Genomic epidemiology of Gram-negative pathogens: from Acinetobacter to E. coli. Professor Mark Pallen, University of Birmingham
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1 Genomic epidemiology of Gram-negative pathogens: from Acinetobacter to E. coli Professor Mark Pallen, University of Birmingham
2 The state we are in: diagnostic microbiology 21st Century problem, but 19th Century techniques! Microscopy and culture on solid media date from the time of Koch and Pasteur
3 Declaration of potential conflict of interest
4
5 Sequencing in Birmingham
6 Case Study Acinetobacter baumannii Gram-negative bacillus Multi-drug resistant colistin and tigecycline as reserve agents moving towards pan-resistance Associated with wound infections and ventilator-associated pneumonia bloodstream infections returning military personnel from Iraq and Afghanistan transmission from military to civilian patients
7 Applications and Questions Epidemiology Q1: Can whole-genome sequencing detect differences between isolates within an outbreak? Q2: Can these differences be used to help determine chains of transmission? Emergence of Resistance Q3: Can it reveal how resistance emerges? Taxonomy and Identification Q4: Can it tell us what defines a species within a genus?
8 Acinetobacter Genomic Epidemiology Outbreak in Birmingham Hospital in 2008 Isolates indistinguishable by current typing methods
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11 Before and after tigecycline therapy Eighteen SNPs detected between AB210 and AB211 nine non-synonymous including a SNP in ades which accounts for resistance phenotype Three contigs in AB210 not covered by reads in AB211, representing three deletions of ~15, 44,17 kb muts truncated; likely increase in mutation rate
12 Phylogenomics of species within genus Acinetobacter 13 new draft genome sequences from 10 species Analyzed genomes from 38 Acinetobacter strains Conclusions 16S sequences not capable of delineating accepted species core-genome phylogenetic tree consistent with currently accepted taxonomy, while also identifying 3 misclassifications average-nucleotide identity (ANI) quickly delivered results consistent with traditional and phylogenetic classifications In this genus it appears to be possible to delineate and define species by genome sequence alone
13 Case Study 2: German E. coli O104:H4 outbreak May-July 2011 >4000 cases >40 deaths Link to sprouting seeds High risk of haemolyticuraemic syndrome Females particularly at risk Frank et al DOI: /NEJMoa
14 Crowd-sourcing the genome UKE Hamburg BGI-Shenzhen The Crowd
15 Crowd-sourcing the genome Within 24 hours of its release, the genome is assembled Within two days, assigned to an existing lineage Within five days, strain-specific diagnostic test released Within a week, two-dozen reports on the biology and evolution of the strain had been filed on an opensource wiki
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17 Squaring the circle A few Tweets and blog posts do not equate to a peer-reviewed research publication Paper for NEJM written up to include case study of a family outbreak Journey, not just destination analyses repeated in house by Nick Loman
18 Take-away messages Infection still threat even to the most advanced societies Pathogens don t bother with passports! Not a new strain: something similar seen in Germany ten years ago closest genome-sequenced strain was isolated from Central African Republic in late 1990s, belongs to an enteroaggregative lineage German STEC from lineage circulating in human populations rather than from animal source (cf E. coli O157) Bacteria evolve quickly Virulence factors in E. coli can jump from one lineage to another on mobile genetic elements Pathotypes can overlap and evolve Antibiotic resistance seen where no obvious prior use of antibiotics
19 Take-away messages Open-Source Genomics a propitious confluence of high-throughput genomics crowd-sourced analyses a liberal approach to data release Social media (blogging, Twitter) can augment usual channels of academic discourse
20 Take-away messages We were not the first other crowdsourced science projects see Jennifer Gardy TEDx on Public Health 2.0 But have we broken the mould? appropriate for public heath emergencies but not for ordinary science The cite or site dilemma remains have we really shown the finger to Ingelfinger?
21 Benchtop sequencers 454Jr Q Ion Torrent MiSeq Q Q3 2011
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23 Assemblies compared
24 Microbiology 2.0
25 Culture-independent diagnosis MiSeq Stats Run 1 Run 2 Reads (million) Read length 1 x x 150 Throughput (mb) 1,106 2,460 Run time (hours) Human reads 80% 33%
26 Sample 1: Laboratory diagnosis: E. coli O104:H4 Coverage depth E. coli O104:H4 280 chromosome Shiga-toxin encoding phage Mean 8.59 SD Median 8
27 Taxonomic classification within Bacteria Sample 2: Laboratory diagnosis: Salmonella enterica
28 Opportunities Genome sequencing brings the advantages of open-endedness (revealing the unknown unknowns ), universal applicability ultimate in resolution Benchtop sequencing poised to revolutionise microbiology Platforms have defined strengths and weaknesses You ain t seen nothing yet! Oxford nanopore: hope or hype?
29 Take-away messages Bacteria evolve quickly Virulence factors in E. coli can jump from one lineage to another on mobile genetic elements Pathotypes can overlap and evolve Antibiotic resistance seen where no obvious prior use of antibiotics
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31 Challenges Genotype does not always predict phenotype Heterogeneity in bacterial populations (cf Amerithrax) Sometimes indistinguishable really does mean identical Sequencing a genome means different things on different platforms Clinical microbiology remains a conservative discipline How to move from ivory tower to real world? How to communicate with clinicians? Radio has survived alongside TV
32 The promised land is in sight, but still many rivers to cross
33 pathogenomics.bham.ac.uk/blog #SAMG12 We were united by a common love of Science, which we thought sufficient to bring together persons of all distinctions, Christians, Jews, Muslims and Heathens, Monarchists and Republicans. Joseph Priestley on the Lunar Society of Birmingham, 1793
34 Acknowledgements Hospital Infection Society, MRC Nick Loman Thomas Lewis, Deborah Mortiboy, Pauline Jumaa, Lewis Bingle, Charles Penn, Chrystala Constantinidou, Mala Patel, George Weinstock, Neil Hall, Jon Wain, Beryl Oppenheim, Jackie Chan, David Wareham, Neil Woodford, Mike Hornsey, Martin Aepfelbacher
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