Recent evolution in invasive

Salmonellosis

Professor Gordon Dougan

Wellcome Trust Sanger Institute and

Cambridge University

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How does evolution deal with dogma

Statements that have launched a thousand grant applications

and thesis introductions:-

• ‘Salmonella typhimurium is a cause of gastroenteritis in

humans whereas Salmonella typhi is responsible for the

systemic disease typhoid……….’

Nature Immunology 3, 1026 - 1032 (2002) Chronic bacterial infections: living

with unwanted guests. Douglas Young, Tracy Hussell & Gordon Dougan

Comparison of pathogenesis of infection associated with

Salmonella typhimurium versus typhi

Human

Restricted

Typhimurium Typhi

Some pathogens are genetically monophyletic and cannot be differentiated by

MLST

Genome diversity in different microbes

0.0 0.1 0.2 0.3-0.4

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C jejuni

E. coliN. meningitidis

S. aureusS. pneumoniae

S. enterica

H. pylori

Y. enterocolitica

mean H

ratio

mean Ds

Y. pestis,

S. typhi

B. anthracis

M. tuberculosis

C. trachomatis

V. cholerae 01

Shigella sonnei

B. pertussis

Mark Achtman

Comparison of the Salmonella Typhi genomes of Ty2

(1912) and CT18 (1992) illustrates a conserved genome

Ty2

PAI

P4 phage

Although variation in Typhi is limited it can be defined and the

consequences assessed – a paradigm for other human

restricted pathogens?

4,700 Genes

MLST

Diversity

What is the evidence for vertical and horizontal

accumulation of mutation and diversity in S. Typhi?

strain 1 strain 2 strain 3 strain 4

Progenitor

Time

~30,000 yrs

IncH1

plasmids

Ab-R

Variation discovery in Typhi using sequencing of

200 genes and over 100 strains

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Found only 88 SNPs! In ~ 2% of the genome

Population structure – SNP analysis of Salmonella Typhi

Roumagnac et al (2006) Science 314 1301

Any new isolate can

be unequivocally

assigned to a

position in the tree

The position in the

tree is irrefutable

Ancestral root

SNP analysis of Salmonella Typhi linked to gyrase SNPs

NalR - gyrA mutations

Expanding SE Asian

Naladixic acid resistant

clones are H58

RootNalidixic acid

resistant

point mutations SNPs

Selection of strains for resequencing S.

Typhi

454 resequencing

Published sequence

Solexa resequencing

AV

AZ

A0

Roumagnac et al., 2006. Science.

Because we have a phylogenetic

Tree we can truly sample diversity

H58

SE Asia

Expansion

New tech re-sequencing of Salmonella Typhi

Kathryn Holt

2736 SNPs identified; 1815 SNPs used

S. Typhi phylogenetic tree after sequence analysis

SNP scale

Variation landscape in typhoid

Typhi is genetically isolated

with no evidence for antigenic

variation

The differential

accumulation of ~92

pseudogenes in different

arms of the tree is giving

rise to different

pathotypes?

Plasmids entering the tree

Accumulation of plasmids and mutation on the tree

We have different typhoid

disease severity in our part

of the world, is this due to

the host or the pathogen

genetics…???

180

~200

Typhi genotyping

resistance

plasmid

Typhi chromosome

SNPs

deletions

Can SNPs framework be used for association style

genetics as in mammals?

Haplotypes circulating in the Jatinegara district of Jakarta

over a two year period

1500 SNPs assayed at once for each sample (ugs) on Illumina

GoldenGate genotyping platform

Distribution of invasive salmonellosis disease

Non-typhoidal

Salmonellosis

The Gambia 1980s and 2005

Rwanda 1980s

Kenya 1980s

DRC 1990s Malawi 1996-2006

Nigeria 1970s

Ghana 1990s

Kenya 2000s

CAR 2002

Reports from tropical Africa of NTS as a major

cause of pediatric bacteraemia

Uganda 2006

Mozambique

Ethiopia

Egypt

NTS are the commonest cause of bacteraemia in

Malawi:

Blood isolates 0-14 years, 1996-2002

1,873

580189

356

152

300

873

557

NTS

S.pneumoniae

H.influenzae

S.aureus

N.meningitidis

Gp B strep

S.typhi

Other strep

Other GNR

Other

Typhimurium

Enteritidis

AIDS 2002, 16:1633–1641

• 77/78 HIV-infected

• 47% inpatient mortality

• 77% dead at 1 year

• 43% recurrence rate

• Recrudescence not reinfection

Death

Recurrence

NTS in HIV-positive adults

• 97% fever, 67% cough, 48% diarrhoea, 72% tachypnoea

• just ~18% of cases are associated with HIV infection

• 31% malaria slide positive

• 27% Anaemia

• 15% <60% weight-for-age, 51% 60-80% weight-for-age

• No diagnosis without

blood culture facility

• Overall mortality 23%

A diagnostic conundrum:

NTS in HIV-negative children

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onella

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ctera

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Age (months)

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log10

ch

ange

in S

alm

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lla (cfu

/ml)

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a16

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100

100

0anti-S

alm

onella

IgG

titer

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b

Cal MacLennan

In vitro models of Salmonella Bacteraemia

Whole blood assay

8 salmonella

Strains

Healthy adult

donor blood

Serum

Heat-inactivated

serum

+ C’

Whole blood

Serum

Washed Blood cells

resuspended

in RMPI

C9 deficient serum

+ purified C9

C9 deficient serum

Complement-mediated killing of salmonella

is dependent on classical pathway activity

CLASSICAL

(IgG, IgM)

MANNOSE

BINDING LECTIN

ALTERNATIVE

C3b

Membrane Attack

Complex

C5b-9

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% R

esis

tan

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Total S typhimurium Total S enteritidis

% amp resistant SEN % chl resistant SEN

% gent resistant SEN % cotrim resistant SEN

The emergence of drug resistant invasive salmonellosis in Malawi

as monitored by Wellcome Trust Laboratories, Blantyre

Many NTS strains circulating in Africa (Kenya/Malawi) have a

novel MLST type

ST313 NTS

Root

Classic gastroenteritis (DT104,

LT2, SL1344)

Plasmid, Pulse Field Gel Electrophoresis and Multi Locus

Sequence Typing Profiles

MDR, Cm-S MDR, Cm-R

Chloramphenicol treatment Fluoroquinolone treatment

Complete sequence of a Malawian MDR NTS S.

Typhimurium ST313

Rob Kingsley and Nick Thomson

S. Typhimurium D23580 has a unique phage repertoire

compared to other sequenced strains

DT104

D23580

1D/G 1D/G 1D/G1L

Most S. Typhimurium NTS isolates from Malawi between 2003-

2004 have identical pulse field and plasmid pattern to D23580V517

4.6

24.042.098.0

chr

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1.7

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Indistinguishable strains of S. typhimurium NTS

in HIV-positive adults and HIV-negative children

Kingsley et al, Figure 3

Antibiotic resistance

genes located on an

integron situated in the

virulence plasmid of S.

Typhimurium D23580

The invasion associated plasmid harbours a sophisticated mobile

cassette encoding antibiotic and disinfect resistance

ββββ-lactamase

aminoglycoside

sulphonamide

trimethoprim

streptomycin

chloramphenicol

quaternary ammonium

2004

Plasmid, Pulse Field Gel Electrophoresis and Multi Locus

Sequence Typing Profiles

MDR, Cm-S MDR, Cm-R

Chloramphenicol treatment Fluoroquinolone treatment

Mer R

Mercuryaminoglycoside quartenary ammonium

B-lactam

sulphonamide

orf121 qaCEblaOintI1 MerPCDSaadA1 sul3 tniA MerCMerAMer DMer EtniBistB istAsul1CDStnA

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Mer T

1997

Salmonella typhimurium isolated before 2004 have a different

element encoding MDR

D23580

SL1344, DT2, E.

coli K12

Lancet Infectious Disease2007, 7:686-93

The pressure for the use of prophylactic antibiotics in

HIV positive children

AcknowledgementsJulian Parkhill

Nick Thomson

Many in the PSU

Stephen Baker

Kathryn Holt

Tim Perkins

Robert Kingsley

Derek Pickard

Others in Team 15

Sam Kariuki

Chisomo Msefula

Malcolm Molyneux

Melita Gordon

Robert Heyderman

Cal MacLennan

Jeremy Farrar

Christiane Dolecek

Duncan Maskell

Bruce Stocker

The Non-typhoidal Salmonellosis problem

• Multi-drug resistance (chloramphenicol, ampicillin,

cotrimoxazole) >90% in Malawi

• Diagnostic difficulty. Non-specific presentation

• 23% case fatality rate with blood-culturing facility and

appropriate antibiotic therapy

• Rapid progression of clinical course

• Currently no vaccine against non-typhoidal salmonella

Clinical Infectious Diseases 2008; 46:963–9