins. Altogether, these data suggest that auranofin and honokiol act via dampening the redox balance and support their repurposing as antifungals against Scedosporium species and L. prolificans

Genes but Not Genomes Reveal Bacterial Domestication of Lactococcus Lactis

BACKGROUND: The population structure and diversity of Lactococcus lactis subsp. lactis, a major industrial bacterium involved in milk fermentation, was determined at both gene and genome level. Seventy-six lactococcal isolates of various origins were studied by different genotyping methods and thirty-six strains displaying unique macrorestriction fingerprints were analyzed by a new multilocus sequence typing (MLST) scheme. This gene-based analysis was compared to genomic characteristics determined by pulsed-field gel electrophoresis (PFGE). METHODOLOGY/PRINCIPAL FINDINGS: The MLST analysis revealed that L. lactis subsp. lactis is essentially clonal with infrequent intra- and intergenic recombination; also, despite its taxonomical classification as a subspecies, it displays a genetic diversity as substantial as that within several other bacterial species. Genome-based analysis revealed a genome size variability of 20%, a value typical of bacteria inhabiting different ecological niches, and that suggests a large pan-genome for this subspecies. However, the genomic characteristics (macrorestriction pattern, genome or chromosome size, plasmid content) did not correlate to the MLST-based phylogeny, with strains from the same sequence type (ST) differing by up to 230 kb in genome size. CONCLUSION/SIGNIFICANCE: The gene-based phylogeny was not fully consistent with the traditional classification into dairy and non-dairy strains but supported a new classification based on ecological separation between "environmental" strains, the main contributors to the genetic diversity within the subspecies, and "domesticated" strains, subject to recent genetic bottlenecks. Comparison between gene- and genome-based analyses revealed little relationship between core and dispensable genome phylogenies, indicating that clonal diversification and phenotypic variability of the "domesticated" strains essentially arose through substantial genomic flux within the dispensable genome

Yeasts as Biopharmaceutical Production Platforms

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Transcriptional profiling of Scedosporium apiospermum enzymatic antioxidant gene battery unravels the involvement of thioredoxin reductases against chemical and phagocytic cells oxidative stress

International audienceScedosporium species rank the second, after Aspergillus fumigatus, among the filamentous fungi colonizing the airways of patients with cystic fibrosis (CF). Development of microorganisms in the respiratory tract depends on their capacity to evade killing by the host immune system, particularly through the oxidative response of macrophages and neutrophils, with the release of reactive oxygen species (ROS) and reactive nitrogen species (RNS). This is particularly true in the airways of CF patients which display an exacerbated inflammatory reaction. To protect themselves, pathogens have developed various enzymatic antioxidant systems implicated in ROS degradation, including superoxide dismutases, catalases, cytochrome C peroxidases, chloroperoxidases and enzymes of the glutathione and thioredoxin systems, or in RNS degradation, that is, flavohemoglobins, nitrate reductases, and nitrite reductases. Here we investigated the transcriptional regulation of the enzymatic antioxidant gene battery in 24-h-old hyphae of Scedosporium apiospermum in response to oxidative stress induced chemically or by exposure to activated phagocytic cells. We showed that 21 out of the 33 genes potentially implicated in the oxidative or nitrosative stress response were over-expressed upon exposure of the fungus to various chemical oxidants, while they were only 13 in co-cultures with macrophages or neutrophils. Among them, genes encoding two thioredoxin reductases and to a lesser extent, a peroxiredoxin and one catalase were found to be overexpressed after chemical oxidative stress as well as in co-cultures. These results suggest that thioredoxin reductases, which are known to be virulence factors in other pathogenic fungi, play a key role in pathogenesis of scedosporiosis, and may be new drug targets

French translation and validation of the Exercise-Induced Leg Pain Questionnaire

peer reviewedIntroduction: The « Exercise-Induced Leg Pain » (EILP) questionnaire has been developed to evaluate the severity of symptoms and sports ability in individuals with exercise-induced leg pain. Purpose: We aimed to translate and cross-culturally adapt this questionnaire into French and to study the reliability and validity of the French-translated version (EILP-F). Methods: Translation and cross-culturally adaptation of the original EILP (EILP-G) was performed according to established guidelines. The translation part was articulated in six stages as recommended by Beaton et al. To validate the EILP-F questionnaire, 84 subjects were recruited (28 pathological patients with a confirmed diagnosis of chronic leg pain, 28 asymptomatic sport students, 28 athletes healthy control). Discriminative power of the questionnaire was tested as well as reliability (internal consistency, test–retest reliability after a 7-10-day interval), construct validity and floor/ceiling effects. Results: the EILP-F version of the questionnaire has been generated without any major difficulties. The ability of the questionnaire to discriminate the three groups of subjects has been showed with a total score of 61.0 ± 18.5 for the pathologic group; 93.9 ± 7.57 for the asymptomatic group and 94.1 ± 9.79 for the control group (p-value adjusted on age = 0.008). A high internal consistency (Cronbach's alpha of 0.93) and an excellent test-retest radiality (ICC of 0.98 (95% CI 0.97-0.99, p<0.001)) indicated that the EILP-F is reliable. The EILP-F also demonstrated a good construct validity against different subscales of the Short Form-36 questionnaire, a generic quality of life questionnaire, with more than 87 % of prespecified hypotheses confirmed. Finally, no floor or ceiling effects were observed. Conclusion: The EILP-F questionnaire is consistent, valid and reliable for evaluating the French-speaking patients with chronic exercise-induced leg pain

French translation and validation of the exercise-induced leg pain Questionnaire

Objective: The “Exercise-Induced Leg Pain” questionnaire was developed (in German) for the evaluation of the severity of symptoms and sports ability in individuals with exercise-induced leg pain. The purpose of the present study was to translate and cross-culturally adapt this questionnaire into French and to study the reliability and validity of this French-language version. Methods: The translation and cross-cultural adaptation of the original “Exercise-Induced Leg Pain” was performed according to established guidelines. The translation part was carried out in six stages: (i) two initial translations from German to French; (ii) synthesis of the two translations; (iii) backward translations; (iv) comparison between the backward translations and the original questionnaire by an expert committee; (v) pretest; and (vi) approval of the final version of the French-language “Exercise-Induced Leg Pain” questionnaire. To validate this questionnaire, 84 subjects were recruited (28 pathological patients with a confirmed diagnosis of chronic leg pain, 28 asymptomatic sport students, and 28 healthy control athletes). The discriminative power of the questionnaire was tested, as well as its reliability (internal consistency and test–retest reliability after a 7–10-day interval), construct validity and floor/ceiling effects. Results: The French version of the “Exercise-Induced Leg Pain” questionnaire was generated without any major difficulties. The ability of the questionnaire to discriminate between the three groups of subjects was demonstrated with a total score of 61.0 ± 18.5 for the pathologic group; 93.9 ± 7.57 for the asymptomatic group and 94.1 ± 9.79 for the control group. A high internal consistency (Cronbach's alpha of 0.93) and an excellent test–retest reliability [intraclass coefficient correlation: 0.98 (95% confidence interval: 0.97–0.99, p < 0.001)] indicated that the “Exercise-Induced Leg Pain” is reliable. The questionnaire also demonstrated good construct validity against different subscales of the Short Form-36 questionnaire, a generic quality of life questionnaire, with more than 87% of the prespecified hypotheses confirmed. Finally, no floor effects or ceiling effects were observed. Conclusion: The French version of the « Exercise-Induced Leg Pain » was successfully translated and cross-culturally adapted. The questionnaire is consistent, valid and reliable for evaluating French-speaking patients with chronic exercise-induced leg pain. Implications for rehabilitation The “Exercise-Induced Leg Pain” questionnaire aims to assess the severity of symptoms that impact the function and sports ability of patients with exercise-induced leg pain; The French version of the « Exercise-Induced Leg Pain » was successfully translated and cross-culturally adapted. The questionnaire is consistent, valid and reliable for evaluating French-speaking patients with chronic exercise-induced leg pain

Repurposing of auranofin and honokiol as antifungals against Scedosporium

peer reviewedThe slowing-down de novo drug-discovery emphasized the importance of repurposing old drugs. This is particularly true when combating infections caused by therapy-refractory microorganisms, such as Scedosporium species and Lomentospora prolificans. Recent studies on Scedosporium responses to oxidative stress underscored the importance of targeting the underlying mechanisms. Auranofin, ebselen, PX-12, honokiol, and to a lesser extent, conoidin A are known to disturb redox-homeostasis systems in many organisms. Their antifungal activity was assessed against 27 isolates belonging to the major Scedosporium species: S. apiospermum, S. aurantiacum, S. boydii, S. dehoogii, S. minutisporum, and Lomentospora prolificans. Auranofin and honokiol were the most active against all Scedosporium species (mean MIC50 values of 2.875 and 6.143 μg/ml, respectively) and against L. prolificans isolates (mean MIC50 values of 4.0 and 3.563μg/ml respectively). Combinations of auranofin with voriconazole or honokiol revealed additive effects against 9/27 and 18/27 isolates, respectively. Synergistic interaction between auranofin and honokiol was only found against one isolate of L. prolificans. The effects of auranofin upon exposure to oxidative stress were also investigated. For all species except S. dehoogii, the maximal growth in the presence of auranofin significantly decreased when adding a sublethal dose of menadione. The analysis of the expression of genes encoding oxidoreductase enzymes upon exposure of S. apiospermum to honokiol unveiled the upregulation of many genes, especially those coding peroxiredoxins, thioredoxin reductases, and glutaredoxins. Altogether, these data suggest that auranofin and honokiol act via dampening the redox balance and support their repurposing as antifungals against Scedosporium species and L. prolificans

Scedosporium boydii CatA1 and SODC recombinant proteins, new tools for serodiagnosis of Scedosporium infection of patients with cystic fibrosis

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Impact of Infection Status and Cyclosporine on Voriconazole Pharmacokinetics in an Experimental Model of Cerebral Scedosporiosis

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