Biology subjects

Ravi, A.

Publications and source records attributed to Ravi, A..

At least 19 recordsLinked to original sources

bioRxiv2025

Modelling cigarette smoke-induced lung vascular dysfunction using an alveolus-on-chip

The alveolus is central to gas exchange in the lung, and alveolar damage is a characteristics of a variety of lung diseases. Understanding alveolar microvascular dynamics and epithelial-endothelial interactions is essential for accurately modeling alveolar physiology and its dysfunction in lung diseases such as Chronic Obstructive Pulmonary Disease (COPD), pulmonary fibrosis and acute respiratory distress syndrome (ARDS). In this study, we present an open-top, membrane-free alveolus-on-chip platform incorporating self-assembled, perfusable 3D vascular networks by primary human lung endothelial cells and pericytes, co-cultured with alveolar epithelial type 2 (AEC2) cells. These vascular networks were developed within 6 days under continuous flow and remained stable for at least 12 days. The inclusion of pericytes supported capillary-like vessel formation and increased gene expression of EDNRB1, a gene enriched in alveolar microvascular endothelial cells. Furthermore, CD31 gene expression was higher in 3D endothelial networks compared to 2D endothelial monolayers, suggesting increased cell-cell adhesion. Monocytes could be successfully perfused through the networks, expanding the platforms potential for studying immune interactions in lung disorders. Culturing AEC2 monolayers directly on the vascularized hydrogel enabled physiologically relevant cell-cell interactions without artificial membranes, while maintaining air-liquid interface conditions. Importantly, exposure of the AEC2 layer to whole cigarette smoke (WCS) led to complete disintegration of the underlying vascular network, an effect not observed in the absence of AEC2. This chip model provides a human-relevant system for investigating vascular-epithelial crosstalk in the alveolus, smoke-induced lung injury, and immune recruitment, offering a valuable platform for future disease modeling and drug testing applications.

bioengineering↗

bioRxiv2025

Ingestion-activated CGRP neurons control learning but not satiety

Calcitonin Gene-Related Peptide (CGRP) neurons in the parabrachial nucleus are critical for sickness and malaise but have also been proposed to control non-aversive (rewarding) satiation. How one cell type can coordinate these opposing processes has not been explained. Here we reinvestigate the function of these cells using single-cell imaging as well as optical and chemogenetic manipulations. Contrary to current models, we show that CGRP neurons do not track cumulative food consumption, and their activity is not necessary for meal termination. Instead, we identify two distinct populations of CGRP cells, one of which responds rapidly to appetitive signals during ingestion and the other of which responds slowly to aversive visceral cues. Surprisingly, the ingestion-activated CGRP neurons are important for learning about post-ingestive effects but have little effect on food consumption. This reveals two populations of CGRP neurons that are sequentially engaged during, and responsible for, the distinct stages of post-ingestive learning.

neuroscience↗

bioRxiv2025

Single-nucleus epigenomic dysregulation unmasks genetic risk-associated neurodegenerative glia states

The accumulation of abnormal tau protein selectively affects distinct brain regions and specific populations of neurons and glial cells in tau-related dementias, such as Alzheimers disease (AD), Picks disease (PiD), and progressive supranuclear palsy (PSP). Although the three disorders share the feature of tau protein pathology, the regulatory circuitry of non-coding genetic variants underlying risk-associated cell states remains to be elucidated. Using paired single-nucleus profiling of chromatin accessibility and gene expression across AD, PiD, and PSP, we define cell-type-specific cis-regulatory elements (CREs) across six cell types and fifty subclasses. Comparing disease-dynamic CREs across three disorders, we find that glia overrepresent disorder-specific gene regulation related to dynamic cellular response to stress. We show that human genetic variants affecting microglial gene regulation converge into distinct and co-regulated modules affecting specific cellular functions. Moreover, polygenic risk modifiers are maximally co-accessible in disorder-specific glial states, modifying distinct pathways such as sphingomyelin regulation in PiD. Our study informs glial regulators linked to polygenic modifiers of primary tauopathy, introducing modifiable pathways governing resilience for therapeutic consideration.

genomics↗

bioRxiv2024

Comprehensive structure-function analysis reveals gain- and loss-of-function mechanisms impacting oncogenic KRAS activity

To dissect variant-function relationships in the KRAS oncoprotein, we performed deep mutational scanning (DMS) screens for both wild-type and KRASG12D mutant alleles. We defined the spectrum of oncogenic potential for nearly all possible KRAS variants, identifying several novel transforming alleles and elucidating a model to describe the frequency of KRAS mutations in human cancer as a function of transforming potential, mutational probability, and tissue-specific mutational signatures. Biochemical and structural analyses of variants identified in a KRASG12D second-site suppressor DMS screen revealed that attenuation of oncogenic KRAS can be mediated by protein instability and conformational rigidity, resulting in reduced binding affinity to effector proteins, such as RAF and PI3-kinases, or reduced SOS-mediated nucleotide exchange activity. These studies define the landscape of single amino acid alterations that modulate the function of KRAS, providing a resource for the clinical interpretation of KRAS variants and elucidating mechanisms of oncogenic KRAS inactivation for therapeutic exploitation.

cancer biology↗

bioRxiv2023

Genetic, transcriptomic, histological, and biochemical analysis of progressive supranuclear palsy implicates glial activation and novel risk genes

Progressive supranuclear palsy (PSP) is a rare Parkinsonian disorder characterized by problems with movement, balance, cognition, and other symptoms. PSP differs from Alzheimers disease (AD) and other neurodegenerative diseases displaying abnormal forms of the microtubule-associated protein tau ("tauopathies") by the presence of pathology not only in neurons, but also in astrocytes and oligodendrocytes. Genetic contributors may mediate these differences, however much of PSP genetics remains unexplained. Here we conducted the largest genome-wide association study (GWAS) of PSP to date including 2,779 cases (2,595 neuropathologically-confirmed) and 5,584 controls and identified six independent PSP susceptibility loci with genome-wide significant (p < 5x10-8) associations including five known (MAPT, MOBP, STX6, RUNX2, SLCO1A2) and one novel locus (C4A). Integration with cell type-specific epigenomic annotations revealed a unique oligodendrocytic signature that distinguishes PSP from AD and Parkinsons disease. Candidate PSP risk gene prioritization using expression quantitative trait loci (eQTLs) identified oligodendrocyte-specific effects on gene expression in half of the genome-wide significant loci, as well as an association with elevated C4A expression in bulk brain tissue which may be driven by increased C4A copy number in PSP cases. Finally, histological studies demonstrated abnormal tau aggregates in oligodendrocytes that colocalize with C4 (complement) deposition. Integrating GWAS with functional studies including epigenomic and eQTL analyses, we identified potential causal roles for variation in MOBP, STX6, RUNX2, SLCO1A2, and C4A in the pathogenesis of PSP.

neuroscience↗

bioRxiv2022

Cell-surface targeting of fluorophores in Drosophila for rapid neuroanatomy visualization

Visualizing neuronal anatomy often requires labor-intensive immunohistochemistry on fixed and dissected brains. To facilitate rapid anatomical staining in live brains, we used genetically targeted membrane tethers that covalently link fluorescent dyes for in vivo neuronal labeling. We generated a series of extracellularly trafficked small molecule tethering proteins, HaloTag-CD41 and SNAPf-CD4, which directly label transgene expressing cells with commercially available ligand substituted fluorescent dyes. We created stable transgenic Drosophila reporter lines which express extracellular HaloTag-CD4 and SNAPf-CD4 with LexA and Gal4 drivers. Expressing these enzymes in live Drosophila brains, we labeled the expression patterns of various Gal4 driver lines recapitulating histological staining in live brain tissue. Pan-neural expression of SNAPf-CD4 enabled registration of live brains to an existing template for anatomical comparisons. We predict that these extracellular platforms will not only become a valuable complement to existing anatomical methods but will also prove useful for future genetic targeting of other small molecule probes, drugs, and actuators.

neuroscience↗

bioRxiv2022

Integrative Analysis of Checkpoint Blockade Response in Advanced Non-Small Cell Lung Cancer

Anti-PD-1/PD-L1 agents have transformed the treatment landscape of advanced non-small cell lung cancer (NSCLC). While our understanding of the biology underlying immune checkpoint blockade in NSCLC is still incomplete, studies to date have established predictive roles for PD-L1 tumor expression and tumor mutational burden (TMB). To expand our understanding of the molecular features underlying response to checkpoint inhibitors in NSCLC, we describe here the first joint analysis of the Stand Up 2 Cancer - Mark Foundation (SU2C-MARK) Cohort, a resource of whole exome and/or RNA sequencing from 393 patients with NSCLC treated with anti-PD-(L)1 therapy, along with matched clinical response annotation. We identify a number of associations between molecular features and outcome, including: 1) favorable (e.g., ATM altered), and unfavorable (e.g., TERT amplified) genomic subgroups, 2) distinct immune infiltration signatures associated with wound healing (unfavorable) and immune activation (favorable), and 3) a novel de-differentiated tumor-intrinsic subtype characterized by expression of endodermal lineage genes, immune activation, and enhanced response rate. Taken together, results from this cohort extend our understanding of NSCLC-specific predictors, providing a rich set of molecular and immunologic hypotheses with which to further our understanding of the biology of checkpoint blockade in NSCLC.

genomics↗

bioRxiv2021

Voltage imaging in Drosophila using a hybrid chemical-genetic rhodamine voltage reporter

We combine a chemically-synthesized, voltage-sensitive fluorophore with a genetically encoded, self-labeling enzyme to enable voltage imaging in Drosophila melanogaster. Previously, we showed that a rhodamine voltage reporter (RhoVR) combined with the HaloTag self-labeling enzyme could be used to monitor membrane potential changes from mammalian neurons in culture and brain slice. Here, we apply this hybrid RhoVR-Halo approach in vivo to achieve selective neuron labeling in intact fly brains. We generate a Drosophila UAS-HaloTag reporter line in which the HaloTag enzyme is expressed on the surface of cells. We validate the voltage sensitivity of this new construct in cell culture before driving expression of HaloTag in specific brain neurons in flies. We show that selective labeling of synapses, cells, and brain regions can be achieved with RhoVR-Halo in either larval neuromuscular junction (NMJ) or in whole adult brains. Finally, we validate the voltage sensitivity of RhoVR-Halo in fly tissue via dual-electrode/imaging at the NMJ, show the efficacy of this approach for measuring synaptic excitatory post-synaptic potentials (EPSPs) in muscle cells, and perform voltage imaging of carbachol-evoked depolarization and osmolarity-evoked hyperpolarization in projection neurons and in interoceptive subesophageal zone neurons in fly brain explants following in vivo labeling. We envision the turn-on response to depolarizations, fast response kinetics, and two-photon compatibility of chemical indicators, coupled with the cellular and synaptic specificity of genetically-encoded enzymes, will make RhoVR-Halo a powerful complement to neurobiological imaging in Drosophila. Significance StatementVoltage imaging is a powerful method for interrogating neurobiology. Chemical indicators possess fast response kinetics, turn-on responses to membrane depolarization, and can be compatible with two-photon excitation. However, selective cell labeling in intact tissues and in vivo remains a challenge for completely synthetic fluorophores. Here, we show that a chemical - genetic hybrid approach in Drosophila enables cell-specific staining in vivo and voltage imaging in whole-brain explants and at neuromuscular junction synapses.

neuroscience↗

bioRxiv2021

Combined tumor and immune signals from genomes or transcriptomes predict outcomes of checkpoint inhibition in melanoma

Cancer immunotherapy with checkpoint blockade (CPB) leads to improved outcomes in melanoma and other tumor types, but a majority of patients do not respond. High tumor mutation burden (TMB) and high levels of tumor-infiltrating T cells have been associated with response to immunotherapy, but integrative models to predict clinical benefit using DNA or RNA alone have not been comprehensively explored. We sequenced DNA and RNA from melanoma patients receiving CPB, and aggregated previously published data, yielding whole exome sequencing data for 189 patients and bulk RNA sequencing data for 178 patients. Using these datasets, we derived genomic and transcriptomic factors that predict overall survival (OS) and response to immunotherapy. Using whole-exome DNA data alone, we calculated T cell burden (TCB) and B cell burden (BCB) based on rearranged TCR/Ig DNA sequences and found that patients whose melanomas have high TMB together with either high TCB or high BCB survived longer and had higher response rates as compared to patients with either low TMB or TCB/BCB. Next, using bulk RNA-Seq data, differential expression analysis identified 83 genes associated with high or low OS. By combining pairs of immune-expressed genes with tumor-expressed genes, we identified three gene pairs associated with response and survival (Bonferroni P<0.05). All three gene pair models were validated in an independent cohort (n=180) (Bonferroni P<0.05). The best performing gene pair model included the lymphocyte-expressed MAP4K1 (Mitogen- Activated Protein Kinase Kinase Kinase Kinase 1) combined with the transcription factor TBX3 (T-Box Transcription Factor 3) which is overexpressed in poorly differentiated melanomas. We conclude that RNA-based (MAP4K1&TBX3) or DNA-based (TCB&TMB) models combining immune and tumor measures improve predictions of outcome after checkpoint blockade in melanoma.

genomics↗

bioRxiv2021

Linking carbohydrate structure with function in the human gut microbiome using hybrid metagenome assemblies

BackgroundComplex carbohydrates that escape digestion in the small intestine, are broken down in the large intestine by enzymes encoded by the gut microbiome. This is a symbiotic relationship between particular microbes and the host, resulting in metabolic products that influence host gut health and are exploited by other microbes. However, the role of carbohydrate structure in directing microbiota community composition and the succession of carbohydrate-degrading microbes is not fully understood. Here we take the approach of combining data from long and short read sequencing allowing recovery of large numbers of high quality genomes, from which we can predict carbohydrate degrading functions, and impact of carbohydrate on microbial communities. ResultsIn this study we evaluate species-level compositional variation within a single microbiome in response to six structurally distinct carbohydrates in a controlled model gut using hybrid metagenome assemblies. We identified 509 high-quality metagenome-assembled genomes (MAGs) belonging to ten bacterial classes and 28 bacterial families. We found dynamic variations in the microbiome amongst carbohydrate treatments, and over time. Using these data, the MAGs were characterised as primary (0h to 6h) and secondary degraders (12h to 24h). Annotating the MAGs with the Carbohydrate Active Enzyme (CAZyme) database we are able to identify species which are enriched through time and have the potential to actively degrade carbohydrate substrates. ConclusionsRecent advances in sequencing technology allowed us to identify significant unexplored diversity amongst starch degrading species in the human gut microbiota including CAZyme profiles and complete MAGs. We have identified changes in microbial community composition in response to structurally distinct carbohydrate substrates, which can be directly related to the CAZyme complement of the enriched MAGs. Through this approach, we have identified a number of species which have not previously been implicated in starch degradation, but which have the potential to play an important role.

microbiology↗

bioRxiv2021

Breathing on Chip: Biomechanical forces change airway epithelial cell biology ina human Airway Lung-Chip

Human lung function is intricately linked to blood flow and breathing cycles, but it remains unknown how these dynamic cues shape human airway epithelial biology. Here we report a state-of-the-art protocol for studying effects of physiological airflow and stretch on differentiation, cellular composition, and mucociliary clearance of human primary airway epithelial cells cultured on a perfused airway chip. Perfused epithelial tissue cultures developed a large airway-like cellular composition with accelerated maturation and polarization of mucociliary clearance when compared to traditional (static) culture methods. Additional application of airflow and stretch to the airway chip resulted in a cellular composition more comparable to the small(er) airways, reduced baseline secretion of interleukin-8 and other inflammatory proteins, and reduced gene expression of matrix metalloproteinase (MMP) 9, fibronectin, and other extracellular matrix factors. These results indicate that breathing-like mechanical stimuli are important modulators of airway epithelial cell differentiation and homeostasis and that their fine-tuned application could generate models of specific epithelial regions, pathologies, and mucociliary (dys)function.

cell biology↗

bioRxiv2021

Metagenomic investigation of the equine faecal microbiome reveals extensive taxonomic and functional diversity

BackgroundThe horse plays crucial roles across the globe, including in horseracing, as a working and companion animal and as a food animal. The horse hindgut microbiome makes a key contribution in turning a high fiber diet into body mass and horsepower. However, despite its importance, the horse hindgut microbiome remains largely undefined. Here, we applied culture-independent shotgun metagenomics to thoroughbred equine faecal samples to deliver novel insights into this complex microbial community. ResultsWe performed metagenomic sequencing on five equine faecal samples to construct 123 high- or medium-quality metagenome-assembled genomes from Bacteria and Archaea. In addition, we recovered nearly 200 bacteriophage genomes. We document surprising taxonomic and functional diversity, encompassing dozens of novel or unnamed bacterial genera and species, to which we have assigned new Candidatus names. Many of these genera are conserved across a range of mammalian gut microbiomes. ConclusionsOur metagenomic analyses provide new insights into the bacterial, archaeal and bacteriophage components of the horse gut microbiome. The resulting datasets provide a key resource for future high-resolution taxonomic and functional studies on the equine gut microbiome.

genomics↗

bioRxiv2020

Genomic diversity of Escherichia coli isolates from backyard chickens and guinea fowl in the Gambia

Chickens and guinea fowl are commonly reared in Gambian homes as affordable sources of protein. Using standard microbiological techniques, we obtained 68 caecal isolates of Escherichia coli from ten chickens and nine guinea fowl in rural Gambia. After Illumina whole-genome sequencing, 28 sequence types were detected in the isolates (four of them novel), of which ST155 was the most common (22/68, 32%). These strains span four of the eight main phylogroups of E. coli, with phylogroups B1 and A being most prevalent. Nearly a third of the isolates harboured at least one antimicrobial resistance gene, while most of the ST155 isolates (14/22, 64%) encoded resistance to [≥]3 classes of clinically relevant antibiotics, as well as putative virulence factors, suggesting pathogenic potential in humans. Furthermore, hierarchical clustering revealed that several Gambian poultry strains were closely related to isolates from humans. Although the ST155 lineage is common in poultry from Africa and South America, the Gambian ST155 isolates belong to a unique cgMLST cluster comprised of closely related (38-39 alleles differences) isolates from poultry and livestock from sub-Saharan Africa--suggesting that strains can be exchanged between poultry and livestock in this setting. Continued surveillance of E. coli and other potential pathogens in rural backyard poultry from sub-Saharan Africa is warranted. Author notesAll supporting data and protocols have been provided within the article or as supplementary data files. Eleven supplementary figures and eight supplementary files are available with the online version of this article. Data summaryThe genomic assemblies for the isolates reported here are available for download from EnteroBase (http://enterobase.warwick.ac.uk/species/index/ecoli) and the EnteroBase assembly barcodes are provided in File S2. Sequences have been deposited in the NCBI SRA, under the BioProject ID: PRJNA616250 and accession numbers SAMN14485281 to SAMN14485348 (File S2). Assemblies have been deposited in GenBank under the BioProject ID: PRJNA616250 and accession numbers CP053258 and CP053259. Impact statementDomestic birds play a crucial role in human society, in particular contributing to food security in low-income countries. Many households in Sub-Saharan Africa rear free-range chickens and guinea fowl, which are often left to scavenge for feed in and around the family compound, where they are frequently exposed to humans, other animals and the environment. Such proximity between backyard poultry and humans is likely to facilitate transmission of pathogens such as Escherichia coli or antimicrobial resistance between the two host species. Little is known about the population structure of E. coli in rural chickens and guinea fowl, although this information is needed to contextualise the potential risks of transmission of bacterial strains between humans and rural backyard poultry. Thus, we sought to investigate the genomic diversity of E. coli in backyard poultry from rural Gambia.

genomics↗

bioRxiv2020

Genomic diversity of Escherichia coli isolates from non-human primates in the Gambia

Increasing contact between humans and non-human primates provides an opportunity for the transfer of potential pathogens or antimicrobial resistance between host species. We have investigated genomic diversity, and antimicrobial resistance in Escherichia coli isolates from four species of non-human primate in the Gambia: Papio papio (n=22), Chlorocebus sabaeus (n=14), Piliocolobus badius (n=6) and Erythrocebus patas (n=1). We performed Illumina whole-genome sequencing on 101 isolates from 43 stools, followed by nanopore long-read sequencing on eleven isolates. We identified 43 sequence types (STs) by the Achtman scheme (ten of which are novel), spanning five of the eight known phylogroups of E. coli. The majority of simian isolates belong to phylogroup B2--characterised by strains that cause human extraintestinal infections--and encode factors associated with extraintestinal disease. A subset of the B2 strains (ST73, ST681 and ST127) carry the pks genomic island, which encodes colibactin, a genotoxin associated with colorectal cancer. We found little antimicrobial resistance and only one example of multi-drug resistance among the simian isolates. Hierarchical clustering showed that simian isolates from ST442 and ST349 are closely related to isolates recovered from human clinical cases (differences in 50 and seven alleles respectively), suggesting recent exchange between the two host species. Conversely, simian isolates from ST73, ST681 and ST127 were distinct from human isolates, while five simian isolates belong to unique core-genome ST complexes--indicating novel diversity specific to the primate niche. Our results are of public health importance, considering the increasing contact between humans and wild non-human primates. Impact statementLittle is known about the population structure, virulence potential and the burden of antimicrobial resistance among Escherichia coli from wild non-human primates, despite increased exposure to humans through the fragmentation of natural habitats. Previous studies, primarily involving captive animals, have highlighted the potential for bacterial exchange between non-human primates and humans living nearby, including strains associated with intestinal pathology. Using multiple-colony sampling and whole-genome sequencing, we investigated the strain distribution and population structure of E. coli from wild non-human primates from the Gambia. Our results indicate that these monkeys harbour strains that can cause extraintestinal infections in humans. We document the transmission of virulent E. coli strains between monkeys of the same species sharing a common habitat and evidence of recent interaction between strains from humans and wild non-human primates. Also, we present complete genome assemblies for five novel sequence types of E. coli. Author notesAll supporting data, code and protocols have been provided within the article or through supplementary data files. Nine supplementary figures and six supplementary files are available with the online version of this article. AbbreviationsExPEC, Extraintestinal pathogenic Escherichia coli; ST, Sequence type; AMR, Antimicrobial resistance; MLST, Multi-locus sequence typing; VFDB, Virulence factors database; SNP, single nucleotide polymorphism; SPRI, Solid phase reversible immobilisation. Data summaryThe raw sequences and polished assemblies from this study are available in the National Center for Biotechnology Information (NCBI) Short Read Archive, under the BioProject accession number PRJNA604701. The full list and characteristics of these strains and other reference strains used in the analyses are presented in Table 1 and Supplementary Files 1-4 (available with the online version of this article).

genomics↗

bioRxiv2019

Within-Host Diversity and Vertical Transmission of Group B Streptococcus Among Mother-infant Dyads in The Gambia

IntroductionUnderstanding mother-to-infant transmission of Group B Streptococcus (GBS) is vital to the prevention and control of GBS disease. We investigated the transmission and phylogenetic relationships of mothers colonised by GBS and their infants in a peri-urban setting in The Gambia.\n\nMethodsWe collected nasopharyngeal swabs from 35 mother-infant dyads at weekly intervals from birth until six weeks post-partum. GBS was isolated by conventional microbiology techniques. Whole-genome sequencing was performed on GBS isolates from one mother-infant dyad (dyad 17).\n\nResultsWe recovered 85 GBS isolates from the 245 nasopharyngeal swabs. GBS was isolated from 16.33% and 18.37% of sampled mothers and infants, respectively. In 87% of cultured swabs, the culture status of an infant agreed with that of the mother (Kappa p-value <0.001). In dyad 17, phylogenetic analysis revealed within-host strain diversity in the mother and clone to her infant.\n\nConclusionGBS colonisation in mothers presents a significant risk of colonisation in their infants. We confirm vertical transmission from mother to child in dyad 17, accompanied by within-host diversity.

microbiology↗

bioRxiv2019

Antibiotics select for novel pathways of resistance in biofilms

Most bacteria in nature exist in aggregated communities known as biofilms. Bacteria within biofilms are inherently highly resistant to antibiotics. Current understanding of the evolution and mechanisms of antibiotic resistance is largely derived from work from cells in liquid culture and it is unclear whether biofilms adapt and evolve in response to sub-inhibitory concentrations of drugs. Here we used a biofilm evolution model to show that biofilms of a model food borne pathogen, Salmonella Typhimurium rapidly evolve in response to exposure to three clinically important antibiotics. Whilst the model strongly selected for improved biofilm formation in the absence of any drug, once antibiotics were introduced the need to adapt to the drug was more important than the selection for improved biofilm formation. Adaptation to antibiotic stress imposed a marked cost in biofilm formation, particularly evident for populations exposed to cefotaxime and azithromycin. We identified distinct resistance phenotypes in biofilms compared to corresponding planktonic control cultures and characterised new mechanisms of resistance to cefotaxime and azithromycin. Novel substitutions within the multidrug efflux transporter, AcrB were identified and validated as impacting drug export as well as changes in regulators of this efflux system. There were clear fitness costs identified and associated with different evolutionary trajectories. Our results demonstrate that biofilms adapt rapidly to low concentrations of antibiotics and the mechanisms of adaptation are novel. This work will be a starting point for studies to further examine biofilm specific pathways of adaptation which inform future antibiotic use.

microbiology↗

bioRxiv2019

Loss of microbial diversity and pathogen domination of the gut microbiota in critically ill patients

BackgroundFor long-stay patients on the adult intensive care unit, the gut microbiota plays a key role in determining the balance between health and disease. However, it remains unclear which ICU patients might benefit from interventions targeting the gut microbiota or the pathogens therein.\n\nMethodsWe undertook a prospective observational study of twenty-four ICU patients, in which serial faecal samples were subjected to shotgun metagenomic sequencing, phylogenetic profiling and microbial genome analyses.\n\nResultsTwo-thirds of patients experienced a marked drop in gut microbial diversity (to an inverse Simpsons index of <4) at some stage during their stay in ICU, often accompanied by absence or loss of beneficial commensal bacteria. Intravenous administration of the broad-spectrum antimicrobial agent meropenem was significantly associated with loss of gut microbial diversity, but administration of other antibiotics, including piperacillin-tazobactam, failed to trigger statistically detectable changes in microbial diversity. In three quarters of ICU patients, we documented episodes of gut domination by pathogenic strains, with evidence of cryptic nosocomial transmission of Enterococcus faecium. In some patients we also saw domination of the gut microbiota by commensal organisms, such as Methanobrevibacter smithii.\n\nConclusionsOur results support a role for metagenomic surveillance of the gut microbiota and pave the way for patient-specific interventions that maintain or restore gut microbial diversity in the ICU.

microbiology↗

bioRxiv2018

Metagenomic profiling of ticks: identification of novel rickettsial genomes and detection of tick-borne canine parvovirus

BackgroundAcross the world, ticks act as vectors of human and animal pathogens. Ticks rely on bacterial endosymbionts, which often share close and complex evolutionary links with tick-borne pathogens. As the prevalence, diversity and virulence potential of tick-borne agents remain poorly understood, there is a pressing need for microbial surveillance of ticks as potential disease vectors.\n\nMethodology/Principal FindingsWe developed a two-stage protocol that includes 16S-amplicon screening of pooled samples of hard ticks collected from dogs, sheep and camels in Palestine, followed by shotgun metagenomics on individual ticks to detect and characterise tick-borne pathogens and endosymbionts. Two ticks isolated from sheep yielded an abundance of reads from the genus Rickettsia, which were assembled into draft genomes. One of the resulting genomes was highly similar to Rickettsia massiliae strain MTU5. Analysis of signature genes showed that the other represents the first genome sequence of the potential pathogen Candidatus Rickettsia barbariae. Ticks from a dog and a sheep yielded draft genome sequences of strains of the Coxiella-like endosymbiont Candidatus Coxeilla mudrowiae. A sheep tick yielded sequences from the sheep pathogen Anaplasma ovis, while Hyalomma ticks from camels yielded sequences belonging to Francisella-like endosymbionts. From the metagenome of a dog tick from Jericho, we generated a genome sequence of a canine parvovirus.\n\nSignificanceHere, we have shown how a cost-effective two-stage protocol can be used to detect and characterise tick-borne pathogens and endosymbionts. In recovering genome sequences from an unexpected pathogen (canine parvovirus) and a previously unsequenced pathogen (Candidatus Rickettsia barbariae), we demonstrate the open-ended nature of metagenomics. We also provide evidence that ticks can carry canine parvovirus, raising the possibility that ticks might contribute to the spread of this troublesome virus.\n\nAuthor SummaryWe have shown how DNA sequencing can be used to detect and characterise potentially pathogenic microorganisms carried by ticks. We surveyed hard ticks collected from domesticated animals across the West Bank territory of Palestine. All the ticks came from species that are also capable of feeding on humans. We detected several important pathogens, including two species of Rickettsia, the sheep pathogen Anaplasma ovis and canine parvovirus. These findings highlight the importance of hard ticks and the hazards they present for human and animal health in Palestine and the opportunities presented by high-throughput sequencing and bioinformatics analyses of DNA sequences in this setting.

microbiology↗