Publications by authors named "Josephine M Bryant"

23 Publications

  • Page 1 of 1

Stepwise pathogenic evolution of .

Science 2021 04;372(6541)

Molecular Immunity Unit, University of Cambridge Department of Medicine, MRC Laboratory of Molecular Biology, Cambridge, UK.

Although almost all mycobacterial species are saprophytic environmental organisms, a few, such as , have evolved to cause transmissible human infection. By analyzing the recent emergence and spread of the environmental organism through the global cystic fibrosis population, we have defined key, generalizable steps involved in the pathogenic evolution of mycobacteria. We show that epigenetic modifiers, acquired through horizontal gene transfer, cause saltational increases in the pathogenic potential of specific environmental clones. Allopatric parallel evolution during chronic lung infection then promotes rapid increases in virulence through mutations in a discrete gene network; these mutations enhance growth within macrophages but impair fomite survival. As a consequence, we observe constrained pathogenic evolution while person-to-person transmission remains indirect, but postulate accelerated pathogenic adaptation once direct transmission is possible, as observed for Our findings indicate how key interventions, such as early treatment and cross-infection control, might restrict the spread of existing mycobacterial pathogens and prevent new, emergent ones.
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http://dx.doi.org/10.1126/science.abb8699DOI Listing
April 2021

Preadaptation of pandemic GII.4 noroviruses in unsampled virus reservoirs years before emergence.

Virus Evol 2020 Jul 21;6(2):veaa067. Epub 2020 Nov 21.

Division of Infection and Immunity, University College London, London WC1E 6BT, UK.

The control of re-occurring pandemic pathogens requires understanding the origins of new pandemic variants and the factors that drive their global spread. This is especially important for GII.4 norovirus, where vaccines under development offer promise to prevent hundreds of millions of annual gastroenteritis cases. Previous studies have hypothesized that new GII.4 pandemic viruses arise when previously circulating pandemic or pre-pandemic variants undergo substitutions in antigenic regions that enable evasion of host population immunity, as described by conventional models of antigenic drift. In contrast, we show here that the acquisition of new genetic and antigenic characteristics cannot be the proximal driver of new pandemics. Pandemic GII.4 viruses diversify and spread over wide geographical areas over several years prior to simultaneous pandemic emergence of multiple lineages, indicating that the necessary sequence changes must have occurred before diversification, years prior to pandemic emergence. We confirm this result through serological assays of reconstructed ancestral virus capsids, demonstrating that by 2003, the ancestral 2012 pandemic strain had already acquired the antigenic characteristics that allowed it to evade prevailing population immunity against the previous 2009 pandemic variant. These results provide strong evidence that viral genetic changes are necessary but not sufficient for GII.4 pandemic spread. Instead, we suggest that it is changes in host population immunity that enable pandemic spread of an antigenically preadapted GII.4 variant. These results indicate that predicting future GII.4 pandemic variants will require surveillance of currently unsampled reservoir populations. Furthermore, a broadly acting GII.4 vaccine will be critical to prevent future pandemics.
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http://dx.doi.org/10.1093/ve/veaa067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7751145PMC
July 2020

Human cytomegalovirus haplotype reconstruction reveals high diversity due to superinfection and evidence of within-host recombination.

Proc Natl Acad Sci U S A 2019 03 28;116(12):5693-5698. Epub 2019 Feb 28.

Division of Infection and Immunity, University College London, London WC1E 6BT, United Kingdom;

Recent sequencing efforts have led to estimates of human cytomegalovirus (HCMV) genome-wide intrahost diversity that rival those of persistent RNA viruses [Renzette N, Bhattacharjee B, Jensen JD, Gibson L, Kowalik TF (2011) 7:e1001344]. Here, we deep sequence HCMV genomes recovered from single and longitudinally collected blood samples from immunocompromised children to show that the observations of high within-host HCMV nucleotide diversity are explained by the frequent occurrence of mixed infections caused by genetically distant strains. To confirm this finding, we reconstructed within-host viral haplotypes from short-read sequence data. We verify that within-host HCMV nucleotide diversity in unmixed infections is no greater than that of other DNA viruses analyzed by the same sequencing and bioinformatic methods and considerably less than that of human immunodeficiency and hepatitis C viruses. By resolving individual viral haplotypes within patients, we reconstruct the timing, likely origins, and natural history of superinfecting strains. We uncover evidence for within-host recombination between genetically distinct HCMV strains, observing the loss of the parental virus containing the nonrecombinant fragment. The data suggest selection for strains containing the recombinant fragment, generating testable hypotheses about HCMV evolution and pathogenesis. These results highlight that high HCMV diversity present in some samples is caused by coinfection with multiple distinct strains and provide reassurance that within the host diversity for single-strain HCMV infections is no greater than for other herpesviruses.
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http://dx.doi.org/10.1073/pnas.1818130116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431178PMC
March 2019

Direct Whole-Genome Sequencing of Sputum Accurately Identifies Drug-Resistant Mycobacterium tuberculosis Faster than MGIT Culture Sequencing.

J Clin Microbiol 2018 08 26;56(8). Epub 2018 Jul 26.

Division of Infection and Immunity, University College London, London, United Kingdom.

The current methods available to diagnose antimicrobial-resistant infections require a positive culture or only test a limited number of resistance-associated mutations. A rapid accurate identification of antimicrobial resistance enables the prompt initiation of effective treatment. Here, we determine the utility of whole-genome sequencing (WGS) of directly from routinely obtained diagnostic sputum samples to provide a comprehensive resistance profile compared to that from mycobacterial growth indicator tube (MGIT) WGS. We sequenced from 43 sputum samples by targeted DNA enrichment using the Agilent SureSelectXT kit, and 43 MGIT positive samples from each participant. Thirty two (74%) sputum samples and 43 (100%) MGIT samples generated whole genomes. The times to antimicrobial resistance profiles and concordance were compared with Xpert MTB/RIF and phenotypic resistance testing from cultures of the same samples. Antibiotic susceptibility could be predicted from WGS of sputum within 5 days of sample receipt and up to 24 days earlier than WGS from MGIT culture and up to 31 days earlier than phenotypic testing. Direct sputum results could be reduced to 3 days with faster hybridization and if only regions encoding drug resistance are sequenced. We show that direct sputum sequencing has the potential to provide comprehensive resistance detection significantly faster than MGIT whole-genome sequencing or phenotypic testing of resistance from cultures in a clinical setting. This improved turnaround time enables prompt appropriate treatment with associated patient and health service benefits. Improvements in sample preparation are necessary to ensure comparable sensitivities and complete resistance profile predictions in all cases.
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http://dx.doi.org/10.1128/JCM.00666-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6062781PMC
August 2018

Genomic epidemiology of a national outbreak of post-surgical wound infections in Brazil.

Microb Genom 2017 05 3;3(5):e000111. Epub 2017 May 3.

1​Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

An epidemic of post-surgical wound infections, caused by a non-tuberculous mycobacterium, has been on-going in Brazil. It has been unclear whether one or multiple lineages are responsible and whether their wide geographical distribution across Brazil is due to spread from a single point source or is the result of human-mediated transmission. 188 isolates, collected from nine Brazilian states, were whole genome sequenced and analysed using phylogenetic and comparative genomic approaches. The isolates from Brazil formed a single clade, which was estimated to have emerged in 2003. We observed temporal and geographic structure within the lineage that enabled us to infer the movement of sub-lineages across Brazil. The genome size of the Brazilian lineage was reduced relative to most strains in the three subspecies of and contained a novel plasmid, pMAB02, in addition to the previously described pMAB01 plasmid. One lineage, which emerged just prior to the initial outbreak, is responsible for the epidemic of post-surgical wound infections in Brazil. Phylogenetic analysis indicates that multiple transmission events led to its spread. The presence of a novel plasmid and the reduced genome size suggest that the lineage has undergone adaptation to the surgical niche.
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http://dx.doi.org/10.1099/mgen.0.000111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562415PMC
May 2017

You are where you live.

Nat Rev Microbiol 2017 Jan;15(2):68

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

This month's Genome Watch discusses how whole-genome sequencing of bacteria from several body sites has provided insights into the spatial diversity of bacteria within patients.
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http://dx.doi.org/10.1038/nrmicro.2016.203DOI Listing
January 2017

Emergence and spread of a human-transmissible multidrug-resistant nontuberculous mycobacterium.

Science 2016 11;354(6313):751-757

Royal Brompton and Harefield NHS Foundation Trust, UK.

Lung infections with Mycobacterium abscessus, a species of multidrug-resistant nontuberculous mycobacteria, are emerging as an important global threat to individuals with cystic fibrosis (CF), in whom M. abscessus accelerates inflammatory lung damage, leading to increased morbidity and mortality. Previously, M. abscessus was thought to be independently acquired by susceptible individuals from the environment. However, using whole-genome analysis of a global collection of clinical isolates, we show that the majority of M. abscessus infections are acquired through transmission, potentially via fomites and aerosols, of recently emerged dominant circulating clones that have spread globally. We demonstrate that these clones are associated with worse clinical outcomes, show increased virulence in cell-based and mouse infection models, and thus represent an urgent international infection challenge.
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http://dx.doi.org/10.1126/science.aaf8156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5142603PMC
November 2016

Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus.

Front Microbiol 2016 9;7:1317. Epub 2016 Sep 9.

Infection, Immunity, Inflammation and Physiological Medicine, Institute of Child Health, University College LondonLondon, UK; Division of Infection and Immunity, University College LondonLondon, UK; Great Ormond Street Hospital for Children NHS Foundation TrustLondon, UK.

Human cytomegalovirus (HCMV) is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed pediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1-27 weeks. Changes in consensus sequence and resistance mutations were analyzed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54) and C480F (UL97). In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of 11 subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome.
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http://dx.doi.org/10.3389/fmicb.2016.01317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016526PMC
September 2016

Phylogenomic exploration of the relationships between strains of Mycobacterium avium subspecies paratuberculosis.

BMC Genomics 2016 Jan 26;17:79. Epub 2016 Jan 26.

Moredun Research Institute, Pentlands Science Park, Penicuik, EH26 0PZ, UK.

Background: Mycobacterium avium subspecies paratuberculosis (Map) is an infectious enteric pathogen that causes Johne's disease in livestock. Determining genetic diversity is prerequisite to understanding the epidemiology and biology of Map. We performed the first whole genome sequencing (WGS) of 141 global Map isolates that encompass the main molecular strain types currently reported. We investigated the phylogeny of the Map strains, the diversity of the genome and the limitations of commonly used genotyping methods.

Results: Single nucleotide polymorphism (SNP) and phylogenetic analyses confirmed two major lineages concordant with the former Type S and Type C designations. The Type I and Type III strain groups are subtypes of Type S, and Type B strains are a subtype of Type C and not restricted to Bison species. We found that the genome-wide SNPs detected provided greater resolution between isolates than currently employed genotyping methods. Furthermore, the SNP used for IS1311 typing is not informative, as it is likely to have occurred after Type S and C strains diverged and does not assign all strains to the correct lineage. Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR) differentiates Type S from Type C but provides limited resolution between isolates within these lineages and the polymorphisms detected do not necessarily accurately reflect the phylogenetic relationships between strains. WGS of passaged strains and coalescent analysis of the collection revealed a very high level of genetic stability, with the substitution rate estimated to be less than 0.5 SNPs per genome per year.

Conclusions: This study clarifies the phylogenetic relationships between the previously described Map strain groups, and highlights the limitations of current genotyping techniques. Map isolates exhibit restricted genetic diversity and a substitution rate consistent with a monomorphic pathogen. WGS provides the ultimate level of resolution for differentiation between strains. However, WGS alone will not be sufficient for tracing and tracking Map infections, yet importantly it can provide a phylogenetic context for affirming epidemiological connections.
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http://dx.doi.org/10.1186/s12864-015-2234-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4729121PMC
January 2016

Personalizing therapy for multidrug resistant TB: the potential of Rapid Whole Genome Sequencing.

Expert Rev Anti Infect Ther 2016 27;14(1):1-3. Epub 2015 Nov 27.

a Division of Infection and Immunity , UCL , London , UK.

Multidrug resistant tuberculosis is an increasing problem globally. The current gold standard in drug sensitivity testing is slow and cumbersome. To tackle drug resistance effectively, a more rapid method of testing is required. Current molecular tests are fast, but only offer information on a limited number of genetic loci. Whole genome sequencing presents an attractive alternative that can provide comprehensive, clinically relevant information on all described loci. Although the standard approach to whole genome sequencing of Mycobacterium tuberculosis is slow due to the requirement of culture, this article will describe recent advances that mean it has the potential to provide results within days.
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http://dx.doi.org/10.1586/14787210.2016.1116385DOI Listing
October 2016

Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

J Clin Microbiol 2015 Jul 13;53(7):2230-7. Epub 2015 May 13.

UCL, Division of Infection and Immunity, London, United Kingdom.

The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.
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http://dx.doi.org/10.1128/JCM.00486-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4473240PMC
July 2015

Genomic Investigations unmask Mycoplasma amphoriforme, a new respiratory pathogen.

Clin Infect Dis 2015 Feb 23;60(3):381-8. Epub 2014 Oct 23.

Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

Background: Mycoplasma amphoriforme has been associated with infection in patients with primary antibody deficiency (PAD). Little is known about the natural history of infection with this organism and its ability to be transmitted in the community.

Methods: The bacterial load was estimated in sequential sputum samples from 9 patients by quantitative polymerase chain reaction. The genomes of all available isolates, originating from patients in the United Kingdom, France, and Tunisia, were sequenced along with the type strain. Genomic data were assembled and annotated, and a high-resolution phylogenetic tree was constructed.

Results: By using high-resolution whole-genome sequencing (WGS) data, we show that patients can be chronically infected with M. amphoriforme manifesting as a relapsing-remitting bacterial load, interspersed by periods when the organism is undetectable. Importantly, we demonstrate transmission of strains within a clinical environment. Antibiotic resistance mutations accumulate in isolates taken from patients who received multiple courses of antibiotics.

Conclusions: Mycoplasma amphoriforme isolates form a closely related species responsible for a chronic relapsing and remitting infection in PAD patients in the United Kingdom and from immunocompetent patients in other countries. We provide strong evidence of transmission between patients attending the same clinic, suggesting that screening and isolation may be necessary for susceptible patients. This work demonstrates the critical role that WGS can play in rapidly unraveling the biology of a novel pathogen.
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http://dx.doi.org/10.1093/cid/ciu820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4293396PMC
February 2015

Pre-Columbian mycobacterial genomes reveal seals as a source of New World human tuberculosis.

Nature 2014 Oct 20;514(7523):494-7. Epub 2014 Aug 20.

1] Department of Archaeological Sciences, University of Tübingen, Ruemelinstraße 23, 72070 Tübingen, Germany [2] Senckenberg Centre for Human Evolution and Palaeoenvironment, University of Tübingen, Tübingen 72070, Germany [3] Max Planck Institute for Science and History, Khalaische Straße 10, 07745 Jena, Germany.

Modern strains of Mycobacterium tuberculosis from the Americas are closely related to those from Europe, supporting the assumption that human tuberculosis was introduced post-contact. This notion, however, is incompatible with archaeological evidence of pre-contact tuberculosis in the New World. Comparative genomics of modern isolates suggests that M. tuberculosis attained its worldwide distribution following human dispersals out of Africa during the Pleistocene epoch, although this has yet to be confirmed with ancient calibration points. Here we present three 1,000-year-old mycobacterial genomes from Peruvian human skeletons, revealing that a member of the M. tuberculosis complex caused human disease before contact. The ancient strains are distinct from known human-adapted forms and are most closely related to those adapted to seals and sea lions. Two independent dating approaches suggest a most recent common ancestor for the M. tuberculosis complex less than 6,000 years ago, which supports a Holocene dispersal of the disease. Our results implicate sea mammals as having played a role in transmitting the disease to humans across the ocean.
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http://dx.doi.org/10.1038/nature13591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4550673PMC
October 2014

Comment on: characterization of the embB gene in Mycobacterium tuberculosis isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array.

J Antimicrob Chemother 2014 Aug 20;69(8):2298-9. Epub 2014 Apr 20.

Department of Medicine, University of Cambridge, Cambridge, UK Wellcome Trust Sanger Institute, Hinxton, UK Clinical Microbiology and Public Health Laboratory, Public Health England, Cambridge, UK Cambridge University Hospitals NHS Foundation Trust, Cambridge, UK.

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http://dx.doi.org/10.1093/jac/dku101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4100706PMC
August 2014

Whole-genome sequencing to establish relapse or re-infection with Mycobacterium tuberculosis: a retrospective observational study.

Lancet Respir Med 2013 Dec 21;1(10):786-92. Epub 2013 Nov 21.

Wellcome Trust Sanger Institute, Hinxton, UK.

Background: Recurrence of tuberculosis after treatment makes management difficult and is a key factor for determining treatment efficacy. Two processes can cause recurrence: relapse of the primary infection or re-infection with an exogenous strain. Although re-infection can and does occur, its importance to tuberculosis epidemiology and its biological basis is still debated. We used whole-genome sequencing-which is more accurate than conventional typing used to date-to assess the frequency of recurrence and to gain insight into the biological basis of re-infection.

Methods: We assessed patients from the REMoxTB trial-a randomised controlled trial of tuberculosis treatment that enrolled previously untreated participants with Mycobacterium tuberculosis infection from Malaysia, South Africa, and Thailand. We did whole-genome sequencing and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing of pairs of isolates taken by sputum sampling: one from before treatment and another from either the end of failed treatment at 17 weeks or later or from a recurrent infection. We compared the number and location of SNPs between isolates collected at baseline and recurrence.

Findings: We assessed 47 pairs of isolates. Whole-genome sequencing identified 33 cases with little genetic distance (0-6 SNPs) between strains, deemed relapses, and three cases for which the genetic distance ranged from 1306 to 1419 SNPs, deemed re-infections. Six cases of relapse and six cases of mixed infection were classified differently by whole-genome sequencing and MIRU-VNTR. We detected five single positive isolates (positive culture followed by at least two negative cultures) without clinical evidence of disease.

Interpretation: Whole-genome sequencing enables the differentiation of relapse and re-infection cases with greater resolution than do genotyping methods used at present, such as MIRU-VNTR, and provides insights into the biology of recurrence. The additional clarity provided by whole-genome sequencing might have a role in defining endpoints for clinical trials.

Funding: Wellcome Trust, European Union, Medical Research Council, Global Alliance for TB Drug Development, European and Developing Country Clinical Trials Partnership.
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http://dx.doi.org/10.1016/S2213-2600(13)70231-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3861685PMC
December 2013

Culture-free club.

Nat Rev Microbiol 2013 Jul 10;11(7):434. Epub 2013 Jun 10.

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

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http://dx.doi.org/10.1038/nrmicro3052DOI Listing
July 2013

Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study.

Lancet 2013 May 29;381(9877):1551-60. Epub 2013 Mar 29.

Wellcome Trust Sanger Institute, Hinxton, UK.

Background: Increasing numbers of individuals with cystic fibrosis are becoming infected with the multidrug-resistant non-tuberculous mycobacterium (NTM) Mycobacterium abscessus, which causes progressive lung damage and is extremely challenging to treat. How this organism is acquired is not currently known, but there is growing concern that person-to-person transmission could occur. We aimed to define the mechanisms of acquisition of M abscessus in individuals with cystic fibrosis.

Method: Whole genome sequencing and antimicrobial susceptibility testing were done on 168 consecutive isolates of M abscessus from 31 patients attending an adult cystic fibrosis centre in the UK between 2007 and 2011. In parallel, we undertook detailed environmental testing for NTM and defined potential opportunities for transmission between patients both in and out of hospital using epidemiological data and social network analysis.

Findings: Phylogenetic analysis revealed two clustered outbreaks of near-identical isolates of the M abscessus subspecies massiliense (from 11 patients), differing by less than ten base pairs. This variation represents less diversity than that seen within isolates from a single individual, strongly indicating between-patient transmission. All patients within these clusters had numerous opportunities for within-hospital transmission from other individuals, while comprehensive environmental sampling, initiated during the outbreak, failed to detect any potential point source of NTM infection. The clusters of M abscessus subspecies massiliense showed evidence of transmission of mutations acquired during infection of an individual to other patients. Thus, isolates with constitutive resistance to amikacin and clarithromycin were isolated from several individuals never previously exposed to long-term macrolides or aminoglycosides, further indicating cross-infection.

Interpretation: Whole genome sequencing has revealed frequent transmission of multidrug resistant NTM between patients with cystic fibrosis despite conventional cross-infection measures. Although the exact transmission route is yet to be established, our epidemiological analysis suggests that it could be indirect.

Funding: The Wellcome Trust, Papworth Hospital, NIHR Cambridge Biomedical Research Centre, UK Health Protection Agency, Medical Research Council, and the UKCRC Translational Infection Research Initiative.
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http://dx.doi.org/10.1016/S0140-6736(13)60632-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3664974PMC
May 2013

Inferring patient to patient transmission of Mycobacterium tuberculosis from whole genome sequencing data.

BMC Infect Dis 2013 Feb 27;13:110. Epub 2013 Feb 27.

Background: Mycobacterium tuberculosis is characterised by limited genomic diversity, which makes the application of whole genome sequencing particularly attractive for clinical and epidemiological investigation. However, in order to confidently infer transmission events, an accurate knowledge of the rate of change in the genome over relevant timescales is required.

Methods: We attempted to estimate a molecular clock by sequencing 199 isolates from epidemiologically linked tuberculosis cases, collected in the Netherlands spanning almost 16 years.

Results: Multiple analyses support an average mutation rate of ~0.3 SNPs per genome per year. However, all analyses revealed a very high degree of variation around this mean, making the confirmation of links proposed by epidemiology, and inference of novel links, difficult. Despite this, in some cases, the phylogenetic context of other strains provided evidence supporting the confident exclusion of previously inferred epidemiological links.

Conclusions: This in-depth analysis of the molecular clock revealed that it is slow and variable over short time scales, which limits its usefulness in transmission studies. However, the superior resolution of whole genome sequencing can provide the phylogenetic context to allow the confident exclusion of possible transmission events previously inferred via traditional DNA fingerprinting techniques and epidemiological cluster investigation. Despite the slow generation of variation even at the whole genome level we conclude that the investigation of tuberculosis transmission will benefit greatly from routine whole genome sequencing.
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http://dx.doi.org/10.1186/1471-2334-13-110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3599118PMC
February 2013

The genome of Mycobacterium africanum West African 2 reveals a lineage-specific locus and genome erosion common to the M. tuberculosis complex.

PLoS Negl Trop Dis 2012 28;6(2):e1552. Epub 2012 Feb 28.

Wellcome Trust Genome Campus, Wellcome Trust Sanger Institute, Hinxton, UK.

Background: M. africanum West African 2 constitutes an ancient lineage of the M. tuberculosis complex that commonly causes human tuberculosis in West Africa and has an attenuated phenotype relative to M. tuberculosis.

Methodology/principal Findings: In search of candidate genes underlying these differences, the genome of M. africanum West African 2 was sequenced using classical capillary sequencing techniques. Our findings reveal a unique sequence, RD900, that was independently lost during the evolution of two important lineages within the complex: the "modern" M. tuberculosis group and the lineage leading to M. bovis. Closely related to M. bovis and other animal strains within the M. tuberculosis complex, M. africanum West African 2 shares an abundance of pseudogenes with M. bovis but also with M. africanum West African clade 1. Comparison with other strains of the M. tuberculosis complex revealed pseudogenes events in all the known lineages pointing toward ongoing genome erosion likely due to increased genetic drift and relaxed selection linked to serial transmission-bottlenecks and an intracellular lifestyle.

Conclusions/significance: The genomic differences identified between M. africanum West African 2 and the other strains of the Mycobacterium tuberculosis complex may explain its attenuated phenotype, and pave the way for targeted experiments to elucidate the phenotypic characteristic of M. africanum. Moreover, availability of the whole genome data allows for verification of conservation of targets used for the next generation of diagnostics and vaccines, in order to ensure similar efficacy in West Africa.
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http://dx.doi.org/10.1371/journal.pntd.0001552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3289620PMC
June 2012

Atypical at skew in Firmicute genomes results from selection and not from mutation.

PLoS Genet 2011 Sep 15;7(9):e1002283. Epub 2011 Sep 15.

Department of Biology and Biochemistry, University of Bath, Bath, United Kingdom.

The second parity rule states that, if there is no bias in mutation or selection, then within each strand of DNA complementary bases are present at approximately equal frequencies. In bacteria, however, there is commonly an excess of G (over C) and, to a lesser extent, T (over A) in the replicatory leading strand. The low G+C Firmicutes, such as Staphylococcus aureus, are unusual in displaying an excess of A over T on the leading strand. As mutation has been established as a major force in the generation of such skews across various bacterial taxa, this anomaly has been assumed to reflect unusual mutation biases in Firmicute genomes. Here we show that this is not the case and that mutation bias does not explain the atypical AT skew seen in S. aureus. First, recently arisen intergenic SNPs predict the classical replication-derived equilibrium enrichment of T relative to A, contrary to what is observed. Second, sites predicted to be under weak purifying selection display only weak AT skew. Third, AT skew is primarily associated with largely non-synonymous first and second codon sites and is seen with respect to their sense direction, not which replicating strand they lie on. The atypical AT skew we show to be a consequence of the strong bias for genes to be co-oriented with the replicating fork, coupled with the selective avoidance of both stop codons and costly amino acids, which tend to have T-rich codons. That intergenic sequence has more A than T, while at mutational equilibrium a preponderance of T is expected, points to a possible further unresolved selective source of skew.
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http://dx.doi.org/10.1371/journal.pgen.1002283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3174206PMC
September 2011