Bacterial pathogenesis

Trevor's research utilizes high-throughput genome sequencing to investigate C. difficile populations and intestinal microbial communities that are associated with health and disease, and murine infection models to identify the host genetic factors that are linked to disease susceptibility. Healthcare-associated pathogens are a major focus of the research programme.

[David Goulding, Genome Research Limited]

Background

Hospital-acquired infections are presenting a major challenge to clinical practice and public health control. There is evidence that the modern hospital environment selects for super-fit variants of commensal, environmental or opportunistic pathogens that confound control and treatment regimens. The evolution and transmissibility of such organisms is tractable by high quality phylogenetic, phenotypic and epidemiological analysis.

Clostridium difficile is a prime example of a significant hospital-acquired pathogen. This bacterial species is a major cause of antibiotic-associated diarrhoea and is a rapidly emerging hospital-acquired infection currently confounding standard medical practices associated with antibiotic prescribing and infection control protocols. The use of antibiotics to treat other infections can trigger C. difficile disease and transmission, whereas antibiotics used to treat C. difficile infections are becoming less effective. Little is known about how C. difficile causes disease or transmits so efficiently within the hospital setting, but this appears to be due to an infective cycle that relies on highly resistant and infectious spores. An improved understanding of C. difficile biology, transmission and pathogenesis will directly benefit the ability to control and treat C. difficile in hospitals.

Research

The goals of the research programme are to investigate the genetic traits that contribute to C. difficile pathogenesis, persistence and transmission. Additional aims are to identify avenues for novel therapeutic intervention and to guide hospital infection control measures.

Collaborations

The Lawley Group has strong internal collaborations with the Microbial pathogenesis group, Pathogen Genomics Group, the Proteomic Mass Spectrometry Laboratory and the Mouse Genetics Programme.

The Group has several productive external collaborations including those with Professor Fiona Powrie's Group at the University of Oxford, Professor Brendan Wren's Group at London School of Hygiene & Tropical Medicine (LSHTM) and Professor Neil Fairweather's Group at Imperial College London as well as close interactions with several NHS hospitals and the Health Protection Agency reference laboratories within the UK and abroad.

Selected Publications

  • Emergence and global spread of epidemic healthcare-associated Clostridium difficile.

    He M, Miyajima F, Roberts P, Ellison L, Pickard DJ, Martin MJ, Connor TR, Harris SR, Fairley D, Bamford KB, D'Arc S, Brazier J, Brown D, Coia JE, Douce G, Gerding D, Kim HJ, Koh TH, Kato H, Senoh M, Louie T, Michell S, Butt E, Peacock SJ, Brown NM, Riley T, Songer G, Wilcox M, Pirmohamed M, Kuijper E, Hawkey P, Wren BW, Dougan G, Parkhill J and Lawley TD

    Nature genetics 2013;45;1;109-13

  • Intestinal colonization resistance.

    Lawley TD and Walker AW

    Immunology 2013;138;1;1-11

  • The Clostridium difficile spo0A gene is a persistence and transmission factor.

    Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, Wren BW, Fairweather NF, Dougan G and Lawley TD

    Infection and immunity 2012;80;8;2704-11

  • Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice.

    Lawley TD, Clare S, Walker AW, Stares MD, Connor TR, Raisen C, Goulding D, Rad R, Schreiber F, Brandt C, Deakin LJ, Pickard DJ, Duncan SH, Flint HJ, Clark TG, Parkhill J and Dougan G

    PLoS pathogens 2012;8;10;e1002995

  • Evolutionary dynamics of Clostridium difficile over short and long time scales.

    He M, Sebaihia M, Lawley TD, Stabler RA, Dawson LF, Martin MJ, Holt KE, Seth-Smith HM, Quail MA, Rance R, Brooks K, Churcher C, Harris D, Bentley SD, Burrows C, Clark L, Corton C, Murray V, Rose G, Thurston S, van Tonder A, Walker D, Wren BW, Dougan G and Parkhill J

    Proceedings of the National Academy of Sciences of the United States of America 2010;107;16;7527-32

  • Antibiotic treatment of clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts.

    Lawley TD, Clare S, Walker AW, Goulding D, Stabler RA, Croucher N, Mastroeni P, Scott P, Raisen C, Mottram L, Fairweather NF, Wren BW, Parkhill J and Dougan G

    Infection and immunity 2009;77;9;3661-9

  • Proteomic and genomic characterization of highly infectious Clostridium difficile 630 spores.

    Lawley TD, Croucher NJ, Yu L, Clare S, Sebaihia M, Goulding D, Pickard DJ, Parkhill J, Choudhary J and Dougan G

    Journal of bacteriology 2009;191;17;5377-86

  • Comparative genome and phenotypic analysis of Clostridium difficile 027 strains provides insight into the evolution of a hypervirulent bacterium.

    Stabler RA, He M, Dawson L, Martin M, Valiente E, Corton C, Lawley TD, Sebaihia M, Quail MA, Rose G, Gerding DN, Gibert M, Popoff MR, Parkhill J, Dougan G and Wren BW

    Genome biology 2009;10;9;R102

  • Host transmission of Salmonella enterica serovar Typhimurium is controlled by virulence factors and indigenous intestinal microbiota.

    Lawley TD, Bouley DM, Hoy YE, Gerke C, Relman DA and Monack DM

    Infection and immunity 2008;76;1;403-16

  • Genome-wide screen for Salmonella genes required for long-term systemic infection of the mouse.

    Lawley TD, Chan K, Thompson LJ, Kim CC, Govoni GR and Monack DM

    PLoS pathogens 2006;2;2;e11

Team

Team members

Blessing Anonye
PhD Student
Sam Forster
Postdoctoral Fellow
Mark Stares
Advanced Research Assistant

Blessing Anonye

- PhD Student

Prior to my PhD, I had an MSc in Industrial and Commercial Biotechnology from Newcastle University, UK. My masters project involved the isolation of bacteria (Streptomyces) from beach and dune sand system and subsequent characterisation and screening of the isolates for the production of potentially novel antibiotics.

Research

My research at the Sanger Institute focuses on understanding the human intestinal microbiota in health and in disease. In particular, my PhD project focuses on the development of a bacteriotherapy for the treatment of severe Clostridium difficile disease using a range of bioinformatics and molecular based techniques. Knowing the bacterial species that can confer colonization resistance against C. difficile is important, as a major cause of this disease, is antibiotic disruption of the intestinal microbiota.

References

  • Vying over spilt oil.

    Pham N TA and Anonye BO

    Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

    Nature reviews. Microbiology 2014;12;3;156

  • Bacteriotherapy for the treatment of intestinal dysbiosis caused by Clostridium difficile infection.

    Adamu BO and Lawley TD

    Bacterial Pathogenesis Laboratory, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

    Faecal microbiota transplantation (FMT) has been used for more than five decades to treat a variety of intestinal diseases associated with pathological imbalances within the resident microbiota, termed dysbiosis. FMT has been particularly effective for treating patients with recurrent Clostridium difficile infection who are left with few clinical options other than continued antibiotic therapy. Our increasing knowledge of the structure and function of the human intestinal microbiota and C. difficile pathogenesis has led to the understanding that FMT promotes intestinal ecological restoration and highlights the microbiota as a viable therapeutic target. However, the use of undefined faecal samples creates a barrier for widespread clinical use because of safety and aesthetic issues. An emerging concept of bacteriotherapy, the therapeutic use of a defined mixture of harmless, health-associated bacteria, holds promise for the treatment of patients with severe C. difficile infection, and possibly represents a paradigm shift for the treatment of diseases linked to intestinal dysbiosis.

    Funded by: Medical Research Council: 93614; Wellcome Trust: 098051

    Current opinion in microbiology 2013;16;5;596-601

Sam Forster

- Postdoctoral Fellow

I trained in computational science and genetics at the University of Melbourne before undertaking Biomedical Science Honours degree and PhD under the supervision of Prof. Paul Hertzog at the Monash Institute, Melbourne Australia. This work examined the intricacy of type 1 interferon signalling, an important component of the innate immune response.

Research

My research combines computational analysis, high throughput genomics and transcriptomics to investigate the interaction between host and microbiota. Optimized microbiota populations are rationally selected and characterized using bioinformatics algorithm development, molecular biology, microbiology, in-vitro cell culture and in-vivo models. My work aims to improve our understanding of the role of microbiota in determining host biology, specifically colonization resistance and infection.

References

  • IFNβ-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage.

    Cheon H, Holvey-Bates EG, Schoggins JW, Forster S, Hertzog P, Imanaka N, Rice CM, Jackson MW, Junk DJ and Stark GR

    Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA.

    A single high dose of interferon-β (IFNβ) activates powerful cellular responses, in which many anti-viral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells downregulate this initial response rapidly. However, the expression of many anti-viral proteins that do no harm is sustained, prolonging a substantial part of the initial anti-viral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNβ-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNβ, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase in other IFNβ-induced proteins, and to constitutive resistance to DNA damage.

    Funded by: NCI NIH HHS: P01 CA062220; NIAID NIH HHS: R01 AI091707; NIDDK NIH HHS: K01 DK095031

    The EMBO journal 2013;32;20;2751-63

  • Structural basis of a unique interferon-β signaling axis mediated via the receptor IFNAR1.

    de Weerd NA, Vivian JP, Nguyen TK, Mangan NE, Gould JA, Braniff SJ, Zaker-Tabrizi L, Fung KY, Forster SC, Beddoe T, Reid HH, Rossjohn J and Hertzog PJ

    Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.

    Type I interferons are important in regulating immune responses to pathogens and tumors. All interferons are considered to signal via the heterodimeric IFNAR1-IFNAR2 complex, yet some subtypes such as interferon-β (IFN-β) can exhibit distinct functional properties, although the molecular basis of this is unclear. Here we demonstrate IFN-β can uniquely and specifically ligate to IFNAR1 in an IFNAR2-independent manner, and we provide the structural basis of the IFNAR1-IFN-β interaction. The IFNAR1-IFN-β complex transduced signals that modulated expression of a distinct set of genes independently of Jak-STAT pathways. Lipopolysaccharide-induced sepsis was ameliorated in Ifnar1(-/-) mice but not Ifnar2(-/-) mice, suggesting that IFNAR1-IFN-β signaling is pathologically relevant. Thus, we provide a molecular basis for understanding specific functions of IFN-β.

    Nature immunology 2013;14;9;901-7

  • RNA-eXpress annotates novel transcript features in RNA-seq data.

    Forster SC, Finkel AM, Gould JA and Hertzog PJ

    Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia, 3168.

    Next-generation sequencing is rapidly becoming the approach of choice for transcriptional analysis experiments. Substantial advances have been achieved in computational approaches to support these technologies. These approaches typically rely on existing transcript annotations, introducing a bias towards known genes, require specific experimental design and computational resources, or focus only on identification of splice variants (ignoring other biologically relevant transcribed features contained within the data that may be important for downstream analysis). Biologically relevant transcribed features also include large and small non-coding RNA, new transcription start sites, alternative promoters, RNA editing and processing of coding transcripts. Also, many existing solutions lack accessible interfaces required for wide scale adoption. We present a user-friendly, rapid and computation-efficient feature annotation framework (RNA-eXpress) that enables identification of transcripts and other genomic and transcriptional features independently of current annotations. RNA-eXpress accepts mapped reads in the standard binary alignment (BAM) format and produces a study-specific feature annotation in GTF format, comparison statistics, sequence extraction and feature counts. The framework is designed to be easily accessible while allowing advanced users to integrate new feature-identification algorithms through simple class extension, thus facilitating expansion to novel feature types or identification of study-specific feature types.

    Bioinformatics (Oxford, England) 2013;29;6;810-2

  • Interferome v2.0: an updated database of annotated interferon-regulated genes.

    Rusinova I, Forster S, Yu S, Kannan A, Masse M, Cumming H, Chapman R and Hertzog PJ

    Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.

    Interferome v2.0 (http://interferome.its.monash.edu.au/interferome/) is an update of an earlier version of the Interferome DB published in the 2009 NAR database edition. Vastly improved computational infrastructure now enables more complex and faster queries, and supports more data sets from types I, II and III interferon (IFN)-treated cells, mice or humans. Quantitative, MIAME compliant data are collected, subjected to thorough, standardized, quantitative and statistical analyses and then significant changes in gene expression are uploaded. Comprehensive manual collection of metadata in v2.0 allows flexible, detailed search capacity including the parameters: range of -fold change, IFN type, concentration and time, and cell/tissue type. There is no limit to the number of genes that can be used to search the database in a single query. Secondary analysis such as gene ontology, regulatory factors, chromosomal location or tissue expression plots of IFN-regulated genes (IRGs) can be performed in Interferome v2.0, or data can be downloaded in convenient text formats compatible with common secondary analysis programs. Given the importance of IFN to innate immune responses in infectious, inflammatory diseases and cancer, this upgrade of the Interferome to version 2.0 will facilitate the identification of gene signatures of importance in the pathogenesis of these diseases.

    Nucleic acids research 2013;41;Database issue;D1040-6

  • Helicobacter pylori VacA suppresses Lactobacillus acidophilus-induced interferon beta signaling in macrophages via alterations in the endocytic pathway.

    Weiss G, Forster S, Irving A, Tate M, Ferrero RL, Hertzog P, Frøkiær H and Kaparakis-Liaskos M

    Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Australia. gudrun.weiss@imperial.ac.uk

    Helicobacter pylori causes chronic gastritis and avoids elimination by the immune system of the infected host. The commensal bacterium Lactobacillus acidophilus has been suggested to exert beneficial effects as a supplement during H. pylori eradication therapy. In the present study, we applied whole-genome microarray analysis to compare the immune responses induced in murine bone marrow-derived macrophages (BMDMs) stimulated with L. acidophilus, H. pylori, or both bacteria in combination. While L. acidophilus induced a Th1-polarizing response characterized by high expression of interferon beta (IFN-β) and interleukin 12 (IL-12), H. pylori strongly induced the innate cytokines IL-1β and IL-1α. In BMDMs prestimulated with L. acidophilus, H. pylori blocked the expression of L. acidophilus-induced IFN-β and IL-12 and suppressed the expression of key regulators of the Rho, Rac, and Cdc42 GTPases. The inhibition of L. acidophilus-induced IFN-β was independent of H. pylori viability and the virulence factor CagPAI; however, a vacuolating cytotoxin (vacA) mutant was unable to block IFN-β. Confocal microscopy demonstrated that the addition of H. pylori to L. acidophilus-stimulated BMDMs redirects intracellular processing, leading to an accumulation of L. acidophilus in the endosomal and lysosomal compartments. Thus, our findings indicate that H. pylori inhibits the development of a strong Th1-polarizing response in BMDMs stimulated with L. acidophilus by blocking the production of IFN-β in a VacA-dependent manner. We suggest that this abrogation is caused by a redirection of the endocytotic pathway in the processing of L. acidophilus. IMPORTANCE Approximately half of the world's population is infected with Helicobacter pylori. The factors that allow this pathogen to persist in the stomach and cause chronic infections have not yet been fully elucidated. In particular, how H. pylori avoids killing by macrophages, one of the main types of immune cell underlying the epithelium, remains elusive. Here we have shown that the H. pylori virulence factor VacA plays a key role by blocking the activation of innate cytokines induced by the probiotic Lactobacillus acidophilus in macrophages and suppresses the expression of key regulators required for the organization and dynamics of the intracellular cytoskeleton. Our results identify potential targets for the treatment of H. pylori infection and vaccination, since specific inhibition of the toxin VacA possibly allows the activation of an efficient immune response and thereby eradication of H. pylori in the host.

    mBio 2013;4;3;e00609-12

  • Silencing of Irf7 pathways in breast cancer cells promotes bone metastasis through immune escape.

    Bidwell BN, Slaney CY, Withana NP, Forster S, Cao Y, Loi S, Andrews D, Mikeska T, Mangan NE, Samarajiwa SA, de Weerd NA, Gould J, Argani P, Möller A, Smyth MJ, Anderson RL, Hertzog PJ and Parker BS

    Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia; Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia.

    Breast cancer metastasis is a key determinant of long-term patient survival. By comparing the transcriptomes of primary and metastatic tumor cells in a mouse model of spontaneous bone metastasis, we found that a substantial number of genes suppressed in bone metastases are targets of the interferon regulatory factor Irf7. Restoration of Irf7 in tumor cells or administration of interferon led to reduced bone metastases and prolonged survival time. In mice deficient in the interferon (IFN) receptor or in natural killer (NK) and CD8(+) T cell responses, metastasis was accelerated, indicating that Irf7-driven suppression of metastasis was reliant on IFN signaling to host immune cells. We confirmed the clinical relevance of these findings in over 800 patients in which high expression of Irf7-regulated genes in primary tumors was associated with prolonged bone metastasis-free survival. This gene signature may identify patients that could benefit from IFN-based therapies. Thus, we have identified an innate immune pathway intrinsic to breast cancer cells, the suppression of which restricts immunosurveillance to enable metastasis.

    Funded by: Worldwide Cancer Research: 10-0510

    Nature medicine 2012;18;8;1224-31

  • Interferon signatures in immune disorders and disease.

    Forster S

    Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia. sam.forster@monash.edu

    The interferon (IFN) family and the type-I IFNs specifically have an important and well-characterized role in antiviral defence, immune modulation and cell-cycle control and are regularly applied in the clinical context. Advances in high-content technologies have facilitated an enhanced understanding of the global IFN response capable of being induced. Recent application of these technologies is improving our understanding of the specificity and subtleties associated with this response. This review considers our current understanding of the temporal gene profile induced through IFN stimulation across a diversity of disease conditions including autoimmune diseases, bacterial and viral infections. Understanding these signatures, the disease-specific differences and the biological effects induced has the potential to facilitate IFN-driven therapeutic development.

    Immunology and cell biology 2012;90;5;520-7

  • Systems biology of interferon responses.

    Hertzog P, Forster S and Samarajiwa S

    Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia. paul.hertzog@med.monash.edu.au

    The interferons (IFNs) are a pleiotropic family of cytokines that perform fundamental functions in protecting host organisms from disease and in maintaining homeostasis. Like other multifunctional cytokines, excessive or inappropriate activity can cause toxicity and even death. Therefore, host organisms have evolved specific and highly regulated mechanisms to control the temporal and tissue specificity of production of IFNs and the selection of pathways and genes to be activated as the effectors of the IFN response in cells. There are now numerous microarray datasets available to enable a "global" analysis of the genes involved in the IFN response. This article describes the INTERFEROME database, which assimilates the available expression profiling data and its contents and enables the definition of IFN-regulated genes, discovery of pathways, regulatory networks, and tissue specificities of the IFN response.

    Funded by: Cancer Research UK: 10825

    Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 2011;31;1;5-11

  • Toll-like receptors as interferon-regulated genes and their role in disease.

    Khoo JJ, Forster S and Mansell A

    Centre for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Victoria, Australia.

    The Toll-like receptors (TLRs) are innate sensors that recognize both microbial and endogenous ligands, initiating the host defense response. TLRs initiate the potent proinflammatory response to infection, are the target for adjuvants, and are essential for the establishment and maturation of adaptive immunity. As such they have been the interest of widespread research and the target of therapeutic intervention on multiple diseases. It has become apparent that expression of a subset of TLRs (TLR1, TLR2, TLR3, TLR5, and TLR7) is induced by Type I interferons (IFN). The role and impact of IFN expression on TLR responses is therefore critical in understanding the role of TLRs in disease, particularly as IFN itself is a downstream gene induced by specific TLRs. In this review we discuss the function and role of IFN-regulated TLRs in disease and how the role of IFN may impact upon TLR induction of the immune response in diseases, particularly in mouse models.

    Journal of interferon & cytokine research : the official journal of the International Society for Interferon and Cytokine Research 2011;31;1;13-25

  • INTERFEROME: the database of interferon regulated genes.

    Samarajiwa SA, Forster S, Auchettl K and Hertzog PJ

    Center for Innate Immunity and Infectious Diseases, Monash Institute of Medical Research, Monash University, Clayton, Victoria, Australia.

    INTERFEROME is an open access database of types I, II and III Interferon regulated genes (http://www.interferome.org) collected from analysing expression data sets of cells treated with IFNs. This database of interferon regulated genes integrates information from high-throughput experiments with annotation, ontology, orthologue sequences from 37 species, tissue expression patterns and gene regulatory information to enable a detailed investigation of the molecular mechanisms underlying IFN biology. INTERFEROME fulfils a need in infection, immunity, development and cancer research by providing computational tools to assist in identifying interferon signatures in gene lists generated by high-throughput expression technologies, and their potential molecular and biological consequences.

    Nucleic acids research 2009;37;Database issue;D852-7

Mark Stares

- Advanced Research Assistant

BSc - Biological Sciences MSc - Biotechnology Sanger Institute - 2008 to present

Research

Lab based Research Assistant, performing: anaerobic microbiology and microbiota/genomic sequencing.

References

  • Diarrhea in young children from low-income countries leads to large-scale alterations in intestinal microbiota composition.

    Pop M, Walker AW, Paulson J, Lindsay B, Antonio M, Hossain MA, Oundo J, Tamboura B, Mai V, Astrovskaya I, Corrada Bravo H, Rance R, Stares M, Levine MM, Panchalingam S, Kotloff K, Ikumapayi UN, Ebruke C, Adeyemi M, Ahmed D, Ahmed F, Alam MT, Amin R, Siddiqui S, Ochieng JB, Ouma E, Juma J, Mailu E, Omore R, Morris JG, Breiman RF, Saha D, Parkhill J, Nataro JP and Stine OC

    Background: Diarrheal diseases continue to contribute significantly to morbidity and mortality in infants and young children in developing countries. There is an urgent need to better understand the contributions of novel, potentially uncultured, diarrheal pathogens to severe diarrheal disease, as well as distortions in normal gut microbiota composition that might facilitate severe disease.

    Results: We use high throughput 16S rRNA gene sequencing to compare fecal microbiota composition in children under five years of age who have been diagnosed with moderate to severe diarrhea (MSD) with the microbiota from diarrhea-free controls. Our study includes 992 children from four low-income countries in West and East Africa, and Southeast Asia. Known pathogens, as well as bacteria currently not considered as important diarrhea-causing pathogens, are positively associated with MSD, and these include Escherichia/Shigella, and Granulicatella species, and Streptococcus mitis/pneumoniae groups. In both cases and controls, there tend to be distinct negative correlations between facultative anaerobic lineages and obligate anaerobic lineages. Overall genus-level microbiota composition exhibit a shift in controls from low to high levels of Prevotella and in MSD cases from high to low levels of Escherichia/Shigella in younger versus older children; however, there was significant variation among many genera by both site and age.

    Conclusions: Our findings expand the current understanding of microbiota-associated diarrhea pathogenicity in young children from developing countries. Our findings are necessarily based on correlative analyses and must be further validated through epidemiological and molecular techniques.

    Funded by: NHGRI NIH HHS: 5R01HG004885, 5R01HG005220; Wellcome Trust: WT098051

    Genome biology 2014;15;6;R76

  • Survey of culture, goldengate assay, universal biosensor assay, and 16S rRNA Gene sequencing as alternative methods of bacterial pathogen detection.

    Lindsay B, Pop M, Antonio M, Walker AW, Mai V, Ahmed D, Oundo J, Tamboura B, Panchalingam S, Levine MM, Kotloff K, Li S, Magder LS, Paulson JN, Liu B, Ikumapayi U, Ebruke C, Dione M, Adeyemi M, Rance R, Stares MD, Ukhanova M, Barnes B, Lewis I, Ahmed F, Alam MT, Amin R, Siddiqui S, Ochieng JB, Ouma E, Juma J, Mailu E, Omore R, O'Reilly CE, Hannis J, Manalili S, Deleon J, Yasuda I, Blyn L, Ranken R, Li F, Housley R, Ecker DJ, Hossain MA, Breiman RF, Morris JG, McDaniel TK, Parkhill J, Saha D, Sampath R, Stine OC and Nataro JP

    University of Maryland, School of Medicine, Baltimore, Maryland, USA.

    Cultivation-based assays combined with PCR or enzyme-linked immunosorbent assay (ELISA)-based methods for finding virulence factors are standard methods for detecting bacterial pathogens in stools; however, with emerging molecular technologies, new methods have become available. The aim of this study was to compare four distinct detection technologies for the identification of pathogens in stools from children under 5 years of age in The Gambia, Mali, Kenya, and Bangladesh. The children were identified, using currently accepted clinical protocols, as either controls or cases with moderate to severe diarrhea. A total of 3,610 stool samples were tested by established clinical culture techniques: 3,179 DNA samples by the Universal Biosensor assay (Ibis Biosciences, Inc.), 1,466 DNA samples by the GoldenGate assay (Illumina), and 1,006 DNA samples by sequencing of 16S rRNA genes. Each method detected different proportions of samples testing positive for each of seven enteric pathogens, enteroaggregative Escherichia coli (EAEC), enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), Shigella spp., Campylobacter jejuni, Salmonella enterica, and Aeromonas spp. The comparisons among detection methods included the frequency of positive stool samples and kappa values for making pairwise comparisons. Overall, the standard culture methods detected Shigella spp., EPEC, ETEC, and EAEC in smaller proportions of the samples than either of the methods based on detection of the virulence genes from DNA in whole stools. The GoldenGate method revealed the greatest agreement with the other methods. The agreement among methods was higher in cases than in controls. The new molecular technologies have a high potential for highly sensitive identification of bacterial diarrheal pathogens.

    Journal of clinical microbiology 2013;51;10;3263-9

  • Targeted restoration of the intestinal microbiota with a simple, defined bacteriotherapy resolves relapsing Clostridium difficile disease in mice.

    Lawley TD, Clare S, Walker AW, Stares MD, Connor TR, Raisen C, Goulding D, Rad R, Schreiber F, Brandt C, Deakin LJ, Pickard DJ, Duncan SH, Flint HJ, Clark TG, Parkhill J and Dougan G

    Wellcome Trust Sanger Institute, Hinxton, United Kingdom. tl2@sanger.ac.uk

    Relapsing C. difficile disease in humans is linked to a pathological imbalance within the intestinal microbiota, termed dysbiosis, which remains poorly understood. We show that mice infected with epidemic C. difficile (genotype 027/BI) develop highly contagious, chronic intestinal disease and persistent dysbiosis characterized by a distinct, simplified microbiota containing opportunistic pathogens and altered metabolite production. Chronic C. difficile 027/BI infection was refractory to vancomycin treatment leading to relapsing disease. In contrast, treatment of C. difficile 027/BI infected mice with feces from healthy mice rapidly restored a diverse, healthy microbiota and resolved C. difficile disease and contagiousness. We used this model to identify a simple mixture of six phylogenetically diverse intestinal bacteria, including novel species, which can re-establish a health-associated microbiota and clear C. difficile 027/BI infection from mice. Thus, targeting a dysbiotic microbiota with a defined mixture of phylogenetically diverse bacteria can trigger major shifts in the microbial community structure that displaces C. difficile and, as a result, resolves disease and contagiousness. Further, we demonstrate a rational approach to harness the therapeutic potential of health-associated microbial communities to treat C. difficile disease and potentially other forms of intestinal dysbiosis.

    Funded by: Medical Research Council: 93614, G0901743; Wellcome Trust: 076964, 098051

    PLoS pathogens 2012;8;10;e1002995

  • Dominant and diet-responsive groups of bacteria within the human colonic microbiota.

    Walker AW, Ince J, Duncan SH, Webster LM, Holtrop G, Ze X, Brown D, Stares MD, Scott P, Bergerat A, Louis P, McIntosh F, Johnstone AM, Lobley GE, Parkhill J and Flint HJ

    Pathogen Genomics, Wellcome Trust Sanger Institute, Cambridge, UK.

    The populations of dominant species within the human colonic microbiota can potentially be modified by dietary intake with consequences for health. Here we examined the influence of precisely controlled diets in 14 overweight men. Volunteers were provided successively with a control diet, diets high in resistant starch (RS) or non-starch polysaccharides (NSPs) and a reduced carbohydrate weight loss (WL) diet, over 10 weeks. Analysis of 16S rRNA sequences in stool samples of six volunteers detected 320 phylotypes (defined at >98% identity) of which 26, including 19 cultured species, each accounted for >1% of sequences. Although samples clustered more strongly by individual than by diet, time courses obtained by targeted qPCR revealed that 'blooms' in specific bacterial groups occurred rapidly after a dietary change. These were rapidly reversed by the subsequent diet. Relatives of Ruminococcus bromii (R-ruminococci) increased in most volunteers on the RS diet, accounting for a mean of 17% of total bacteria compared with 3.8% on the NSP diet, whereas the uncultured Oscillibacter group increased on the RS and WL diets. Relatives of Eubacterium rectale increased on RS (to mean 10.1%) but decreased, along with Collinsella aerofaciens, on WL. Inter-individual variation was marked, however, with >60% of RS remaining unfermented in two volunteers on the RS diet, compared to <4% in the other 12 volunteers; these two individuals also showed low numbers of R-ruminococci (<1%). Dietary non-digestible carbohydrate can produce marked changes in the gut microbiota, but these depend on the initial composition of an individual's gut microbiota.

    Funded by: Wellcome Trust: 076964, WT 76964

    The ISME journal 2011;5;2;220-30

* quick link - http://q.sanger.ac.uk/bactpath