Magali Chabé

The Airway Microbiota in Cystic Fibrosis: A Complex Fungal and Bacterial Community—Implications for Therapeutic Management

Background Given the polymicrobial nature of pulmonary infections in patients with cystic fibrosis (CF), it is essential to enhance our knowledge on the composition of the microbial community to improve patient management. In this study, we developed a pyrosequencing approach to extensively explore the diversity and dynamics of fungal and prokaryotic populations in CF lower airways. Methodology and Principal Findings Fungi and bacteria diversity in eight sputum samples collected from four adult CF patients was investigated using conventional microbiological culturing and high-throughput pyrosequencing approach targeting the ITS2 locus and the 16S rDNA gene. The unveiled microbial community structure was compared to the clinical profile of the CF patients. Pyrosequencing confirmed recently reported bacterial diversity and observed complex fungal communities, in which more than 60% of the species or genera were not detected by cultures. Strikingly, the diversity and species richness of fungal and bacterial communities was significantly lower in patients with decreased lung function and poor clinical status. Values of Chao1 richness estimator were statistically correlated with values of the Shwachman-Kulczycki score, body mass index, forced vital capacity, and forced expiratory volume in 1 s (p = 0.046, 0.047, 0.004, and 0.001, respectively for fungal Chao1 indices, and p = 0.010, 0.047, 0.002, and 0.0003, respectively for bacterial Chao1 values). Phylogenetic analysis showed high molecular diversities at the sub-species level for the main fungal and bacterial taxa identified in the present study. Anaerobes were isolated with Pseudomonas aeruginosa, which was more likely to be observed in association with Candida albicans than with Aspergillus fumigatus. Conclusions In light of the recent concept of CF lung microbiota, we viewed the microbial community as a unique pathogenic entity. We thus interpreted our results to highlight the potential interactions between microorganisms and the role of fungi in the context of improving survival in CF.

Exploring and quantifying fungal diversity in freshwater lake ecosystems using rDNA cloning/sequencing and SSU tag pyrosequencing.

Water samples were collected along transects from the shore to the centre of two French lakes: the deep, volcanic, oligomesotrophic and low allochthonic-impacted Lake Pavin, and the productive and higher allochthonic-impacted Lake Aydat. The biodiversity was analysed using two approaches: the classical approach consisting of cloning/sequencing of the 18S, ITS1, 5.8S, ITS2 and partial 28S region using primers designed for fungus sequences, and the pyrosequencing of 18S rRNA hypervariable V2, V3 and V5 regions using two primer sets (one universal for eukaryotes and one for fungi). The classical approach yielded 146 (Lake Pavin) and 143 (Lake Aydat) sequences, corresponding to 46 and 63 operational taxonomic units (OTUs) respectively. Fungi represented half of the OTUs identified in Lake Pavin and 30% in Lake Aydat, and were dominated by sequences from Chytridiomycota found throughout Lake Pavin but mostly in the central pelagic zone of Lake Aydat. The pyrosequencing approach yielded 42,064 (Pavin) and 61,371 (Aydat) reads, of which 12-15% and 9-19% reads were assigned to fungi in Lakes Pavin and Aydat respectively. Chytridiomycota members were also dominant among these reads, with OTUs displaying up to > 33-fold overrepresentation in the centre compared with the riparian areas of Lake Aydat. Besides fungi, both approaches revealed other major eukaryote groups, with the highest diversity in the central areas of lakes. One of the major findings of our study was that the two lakes displayed contrasting spatial distributions, homogenous for Lake Pavin and heterogeneous for Lake Aydat, which may be related to their peculiarities. This study represents the first unveiling of microbial eukaryote and fungus diversity assessed with two complementary molecular methods, and is considered a major milestone towards understanding the dynamics and ecology of fungi in freshwater lake ecosystems, which are directly link to the abundance and distribution of taxa.

Immunocompetent Hosts as a Reservoir of Pneumocystis Organisms: Histological and RT-PCR Data Demonstrate Active Replication

Abstract The present study was conducted to further examine recent data suggesting that pneumocystosis could be transmitted between patients and healthcare workers in the hospital environment, as has been proven with Pneumocystis-infected SCID mice and immunocompetent Balb/c mice. Using an experimental design (i.e., SCID–Balb/c mouse airborne transmission system), the present work found that healthy host-to-healthy host transmission of Pneumocystis organisms can occur, and that ‘second’ healthy contacts are able to transmit the infectious organisms to immunocompromised hosts. Further tests designed to explore the behavior of Pneumocystis organisms in the lungs of immunocompetent hosts were performed using histological and molecular approaches (e.g. testing the expression of both cyclin-dependent serine-threonine kinase and heat-shock 70 protein in Pneumocystis). The results showed Pneumocystis organisms were able to replicate in the lungs of immunocompetent hosts, which indicates these hosts are a reservoir for Pneumocystis spp.

Microplanktonic Community Structure in a Coastal System Relative to a Phaeocystis Bloom Inferred from Morphological and Tag Pyrosequencing Methods

Background Massive phytoplankton blooms, like the recurrent Phaeocystis proliferation observed every year in the Eastern English Channel (EEC), have a significant influence on the overall planktonic community structure and their food web dynamics. As well as being an important area for local fisheries, the EEC is an ideal ecosystem for work on microbial diversity. This is because, although its environmental context is relatively complex, it is reasonably well understood due to several years of monitoring and morphological observations of its planktonic organisms. The objective of our study was to better understand the under-explored microbial eukaryotic diversity relative to the Phaeocystis bloom. Methodology and Principal Findings The community structure of microplankton (diatoms, haptophytes, ciliates and dinoflagellates) was studied through morphological observations and tag pyrosequencing. During the annual Phaeocystis spring bloom, the phytoplankton biomass increased by 34-fold, while the microzooplankton biomass showed a 4-fold increase, representing on average about 4.6% of the biomass of their phytoplankton prey. Tag pyrosequencing unveiled an extensive diversity of Gymnodiniaceae, with G. spirale and G. fusiformis representing the most abundant reads. An extended diversity of Phaeocystales, with partial 18S rDNA genes sequence identity as low as 85% was found, with taxa corresponding to P. globosa, but also to unknown Phaeocystaceae. Conclusions Morphological analyses and pyrosequencing were generally in accordance with capturing frequency shifts of abundant taxa. Tag pyrosequencing allowed highlighting the maintenance of microplankton diversity during the Phaeocystis bloom and the increase of the taxa presenting low number of reads (minor taxa) along with the dominant ones in response to biotic and/or abiotic changing conditions. Although molecular approaches have enhanced our perception on diversity, it has come to light that the challenge of modelling and predicting ecological change requires the use of different complementary approaches, to link taxonomic data with the functional roles of microbes in biogeochemical cycles.

Molecular Identification of Tritrichomonas foetus-Like Organisms as Coinfecting Agents of Human Pneumocystis Pneumonia

ABSTRACT Trichomonads closely related to the bovid parasite Tritrichomonas foetus were identified in the bronchoalveolar lavage sample from a patient with AIDS in association with Pneumocystis pneumonia. This human case of T. foetus-like infection emphasizes the zoonotic potential of trichomonads, although the existence of a human-host-adapted T. foetus strain cannot be excluded.

Colonization with the enteric protozoa Blastocystis is associated with increased diversity of human gut bacterial microbiota

Alterations in the composition of commensal bacterial populations, a phenomenon known as dysbiosis, are linked to multiple gastrointestinal disorders, such as inflammatory bowel disease and irritable bowel syndrome, or to infections by diverse enteric pathogens. Blastocystis is one of the most common single-celled eukaryotes detected in human faecal samples. However, the clinical significance of this widespread colonization remains unclear, and its pathogenic potential is controversial. To address the issue of Blastocystis pathogenicity, we investigated the impact of colonization by this protist on the composition of the human gut microbiota. For that purpose, we conducted a cross-sectional study including 48 Blastocystis-colonized patients and 48 Blastocystis-free subjects and performed an Ion Torrent 16S rDNA gene sequencing to decipher the Blastocystis-associated gut microbiota. Here, we report a higher bacterial diversity in faecal microbiota of Blastocystis colonized patients, a higher abundance of Clostridia as well as a lower abundance of Enterobacteriaceae. Our results contribute to suggesting that Blastocystis colonization is usually associated with a healthy gut microbiota, rather than with gut dysbiosis generally observed in metabolic or infectious inflammatory diseases of the lower gastrointestinal tract.

Long-Term Colonization with Pneumocystis jirovecii in Hospital Staffs: A Challenge to Prevent Nosocomial Pneumocystosis

In a recent work we explored the ability of healthy hosts to contract Pneumocystis transient infections from hosts with Pneumocystis pneumonia (PcP) for transmitting it to immunocompromised hosts [3]. We showed that Balb/c mice transiently infected by contact with Pneumocystis-infected SCID mice were able to transmit Pneumocystis to other SCID mice, who developed PcP. Using the same model, another team has recently confirmed these results [2,5]. Since Pneumocystis DNA has been detected in health care workers in contact with PcP patients [6,10], this finding is relevant for at least two reasons. Firstly, these health care workers, like the Balb/c mouse carriers [2,3,5], may play a role in the circulation of P. jirovecii in the hospital environment. Secondly, health care worker carriers could play a role as reservoirs that contributed to the survival of Pneumocystis within human populations. In the present work we explored the impact of P. jirovecii carriage among the staff members of our teaching hospital. We examined the prevalence of Pneumocystis carriage, evaluated the duration of carriage as well as the potential influence of barrier systems. The methodology described herein provides a way for approaching the question of the source of Pneumocystis infection in hospitals.

Pneumocystis: from a doubtful unique entity to a group of highly diversified fungal species

At the end of the 20th century the unique taxonomically enigmatic entity called Pneumocystis carinii was identified as a heterogeneous group of microscopic Fungi, constituted of multiple stenoxenic biological entities largely spread across ecosystems, closely adapted to, and coevolving in parallel with, mammal species. The discoveries and reasoning that led to the current conceptions about the taxonomy of Pneumocystis at the species level are examined here. The present review also focuses on the biological, morphological and phylogenetical features of Pneumocystis jirovecii, Pneumocystis oryctolagi, Pneumocystis murina, P. carinii and Pneumocystis wakefieldiae, the five Pneumocystis species described until now, mainly on the basis of the phylogenetic species concept. Interestingly, Pneumocystis organisms exhibit a successful adaptation enabling them to dwell and replicate in the lungs of both immunocompromised and healthy mammals, which can act as infection reservoirs. The role of healthy carriers in aerial disease transmission is nowadays recognized as a major contribution to Pneumocystis circulation, and Pneumocystis infection of nonimmunosuppressed hosts has emerged as a public health issue. More studies need to be undertaken both on the clinical consequences of the presence of Pneumocystis in healthy carriers and on the intricate Pneumocystis life cycle to better define its epidemiology, to adapt existing therapies to each clinical context and to discover new drug targets.

Near-Universal Prevalence of Pneumocystis and Associated Increase in Mucus in the Lungs of Infants With Sudden Unexpected Death

We demonstrate that Pneumocystis reaches a >90% prevalence peak at 3–5 months of age and associates with increased mucus (MUC5AC), suggesting airway epithelium stimulation in infants during this age range. Host ability to clear mucus would determine pathogenic expression.

Growth and Airborne Transmission of Cell-Sorted Life Cycle Stages of Pneumocystis carinii

Pneumocystis organisms are airborne opportunistic pathogens that cannot be continuously grown in culture. Consequently, the follow-up of Pneumocystis stage-to-stage differentiation, the sequence of their multiplication processes as well as formal identification of the transmitted form have remained elusive. The successful high-speed cell sorting of trophic and cystic forms is paving the way for the elucidation of the complex Pneumocystis life cycle. The growth of each sorted Pneumocystis stage population was followed up independently both in nude rats and in vitro. In addition, by setting up a novel nude rat model, we attempted to delineate which cystic and/or trophic forms can be naturally aerially transmitted from host to host. The results showed that in axenic culture, cystic forms can differentiate into trophic forms, whereas trophic forms are unable to evolve into cystic forms. In contrast, nude rats inoculated with pure trophic forms are able to produce cystic forms and vice versa. Transmission experiments indicated that 12 h of contact between seeder and recipient nude rats was sufficient for cystic forms to be aerially transmitted. In conclusion, trophic- to cystic-form transition is a key step in the proliferation of Pneumocystis microfungi because the cystic forms (but not the trophic forms) can be transmitted by aerial route from host to host.