Genomic epidemiology of Gram-negative

pathogens: from Acinetobacter to E. coli

Professor Mark Pallen, University of Birmingham

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

Declaration of potential

conflict of interest

Sequencing in Birmingham

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

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?

Acinetobacter Genomic Epidemiology

Outbreak in Birmingham Hospital in 2008

Isolates indistinguishable by current typing methods

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

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

Case Study 2: German E. coli O104:H4 outbreak

May-July 2011

>4000 cases

>40 deaths

Link to sprouting seeds

High risk of haemolytic-

uraemic syndrome

Females particularly at risk

Frank et al DOI: 10.1056/NEJMoa1106483

Crowd-sourcing the genome

BGI-Shenzhen UKE Hamburg The Crowd

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 open-

source wiki

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

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

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

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?

Benchtop sequencers

Ion Torrent MiSeq Q1 2011 Q3 2011

454Jr Q1 2010

Assemblies compared

Microbiology 2.0

MiSeq Stats Run 1 Run 2

Reads (million) 7.3 8.3

Read length 1 x 150 2 x 150

Throughput (mb) 1,106 2,460

Run time (hours) 14.5 27

Human reads 80% 33%

Culture-independent diagnosis

Mean 8.59

SD 18.32

Median 8

Coverage depth E. coli O104:H4 280

chromosome

Shiga-toxin encoding phage

Sample 1:

Laboratory

diagnosis:

E. coli O104:H4

Sample 2:

Laboratory diagnosis:

Salmonella enterica

Taxonomic

classification

within Bacteria

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?

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

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…

The promised land is in sight,

but still many rivers to cross… http://genomemedicine.com/content/3/8/53

pathogenomics.bham.ac.uk/blog

@mjpallen

@pathogenomenick

www.youtube.com/user/pallenm

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

#SAMG12

Acknowledgements

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

Nick Loman

Hospital Infection Society, MRC