Elliott F. Drabek

Analysis of the Gut Microbiota in the Old Order Amish and Its Relation to the Metabolic Syndrome

Obesity has been linked to the human gut microbiota; however, the contribution of gut bacterial species to the obese phenotype remains controversial because of conflicting results from studies in different populations. To explore the possible dysbiosis of gut microbiota in obesity and its metabolic complications, we studied men and women over a range of body mass indices from the Old Order Amish sect, a culturally homogeneous Caucasian population of Central European ancestry. We characterized the gut microbiota in 310 subjects by deep pyrosequencing of bar-coded PCR amplicons from the V1–V3 region of the 16S rRNA gene. Three communities of interacting bacteria were identified in the gut microbiota, analogous to previously identified gut enterotypes. Neither BMI nor any metabolic syndrome trait was associated with a particular gut community. Network analysis identified twenty-two bacterial species and four OTUs that were either positively or inversely correlated with metabolic syndrome traits, suggesting that certain members of the gut microbiota may play a role in these metabolic derangements.

Simultaneous Transcriptional Profiling of Bacteria and Their Host Cells

We developed an RNA-Seq-based method to simultaneously capture prokaryotic and eukaryotic expression profiles of cells infected with intracellular bacteria. As proof of principle, this method was applied to Chlamydia trachomatis-infected epithelial cell monolayers in vitro, successfully obtaining transcriptomes of both C. trachomatis and the host cells at 1 and 24 hours post-infection. Chlamydiae are obligate intracellular bacterial pathogens that cause a range of mammalian diseases. In humans chlamydiae are responsible for the most common sexually transmitted bacterial infections and trachoma (infectious blindness). Disease arises by adverse host inflammatory reactions that induce tissue damage & scarring. However, little is known about the mechanisms underlying these outcomes. Chlamydia are genetically intractable as replication outside of the host cell is not yet possible and there are no practical tools for routine genetic manipulation, making genome-scale approaches critical. The early timeframe of infection is poorly understood and the host transcriptional response to chlamydial infection is not well defined. Our simultaneous RNA-Seq method was applied to a simplified in vitro model of chlamydial infection. We discovered a possible chlamydial strategy for early iron acquisition, putative immune dampening effects of chlamydial infection on the host cell, and present a hypothesis for Chlamydia-induced fibrotic scarring through runaway positive feedback loops. In general, simultaneous RNA-Seq helps to reveal the complex interplay between invading bacterial pathogens and their host mammalian cells and is immediately applicable to any bacteria/host cell interaction.

Functional Dynamics of the Gut Microbiome in Elderly People during Probiotic Consumption

ABSTRACT A mechanistic understanding of the purported health benefits conferred by consumption of probiotic bacteria has been limited by our knowledge of the resident gut microbiota and its interaction with the host. Here, we detail the impact of a single-organism probiotic, Lactobacillus rhamnosus GG ATCC 53103 (LGG), on the structure and functional dynamics (gene expression) of the gut microbiota in a study of 12 healthy individuals, 65 to 80 years old. The analysis revealed that while the overall community composition was stable as assessed by 16S rRNA profiling, the transcriptional response of the gut microbiota was modulated by probiotic treatment. Comparison of transcriptional profiles based on taxonomic composition yielded three distinct transcriptome groups that displayed considerable differences in functional dynamics. The transcriptional profile of LGG in vivo was remarkably concordant across study subjects despite the considerable interindividual nature of the gut microbiota. However, we identified genes involved in flagellar motility, chemotaxis, and adhesion from Bifidobacterium and the dominant butyrate producers Roseburia and Eubacterium whose expression was increased during probiotic consumption, suggesting that LGG may promote interactions between key constituents of the microbiota and the host epithelium. These results provide evidence for the discrete functional effects imparted by a specific single-organism probiotic and challenge the prevailing notion that probiotics substantially modify the resident microbiota within nondiseased individuals in an appreciable fashion. IMPORTANCE Probiotic bacteria have been used for over a century to promote digestive health. Many individuals report that probiotics alleviate a number of digestive issues, yet little evidence links how probiotic microbes influence human health. Here, we show how the resident microbes that inhabit the healthy human gut respond to a probiotic. The well-studied probiotic Lactobacillus rhamnosus GG ATCC 53103 (LGG) was administered in a clinical trial, and a suite of measurements of the resident microbes were taken to evaluate potential changes over the course of probiotic consumption. We found that LGG transiently enriches for functions to potentially promote anti-inflammatory pathways in the resident microbes. Probiotic bacteria have been used for over a century to promote digestive health. Many individuals report that probiotics alleviate a number of digestive issues, yet little evidence links how probiotic microbes influence human health. Here, we show how the resident microbes that inhabit the healthy human gut respond to a probiotic. The well-studied probiotic Lactobacillus rhamnosus GG ATCC 53103 (LGG) was administered in a clinical trial, and a suite of measurements of the resident microbes were taken to evaluate potential changes over the course of probiotic consumption. We found that LGG transiently enriches for functions to potentially promote anti-inflammatory pathways in the resident microbes.

Inter- and intra-specific pan-genomes of Borrelia burgdorferi sensu lato: genome stability and adaptive radiation

BackgroundLyme disease is caused by spirochete bacteria from the Borrelia burgdorferi sensu lato (B. burgdorferi s.l.) species complex. To reconstruct the evolution of B. burgdorferi s.l. and identify the genomic basis of its human virulence, we compared the genomes of 23 B. burgdorferi s.l. isolates from Europe and the United States, including B. burgdorferi sensu stricto (B. burgdorferi s.s., 14 isolates), B. afzelii (2), B. garinii (2), B. “bavariensis” (1), B. spielmanii (1), B. valaisiana (1), B. bissettii (1), and B. “finlandensis” (1).ResultsRobust B. burgdorferi s.s. and B. burgdorferi s.l. phylogenies were obtained using genome-wide single-nucleotide polymorphisms, despite recombination. Phylogeny-based pan-genome analysis showed that the rate of gene acquisition was higher between species than within species, suggesting adaptive speciation. Strong positive natural selection drives the sequence evolution of lipoproteins, including chromosomally-encoded genes 0102 and 0404, cp26-encoded ospC and b08, and lp54-encoded dbpA, a07, a22, a33, a53, a65. Computer simulations predicted rapid adaptive radiation of genomic groups as population size increases.ConclusionsIntra- and inter-specific pan-genome sizes of B. burgdorferi s.l. expand linearly with phylogenetic diversity. Yet gene-acquisition rates in B. burgdorferi s.l. are among the lowest in bacterial pathogens, resulting in high genome stability and few lineage-specific genes. Genome adaptation of B. burgdorferi s.l. is driven predominantly by copy-number and sequence variations of lipoprotein genes. New genomic groups are likely to emerge if the current trend of B. burgdorferi s.l. population expansion continues.

Sparse distance-based learning for simultaneous multiclass classification and feature selection of metagenomic data

MOTIVATION Direct sequencing of microbes in human ecosystems (the human microbiome) has complemented single genome cultivation and sequencing to understand and explore the impact of commensal microbes on human health. As sequencing technologies improve and costs decline, the sophistication of data has outgrown available computational methods. While several existing machine learning methods have been adapted for analyzing microbiome data recently, there is not yet an efficient and dedicated algorithm available for multiclass classification of human microbiota. RESULTS By combining instance-based and model-based learning, we propose a novel sparse distance-based learning method for simultaneous class prediction and feature (variable or taxa, which is used interchangeably) selection from multiple treatment populations on the basis of 16S rRNA sequence count data. Our proposed method simultaneously minimizes the intraclass distance and maximizes the interclass distance with many fewer estimated parameters than other methods. It is very efficient for problems with small sample sizes and unbalanced classes, which are common in metagenomic studies. We implemented this method in a MATLAB toolbox called MetaDistance. We also propose several approaches for data normalization and variance stabilization transformation in MetaDistance. We validate this method on several real and simulated 16S rRNA datasets to show that it outperforms existing methods for classifying metagenomic data. This article is the first to address simultaneous multifeature selection and class prediction with metagenomic count data. AVAILABILITY The MATLAB toolbox is freely available online at http://metadistance.igs.umaryland.edu/. CONTACT zliu@umm.edu SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Impact of Oral Typhoid Vaccination on the Human Gut Microbiota and Correlations with S. Typhi-Specific Immunological Responses

The resident microbial consortia of the human gastrointestinal tract play an integral role in modulating immune responses both locally and systemically. However, detailed information regarding the effector immune responses after vaccine administration in relation to the gastrointestinal microbiota is absent. In this study, the licensed oral live-attenuated typhoid vaccine Ty21a was administered in a clinical study to investigate whether oral immunization resulted in alterations of the microbiota and to identify whether a given microbiota composition, or subsets of the community, are associated with defined S. Typhi-specific immunological responses. The fecal microbiota composition and temporal dynamics were characterized using bacterial 16S rRNA pyrosequencing from individuals who were either immunized with the Ty21a typhoid vaccine (n = 13) or served as unvaccinated controls (n = 4). The analysis revealed considerable inter- and intra-individual variability, yet no discernible perturbations of the bacterial assemblage related to vaccine administration were observed. S. Typhi-specific cell mediated immune (CMI) responses were evaluated by measurement of intracellular cytokine production using multiparametric flow cytometry, and humoral responses were evaluated by measurement of serum anti-LPS IgA and IgG titers. Volunteers were categorized according to the kinetics and magnitude of their responses. While differences in microbial composition, diversity, or temporal stability were not observed among individuals able to mount a positive humoral response, individuals displaying multiphasic CMI responses harbored more diverse, complex communities. In line with this preliminary observation, over two hundred operational taxonomic units (OTUs) were found to differentiate multiphasic and late CMI responders, the vast majority of which classified within the order Clostridiales. These results provide an unprecedented view into the dramatic temporal heterogeneity of both the gut microbiota and host immune responses.

Genome Sequences of the Zoonotic Pathogens Chlamydia psittaci 6BC and Cal10

Chlamydia psittaci is a highly prevalent avian pathogen and the cause of a potentially lethal zoonosis, causing life-threatening pneumonia in humans. We report the genome sequences of C. psittaci 6BC, the prototype strain of the species, and C. psittaci Cal10, a widely used laboratory strain.

Transcriptional Attenuation Controls Macrolide Inducible Efflux and Resistance in Streptococcus pneumoniae and in Other Gram-Positive Bacteria Containing mef/mel(msr(D)) Elements

Macrolide resistance, emerging in Streptococcus pneumoniae and other Gram-positive bacteria, is increasingly due to efflux pumps encoded by mef/mel(msr) operons found on discrete mobile genetic elements. The regulation of mef/mel(msr) in these elements is not well understood. We identified the mef(E)/mel transcriptional start, localized the mef(E)/mel promoter, and demonstrated attenuation of transcription as a mechanism of regulation of macrolide-inducible mef-mediated macrolide resistance in S. pneumoniae. The mef(E)/mel transcriptional start site was a guanine 327 bp upstream of mef(E). Consensus pneumococcal promoter -10 (5′-TATACT-3′) and -35 (5′-TTGAAC-3′) boxes separated by 17 bp were identified 7 bp upstream of the start site. Analysis of the predicted secondary structure of the 327 5’ region identified four pairs of inverted repeats R1-R8 predicted to fold into stem-loops, a small leader peptide [MTASMRLR, (Mef(E)L)] required for macrolide induction and a Rho-independent transcription terminator. RNA-seq analyses provided confirmation of transcriptional attenuation. In addition, expression of mef(E)L was also influenced by mef(E)L-dependent mRNA stability. The regulatory region 5’ of mef(E) was highly conserved in other mef/mel(msr)-containing elements including Tn1207.1 and the 5612IQ complex in pneumococci and Tn1207.3 in Group A streptococci, indicating a regulatory mechanism common to a wide variety of Gram-positive bacteria containing mef/mel(msr) elements.

Association of a Novel Mutation in the Plasmodium falciparum Chloroquine Resistance Transporter With Decreased Piperaquine Sensitivity

Background Amplified copy number in the plasmepsin II/III genes within Plasmodium falciparum has been associated with decreased sensitivity to piperaquine. To examine this association and test whether additional loci might also contribute, we performed a genome-wide association study of ex vivo P. falciparum susceptibility to piperaquine. Methods Plasmodium falciparum DNA from 183 samples collected primarily from Cambodia was genotyped at 33716 genome-wide single nucleotide polymorphisms (SNPs). Linear mixed models and random forests were used to estimate associations between parasite genotypes and piperaquine susceptibility. Candidate polymorphisms were evaluated for their association with dihydroartemisinin-piperaquine treatment outcomes in an independent dataset. Results Single nucleotide polymorphisms on multiple chromosomes were associated with piperaquine 90% inhibitory concentrations (IC90) in a genome-wide analysis. Fine-mapping of genomic regions implicated in genome-wide analyses identified multiple SNPs in linkage disequilibrium with each other that were significantly associated with piperaquine IC90, including a novel mutation within the gene encoding the P. falciparum chloroquine resistance transporter, PfCRT. This mutation (F145I) was associated with dihydroartemisinin-piperaquine treatment failure after adjusting for the presence of amplified plasmepsin II/III, which was also associated with decreased piperaquine sensitivity. Conclusions Our data suggest that, in addition to plasmepsin II/III copy number, other loci, including pfcrt, may also be involved in piperaquine resistance.

Genome Sequence of Mycoplasma feriruminatoris sp. nov., a Fast-Growing Mycoplasma Species.

ABSTRACT Members of the “Mycoplasma mycoides cluster” represent important livestock pathogens worldwide. We report the genome sequence of Mycoplasma feriruminatoris sp. nov., the closest relative to the “Mycoplasma mycoides cluster” and the fastest-growing Mycoplasma species described to date.