Conor J. Meehan

A Phylogenomic View of Ecological Specialization in the Lachnospiraceae, a Family of Digestive Tract-Associated Bacteria

Several bacterial families are known to be highly abundant within the human microbiome, but their ecological roles and evolutionary histories have yet to be investigated in depth. One such family, Lachnospiraceae (phylum Firmicutes, class Clostridia) is abundant in the digestive tracts of many mammals and relatively rare elsewhere. Members of this family have been linked to obesity and protection from colon cancer in humans, mainly due to the association of many species within the group with the production of butyric acid, a substance that is important for both microbial and host epithelial cell growth. We examined the genomes of 30 Lachnospiraceae isolates to better understand the origin of butyric acid capabilities and other ecological adaptations within this group. Butyric acid production-related genes were detected in fewer than half of the examined genomes with the distribution of this function likely arising in part from lateral gene transfer (LGT). An investigation of environment-specific functional signatures indicated that human gut-associated Lachnospiraceae possess genes for endospore formation, whereas other members of this family lack key sporulation-associated genes, an observation supported by analysis of metagenomes from the human gut, oral cavity, and bovine rumen. Our analysis demonstrates that adaptation to an ecological niche and acquisition of defining functional roles within a microbiome can arise through a combination of both habitat-specific gene loss and LGT.

Interactions in the microbiome: communities of organisms and communities of genes

A central challenge in microbial community ecology is the delineation of appropriate units of biodiversity, which can be taxonomic, phylogenetic, or functional in nature. The term ‘community’ is applied ambiguously; in some cases, the term refers simply to a set of observed entities, while in other cases, it requires that these entities interact with one another. Microorganisms can rapidly gain and lose genes, potentially decoupling community roles from taxonomic and phylogenetic groupings. Trait-based approaches offer a useful alternative, but many traits can be defined based on gene functions, metabolic modules, and genomic properties, and the optimal set of traits to choose is often not obvious. An analysis that considers taxon assignment and traits in concert may be ideal, with the strengths of each approach offsetting the weaknesses of the other. Individual genes also merit consideration as entities in an ecological analysis, with characteristics such as diversity, turnover, and interactions modeled using genes rather than organisms as entities. We identify some promising avenues of research that are likely to yield a deeper understanding of microbial communities that shift from observation-based questions of ‘Who is there?’ and ‘What are they doing?’ to the mechanistically driven question of ‘How will they respond?’

Specific gyrA gene mutations predict poor treatment outcome in MDR-TB

Objectives Mutations in the gyrase genes cause fluoroquinolone resistance in Mycobacterium tuberculosis. However, the predictive value of these markers for clinical outcomes in patients with MDR-TB is unknown to date. The objective of this study was to determine molecular markers and breakpoints predicting second-line treatment outcomes in M. tuberculosis patients treated with fourth-generation fluoroquinolones. Methods We analysed treatment outcome data in relation to the gyrA and gyrB sequences and MICs of ofloxacin, gatifloxacin and moxifloxacin for pretreatment M. tuberculosis isolates from 181 MDR-TB patients in Bangladesh whose isolates were susceptible to injectable drugs. Results The gyrA 90Val, 94Gly and 94Ala mutations were most frequent, with the highest resistance levels for 94Gly mutants. Increased pretreatment resistance levels (>2 mg/L), related to specific mutations, were associated with lower cure percentages, with no cure in patients whose isolates were resistant to gatifloxacin at 4 mg/L. Any gyrA 94 mutation, except 94Ala, predicted a significantly lower proportion of cure compared with all other gyrA mutations taken together (all non-94 mutants + 94Ala) [OR = 4.3 (95% CI 1.4–13.0)]. The difference in treatment outcome was not explained by resistance to the other drugs. Conclusions Our study suggests that gyrA mutations at position 94, other than Ala, predict high-level resistance to gatifloxacin and moxifloxacin, as well as poor treatment outcome, in MDR-TB patients in whom an injectable agent is still effective.

The evolution of the class A scavenger receptors

BackgroundThe class A scavenger receptors are a subclass of a diverse family of proteins defined based on their ability to bind modified lipoproteins. The 5 members of this family are strikingly variable in their protein structure and function, raising the question as to whether it is appropriate to group them as a family based on their ligand binding abilities.ResultsTo investigate these relationships, we defined the domain architecture of each of the 5 members followed by collecting and annotating class A scavenger receptor mRNA and amino acid sequences from publicly available databases. Phylogenetic analyses, sequence alignments, and permutation tests revealed a common evolutionary ancestry of these proteins, indicating that they form a protein family. We postulate that 4 distinct gene duplication events and subsequent domain fusions, internal repeats, and deletions are responsible for the diverse protein structures and functions of this family. Despite variation in domain structure, there are highly conserved regions across all 5 members, indicating the possibility that these regions may represent key conserved functional motifs.ConclusionsWe have shown with significant evidence that the 5 members of the class A scavenger receptors form a protein family. We have indicated that these receptors have a common origin which may provide insight into future functional work with these proteins.

Unexpected high prevalence of resistance-associated Rv0678 variants in MDR-TB patients without documented prior use of clofazimine or bedaquiline

Objectives: Resistance-associated variants (RAVs) in Rv0678, a regulator of the MmpS5-MmpL5 efflux pump, have been shown to lead to increased MICs of bedaquiline (2- to 8- fold) and clofazimine (2- to 4-fold). The prevalence of these Rv0678 RAVs in clinical isolates and their impact on treatment outcomes are important factors to take into account in bedaquiline treatment guidelines. Methods: Baseline isolates from two bedaquiline MDR-TB clinical trials were sequenced for Rv0678 RAVs and corresponding bedaquiline MICs were determined on 7H11 agar. Rv0678 RAVs were also investigated in non-MDR-TB sequences of a population-based cohort. Results: Rv0678 RAVs were identified in 23/347 (6.3%) of MDR-TB baseline isolates. Surprisingly, bedaquiline MICs for these isolates were high (> 0.24 mg/L, n = 8), normal (0.03−0.24 mg/L, n = 11) or low (< 0.03 mg/L, n = 4). A variant at position −11 in the intergenic region mmpS5–Rv0678 was identified in 39 isolates (11.3%) and appeared to increase the susceptibility to bedaquiline. In non-MDR-TB isolates, the frequency of Rv0678 RAVs was lower (6/852 or 0.7%). Competition experiments suggested that rifampicin was not the drug selecting for Rv0678 RAVs. Conclusions: RAVs in Rv0678 occur more frequently in MDR-TB patients than previously anticipated, are not associated with prior use of bedaquiline or clofazimine, and in the majority of cases do not lead to bedaquiline MICs above the provisional breakpoint (0.24 mg/L). Their origin remains unknown. Given the variety of RAVs in Rv0678 and their variable effects on the MIC, only phenotypic drug-susceptibility methods can currently be used to assess bedaquiline susceptibility.

Genomic characterization of Nontuberculous Mycobacteria

Mycobacterium tuberculosis and Mycobacterium leprae have remained, for many years, the primary species of the genus Mycobacterium of clinical and microbiological interest. The other members of the genus, referred to as nontuberculous mycobacteria (NTM), have long been underinvestigated. In the last decades, however, the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged. Despite the availability of whole genome sequencing technologies, limited effort has been devoted to the genetic characterization of NTM species. As a consequence, the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting. In this work, we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species. We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus.

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression

ABSTRACT Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness. IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania—a protozoan parasite that kills more than 30,000 people each year—is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo. We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this. IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania—a protozoan parasite that kills more than 30,000 people each year—is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo. We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this.

Diagnosis of Bacterial Bloodstream Infections: A 16S Metagenomics Approach

Background Bacterial bloodstream infection (bBSI) is one of the leading causes of death in critically ill patients and accurate diagnosis is therefore crucial. We here report a 16S metagenomics approach for diagnosing and understanding bBSI. Methodology/Principal Findings The proof-of-concept was delivered in 75 children (median age 15 months) with severe febrile illness in Burkina Faso. Standard blood culture and malaria testing were conducted at the time of hospital admission. 16S metagenomics testing was done retrospectively and in duplicate on the blood of all patients. Total DNA was extracted from the blood and the V3–V4 regions of the bacterial 16S rRNA genes were amplified by PCR and deep sequenced on an Illumina MiSeq sequencer. Paired reads were curated, taxonomically labeled, and filtered. Blood culture diagnosed bBSI in 12 patients, but this number increased to 22 patients when combining blood culture and 16S metagenomics results. In addition to superior sensitivity compared to standard blood culture, 16S metagenomics revealed important novel insights into the nature of bBSI. Patients with acute malaria or recovering from malaria had a 7-fold higher risk of presenting polymicrobial bloodstream infections compared to patients with no recent malaria diagnosis (p-value = 0.046). Malaria is known to affect epithelial gut function and may thus facilitate bacterial translocation from the intestinal lumen to the blood. Importantly, patients with such polymicrobial blood infections showed a 9-fold higher risk factor for not surviving their febrile illness (p-value = 0.030). Conclusions/Significance Our data demonstrate that 16S metagenomics is a powerful approach for the diagnosis and understanding of bBSI. This proof-of-concept study also showed that appropriate control samples are crucial to detect background signals due to environmental contamination.

Characterizing the Diverse Mutational Pathways Associated with R5-Tropic Maraviroc Resistance: HIV-1 That Uses the Drug-Bound CCR5 Coreceptor

ABSTRACT Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinants phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable. IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5 unavailable for use by HIV-1 and is now used in combination antiretroviral therapy. Treatment failure with drug-resistant virus is particularly interesting because it tends to be rare, with lack of sensitivity usually associated with the presence of CXCR4-using virus (CXCR4 is the main alternative coreceptor HIV-1 uses, in addition to CD4). We analyzed envelope sequences from HIV-1, obtained from 20 patients who enrolled in maraviroc clinical trials and experienced treatment failure, without detection of CXCR4-using virus. Evolutionary analysis was employed to identify molecular changes that confer maraviroc resistance. We found that in these individuals, resistant viruses form a distinct population that evolved once and was successful as a result of drug pressure. Further evolutionary analysis placed the complex network of interdependent mutational changes into functional groups that help explain the impediments to the emergence of maraviroc-associated R5 drug resistance.

Lateral gene transfer of an ABC transporter complex between major constituents of the human gut microbiome

BackgroundSeveral links have been established between the human gut microbiome and conditions such as obesity and inflammatory bowel syndrome. This highlights the importance of understanding what properties of the gut microbiome can affect the health of the human host. Studies have been undertaken to determine the species composition of this microbiome and infer functional profiles associated with such host properties. However, lateral gene transfer (LGT) between community members may result in misleading taxonomic attributions for the recipient organisms, thus making species-function links difficult to establish.ResultsWe identified a peptides/nickel transport complex whose components differed in abundance based upon levels of host obesity, and assigned the encoded proteins to members of the microbial community. Each protein was assigned to several distinct taxonomic groups, with moderate levels of agreement observed among different proteins in the complex. Phylogenetic trees of these proteins produced clusters that differed greatly from taxonomic attributions and indicated that habitat-directed LGT of this complex is likely to have occurred, though not always between the same partners.ConclusionsThese findings demonstrate that certain membrane transport systems may be an important factor within an obese-associated gut microbiome and that such complexes may be acquired several times by different strains of the same species. Additionally, an example of individual proteins from different organisms being transferred into one operon was observed, potentially demonstrating a functional complex despite the donors of the subunits being taxonomically disparate. Our results also highlight the potential impact of habitat-directed LGT on the resident microbiota.