Epidemiology of airway colonization by Scedosporium apiospermum during cystic fibrosis

With a frequency of about 10%, species of the Scedosporium apiospermum complex (which comprises at least five distinct species with different antifungal susceptibility patterns) rank the second among the filamentous fungi colonizing the airways in cystic fibrosis (CF). Additionally, it is clearly established that these fungi may disseminate in case of immunodeficiency and that a chronic colonization of the airways by these pathogens may hinder the success of lung transplantation. In this study, we develop a new genotyping method to investigate the epidemiology of the airway colonization by these fungi. 63 multiple and sequential isolates of S. apiospermum collected from 9 CF patients, and selected among those previously studied by random amplification of polymorphic DNA (RAPD), were analyzed using the automated typing system DiversiLab (bioMérieux) based on PCR amplification of repetitive sequences. The DiversiLab Aspergillus rep-PCR kit which uses specific primers designed for Aspergillus fumigatus, was compared with the pan-fungus DiversiLab Fungal kit. Amplification products were separated by capillary electrophoresis on Agilent B2100 bioanalyzer, leading to single profiles for each isolate which were then compared using the DiversiLab software. In addition, species identification of these isolates was clarified by sequencing the betatubulin gene. Results obtained with both kits were comparable. Nevertheless, differentiation was easier using the DiversiLab Fungal kit. Additionally, rep-PCR usually confirmed the colonization patterns described by RAPD. Only two patients showed distinct genotypes. For Patient 2, two isolates were analyzed which were undistinguishable by RAPD, but rep-PCR revealed that they belonged to distinct genotypes, suggesting a transient colonization. For Patient 8 which showed by RAPD two distinct genotypes, 5 genotypes were found by rep-PCR with a dominant one represented by 5 isolates and two very close genotypes (corresponding to 3 isolates), while 4 other isolates belonged to two distant genotypes. In conclusion, the automated typing system DiversiLab proved to be an easy and efficient method to investigate the molecular epidemiology of the airway colonization by S. apiospermum in CF. Our results also confirm the capacity of the different species from the S. apiospermum complex to chronically colonize the airways of CF patients

Clinical and microbiological efficacy of micafungin on Geosmithia argillacea infection in a cystic fibrosis patient

Cystic fibrosis are at risk of colonization by a number of fungi, including Geosmithiaargillacea which appears to be an emerging pathogen in these patients. This pathogen has been recently reported as a cause of invasive/systemic mycosis in immunocompromized patients such as colonized patients who are immunosuppressed for lung transplantation. In this context, we report here a case of clinical and microbiological efficacy of micafungin in a French cystic fibrosis patient chronically colonized with G. argillacea. O.D., a female F508Del-CFTR homozygous patient was diagnosed at birth with cystic fibrosis in January 1996. She was found chronically colonised with multi-resistant Staphylococcus aureus (MRSA) from 1997 to 2011, and with Aspergillus fumigatus from 2001 to 2006. She was treated alternatively with oral voriconazole and itraconazole from 2004 to 2008, and with posaconazole since february 2008. Geosmithia argillacea was first diagnosed in May 2007, and chronic colonisation was persistent from this date to August 2010 with 23/28 fungus positive sputum samples, in spite of posaconazole therapy. For an isolate obtained in October 2008, minimal inhibitory/effective concentrations (MIC/MEC, mg/ml) determined using the Eucast method were 2.0, 2.0, 16.0, 2,0, 0.25 and 0.015 for amphotericin B, itraconazole, voriconazole, posaconazole, caspofungin and micafungin, respectively. The FEV1 predicted value was 73% at the time of first fungus isolation and was decreased to 47% in October 2009. She then was given caspofungin for 21 days ((70 mg/day, later reduced to 50 mg) which resulted in clinical improvement (FEV1 = 64% in January 2010) without eradication of G. argillacea. In June 2010, treatment with micafungin (75 mg, 21days) was realized owing to deterioration of the respiratory function (FEV1 = 56%),without clinical improvement ( FEV1 = 47% in August 2010). O.D. was then treated from September, 23 to November 3, 2010 with micafungin (100mg bid for 21 days and 100mg/day for the following 21 days) which resulted in clinical and microbiological improvement. FEV1 predicted ranged 67-68% in October and December 2010, and February and May 2011, and from the end of treatment to December 2010, 5/6 sputum samples were found negative for G. argillacea. The positive sample contained fungus of the same genotype as previous isolates. The present case is to our knowledge the first description of G. argillacea eradication in a chronically colonized cystic fibrosis patient. Similar to previous studies, G. argillacea colonization was detected in the presence of chronic MRSA after A. fumigatus eradication. Since no change in bacterial colonization was observed before, during, and after G. argillacea colonization, the present case is consistent with a pathogenic role of the fungus in cystic fibrosis patients. In vitro antifungal susceptibility assays suggested that echinocandins are most effective agents against this fungus with a lowest MEC for micafungin (7 isolates studied, MEC range: 0.015-0.03), although eradication could only be obtained with high dose micafungin for a long time (6 weeks)

Unexpected Role for Helicobacter pylori DNA Polymerase I As a Source of Genetic Variability

Helicobacter pylori, a human pathogen infecting about half of the world population, is characterised by its large intraspecies variability. Its genome plasticity has been invoked as the basis for its high adaptation capacity. Consistent with its small genome, H. pylori possesses only two bona fide DNA polymerases, Pol I and the replicative Pol III, lacking homologues of translesion synthesis DNA polymerases. Bacterial DNA polymerases I are implicated both in normal DNA replication and in DNA repair. We report that H. pylori DNA Pol I 5′- 3′ exonuclease domain is essential for viability, probably through its involvement in DNA replication. We show here that, despite the fact that it also plays crucial roles in DNA repair, Pol I contributes to genomic instability. Indeed, strains defective in the DNA polymerase activity of the protein, although sensitive to genotoxic agents, display reduced mutation frequencies. Conversely, overexpression of Pol I leads to a hypermutator phenotype. Although the purified protein displays an intrinsic fidelity during replication of undamaged DNA, it lacks a proofreading activity, allowing it to efficiently elongate mismatched primers and perform mutagenic translesion synthesis. In agreement with this finding, we show that the spontaneous mutator phenotype of a strain deficient in the removal of oxidised pyrimidines from the genome is in part dependent on the presence of an active DNA Pol I. This study provides evidence for an unexpected role of DNA polymerase I in generating genomic plasticity

Chlorine transport processes through a 2000 m aquifer/aquitard system

International audienceIn the Paris Basin, in France, the Callovo-Oxfordian (COx) is currently studied over a 250 km2 surface area by the French national radioactive waste management agency in order to assess the feasibility of long-term underground nuclear waste repository. The COx is a 140 m thick clay-rich layer, which is part of the 2000 m aquitard/aquifer system constituting the sedimentary cover. In such sedimentary context, the transport processes of potential contaminants can be represented by both vertical diffusion and horizontal advection through the most permeable layers. Chloride is used as a natural conservative tracer, and is monitored in term of concentrations and isotopic composition (δ37Cl) for both pore and groundwater. During this study, the samples were collected from three boreholes located in the center of the studied zone, one of them (EST433) going down to 2000 m depth. The main solute transport process is shown to be vertical diffusion from the massive Keuper halite level to the rest of the sedimentary pile. This global diffusive system can be occasionally disturbed by horizontal circulation of groundwater occurring in the Oxfordian and Dogger limestone formations. Therefore, these circulations cut the global diffusive system in a succession of independent diffusive systems. In this study the data set was implemented in a simplified 2D solute transport model and scenarii reproducing known history in term of paleo circulations inside the system, were applied and allowed to obtained a good fit of the data. Model results showed that paleo circulations, occurring between −145 Ma and −110 Ma, still have an impact on current distribution of chloride in the system, especially for δ37Cl. The model highlights the need of the presence of a circulation spatially limited at the base of the Liassic formation to fit the data. The best fit obtained indicated current residence time of 500 ka in the Dogger and Oxfordian, with respective onset of the circulations at −20 Ma and −5 Ma