Weihong Qi

Nasopharyngeal Microbiota in Infants With Acute Otitis Media

Abstract Background. Interspecies interactions of the nasopharyngeal microbiota are likely to be involved in the pathogenesis of acute otitis media (AOM). Capturing the breadth of microbial interactions requires a detailed description of the microbiota during health and AOM. Methods. The nasopharyngeal microbiota of 163 infants with (n = 153) or without (n = 10) AOM was characterized using nasopharyngeal swabs and multiplexed pyrosequencing of 16S rRNA. Nasopharyngeal swab specimens were collected during 4 winter seasons from 2004 through 2010 for infants with AOM and during 2010 for controls. Results. Fifty-eight bacterial families were identified, of which Moraxellaceae, Streptococcaceae, and Pasteurellaceae were the most frequent. Commensal families were less prevalent in infants with AOM than in controls. In infants with AOM, prior exposure to antimicrobials and administration of the heptavalent conjugated pneumococcal polysaccharide vaccine (PCV7) were also associated with reduced prevalence of distinct commensal families (Streptococcaceae and Corynebacteriaceae). In addition, antimicrobial exposure increased the prevalence of Enterobacteriaceae and the abundance of Pasteurellaceae. Other factors, such as age, sex, day care, and a history of recurrent AOM, did not influence the microbiota. Conclusions. Infants’ nasopharyngeal microbiota undergoes significant changes during AOM and after exposure to antimicrobials and PCV7, which is mainly attributable to reduced prevalence of commensal bacterial families.

Microbial communities in the respiratory tract of patients with interstitial lung disease

Background Molecular methods based on phylogenetic differences in the 16S rRNA gene are able to characterise the microbiota of the respiratory tract in health and disease. Objectives Our goals were (1) to characterise bacterial communities in lower and upper airways of patients with interstitial lung disease (ILD) and (2) to compare the results with the microbiota of patients with Pneumocystis pneumonia (PCP) and normal controls. Methods We examined the upper and lower respiratory tract of 18 patients with ILD of whom 5, 6, and 7 had idiopathic interstitial pneumonia (IIP), non-IIP and sarcoidosis, respectively. In addition, six immune-compromised patients with PCP and nine healthy subjects were included as controls. Exclusion criteria were recent bacterial/viral respiratory tract infection, HIV-positivity and subjects receiving antibiotic therapy. Bronchoalveolar lavage fluid and oropharyngeal swabs were simultaneously collected, and microbiota was characterised by ultra-deep 16S rRNA gene sequencing. Results The microbiota in lower airways of the majority of patients (30; 90%) primarily consisted of Prevotellaceae, Streptococcaceae and Acidaminococcaceae. α and β diversity measurements revealed no significant differences in airway microbiota composition between the five different groups of patients. Comparison of bacterial populations in upper and lower respiratory tract showed significant topographical discontinuities for 7 (23%) individuals. Conclusions IIP, non-IIP and sarcoidosis are not associated with disordered airway microbiota and a pathogenic role of commensals in the disease process is therefore unlikely. Nevertheless, molecular analysis of the topographical microbiota continuity along the respiratory tract may provide additional information to assist management of individual patients.

The nasal microbiota in infants with cystic fibrosis in the first year of life: a prospective cohort study

BACKGROUND Respiratory tract infections and subsequent airway inflammation occur early in the life of infants with cystic fibrosis. However, detailed information about the microbial composition of the respiratory tract in infants with this disorder is scarce. We aimed to undertake longitudinal in-depth characterisation of the upper respiratory tract microbiota in infants with cystic fibrosis during the first year of life. METHODS We did this prospective cohort study at seven cystic fibrosis centres in Switzerland. Between Feb 1, 2011, and May 31, 2014, we enrolled 30 infants with a diagnosis of cystic fibrosis. Microbiota characterisation was done with 16S rRNA gene pyrosequencing and oligotyping of nasal swabs collected every 2 weeks from the infants with cystic fibrosis. We compared these data with data for an age-matched cohort of 47 healthy infants. We additionally investigated the effect of antibiotic treatment on the microbiota of infants with cystic fibrosis. Statistical methods included regression analyses with a multivariable multilevel linear model with random effects to correct for clustering on the individual level. FINDINGS We analysed 461 nasal swabs taken from the infants with cystic fibrosis; the cohort of healthy infants comprised 872 samples. The microbiota of infants with cystic fibrosis differed compositionally from that of healthy infants (p=0·001). This difference was also found in exclusively antibiotic-naive samples (p=0·001). The disordering was mainly, but not solely, due to an overall increase in the mean relative abundance of Staphylococcaceae in infants with cystic fibrosis compared with healthy infants (multivariable linear regression model stratified by age and adjusted for season; second month: coefficient 16·2 [95% CI 0·6-31·9]; p=0·04; third month: 17·9 [3·3-32·5]; p=0·02; fourth month: 21·1 [7·8-34·3]; p=0·002). Oligotyping analysis enabled differentiation between Staphylococcus aureus and coagulase-negative Staphylococci. Whereas the analysis showed a decrease in S aureus at and after antibiotic treatment, coagulase-negative Staphylococci increased. INTERPRETATION Our study describes compositional differences in the microbiota of infants with cystic fibrosis compared with healthy controls, and disordering of the microbiota on antibiotic administration. Besides S aureus, coagulase-negative Staphylococci also contributed to the disordering identified in these infants. These findings are clinically important in view of the crucial role that bacterial pathogens have in the disease progression of cystic fibrosis in early life. Our findings could be used to inform future studies of the effect of antibiotic treatment on the microbiota in infants with cystic fibrosis, and could assist in the prevention of early disease progression in infants with this disorder. FUNDING Swiss National Science Foundation, Fondation Botnar, the Swiss Society for Cystic Fibrosis, and the Swiss Lung Association Bern.

Environmental marine pathogen isolation using mesocosm culture of sharpsnout seabream: striking genomic and morphological features of novel Endozoicomonas sp.

Aquaculture is a burgeoning industry, requiring diversification into new farmed species, which are often at risk from infectious disease. We used a mesocosm technique to investigate the susceptibility of sharpsnout seabream (Diplodus puntazzo) larvae to potential environmental pathogens in seawater compared to control borehole water. Fish exposed to seawater succumbed to epitheliocystis from 21 days post hatching, causing mortality in a quarter of the hosts. The pathogen responsible was not chlamydial, as is often found in epitheliocystis, but a novel species of the γ-proteobacterial genus Endozoicomonas. Detailed characterisation of this pathogen within the infectious lesions using high resolution fluorescent and electron microscopy showed densely packed rod shaped bacteria. A draft genome sequence of this uncultured bacterium was obtained from preserved material. Comparison with the genome of the Endozoicomonas elysicola type strain shows that the genome of Ca. Endozoicomonas cretensis is undergoing decay through loss of functional genes and insertion sequence expansion, often indicative of adaptation to a new niche or restriction to an alternative lifestyle. These results demonstrate the advantage of mesocosm studies for investigating the effect of environmental bacteria on susceptible hosts and provide an important insight into the genome dynamics of a novel fish pathogen.

Emerging pathogens of gilthead seabream: characterisation and genomic analysis of novel intracellular β-proteobacteria

New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel β-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

SUSHI: an exquisite recipe for fully documented, reproducible and reusable NGS data analysis

BackgroundNext generation sequencing (NGS) produces massive datasets consisting of billions of reads and up to thousands of samples. Subsequent bioinformatic analysis is typically done with the help of open source tools, where each application performs a single step towards the final result. This situation leaves the bioinformaticians with the tasks to combine the tools, manage the data files and meta-information, document the analysis, and ensure reproducibility.ResultsWe present SUSHI, an agile data analysis framework that relieves bioinformaticians from the administrative challenges of their data analysis. SUSHI lets users build reproducible data analysis workflows from individual applications and manages the input data, the parameters, meta-information with user-driven semantics, and the job scripts. As distinguishing features, SUSHI provides an expert command line interface as well as a convenient web interface to run bioinformatics tools. SUSHI datasets are self-contained and self-documented on the file system. This makes them fully reproducible and ready to be shared. With the associated meta-information being formatted as plain text tables, the datasets can be readily further analyzed and interpreted outside SUSHI.ConclusionSUSHI provides an exquisite recipe for analysing NGS data. By following the SUSHI recipe, SUSHI makes data analysis straightforward and takes care of documentation and administration tasks. Thus, the user can fully dedicate his time to the analysis itself. SUSHI is suitable for use by bioinformaticians as well as life science researchers. It is targeted for, but by no means constrained to, NGS data analysis. Our SUSHI instance is in productive use and has served as data analysis interface for more than 1000 data analysis projects. SUSHI source code as well as a demo server are freely available.

An ER‐directed transcriptional response to unfolded protein stress in the absence of conserved sensor‐transducer proteins in Giardia lamblia

The protozoan Giardia lamblia has a minimized organelle repertoire, and most strikingly lacks a classical stacked Golgi apparatus. Nevertheless, Giardia trophozoites constitutively secrete variant surface proteins, and dramatically increase the volume of protein secretion during differentiation to cysts. Eukaryotic cells have evolved an elaborate system for quality control (QC) of protein folding and capacity in the endoplasmic reticulum (ER). Upon ER‐overload, an unfolded protein response (UPR) is triggered on transcriptional/translational level aiming at alleviating ER stress. In Giardia, a minimized secretory machinery and absence of glycan‐dependent QC suggests that a genetically conserved UPR (or functional equivalent) to cope with insults to the secretory system has been eliminated. We tested this hypothesis of UPR elimination by profiling the transcriptional response during induced ER‐folding stress. We show that on the contrary, ER‐folding stress triggers a stressor‐specific, ER‐directed response with upregulation of only ∼ 30 genes, with different kinetics and scope compared with the UPR of other eukaryotes. Computational genomics revealed conserved cis‐acting motifs in upstream regions of responder genes capable of stressor‐specific gene regulation in transfected cells. Interestingly, the sensors/transducers of folding stress, well conserved in model eukaryotes, are absent in Giardia suggesting the presence of a novel version of this essential eukaryotic function.

Culture-independent genomics of a novel chlamydial pathogen of fish provides new insight into host-specific adaptations utilized by these intracellular bacteria

Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae. Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange-spotted grouper (Epinephelus coioides) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper (Epinephelus lanceolatus) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca. Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae, including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca. S. epinephelii provides new opportunities to study the biology of distantly-related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae.

Host-associated genomic features of the novel uncultured intracellular pathogen Ca. Ichthyocystis revealed by direct sequencing of epitheliocysts

Advances in single-cell and mini-metagenome sequencing have enabled important investigations into uncultured bacteria. In this study, we applied the mini-metagenome sequencing method to assemble genome drafts of the uncultured causative agents of epitheliocystis, an emerging infectious disease in the Mediterranean aquaculture species gilthead seabream. We sequenced multiple cyst samples and constructed 11 genome drafts from a novel beta-proteobacterial lineage, Candidatus Ichthyocystis. The draft genomes demonstrate features typical of pathogenic bacteria with an obligate intracellular lifestyle: a reduced genome of up to 2.6 Mb, reduced G + C content, and reduced metabolic capacity. Reconstruction of metabolic pathways reveals that Ca. Ichthyocystis genomes lack all amino acid synthesis pathways, compelling them to scavenge from the fish host. All genomes encode type II, III, and IV secretion systems, a large repertoire of predicted effectors, and a type IV pilus. These are all considered to be virulence factors, required for adherence, invasion, and host manipulation. However, no evidence of lipopolysaccharide synthesis could be found. Beyond the core functions shared within the genus, alignments showed distinction into different species, characterized by alternative large gene families. These comprise up to a third of each genome, appear to have arisen through duplication and diversification, encode many effector proteins, and are seemingly critical for virulence. Thus, Ca. Ichthyocystis represents a novel obligatory intracellular pathogenic beta-proteobacterial lineage. The methods used: mini-metagenome analysis and manual annotation, have generated important insights into the lifestyle and evolution of the novel, uncultured pathogens, elucidating many putative virulence factors including an unprecedented array of novel gene families.

The Chlamydia suis Genome Exhibits High Levels of Diversity, Plasticity, and Mobile Antibiotic Resistance: Comparative Genomics of a Recent Livestock Cohort Shows Influence of Treatment Regimes

Chlamydia suis is an endemic pig pathogen, belonging to a fascinating genus of obligate intracellular pathogens. Of particular interest, this is the only chlamydial species to have naturally acquired genes encoding for tetracycline resistance. To date, the distribution and mobility of the Tet-island are not well understood. Our study focused on whole genome sequencing of 29 C. suis isolates from a recent porcine cohort within Switzerland, combined with data from USA tetracycline-resistant isolates. Our findings show that the genome of C. suis is very plastic, with unprecedented diversity, highly affected by recombination and plasmid exchange. A large diversity of isolates circulates within Europe, even within individual Swiss farms, suggesting that C. suis originated around Europe. New World isolates have more restricted diversity and appear to derive from European isolates, indicating that historical strain transfers to the United States have occurred. The architecture of the Tet-island is variable, but the tetA(C) gene is always intact, and recombination has been a major factor in its transmission within C. suis. Selective pressure from tetracycline use within pigs leads to a higher number of Tet-island carrying isolates, which appear to be lost in the absence of such pressure, whereas the loss or gain of the Tet-island from individual strains is not observed. The Tet-island appears to be a recent import into the genome of C. suis, with a possible American origin.