“Whats new in Campylobacter
infection”
Andrew Fox
Health Protection /Agency
NorthWest
Laboratory reporting of selected GI pathogens
in England & Wales - 1977 to 2002.
60
Lab reports (1000's)
50
40
Campylobacters
Salmonellas
Rotavirus
Shigellas
Cryptosporidium
Norovirus
30
20
10
0
1977 1979 1981 1983 1985 1987 1989 1991 1993 1995 1997 1999 2001
Year
Campylobacter Statistics
50,000+ confirmed cases in England and Wales (CDR)
Estimated 400,000+ infections annually (IID study)
0.1% cases develop GBS (US estimate)
0-5% cases develop reactive arthritis (Scandinavia)
0.3-5.9% case develop bacteraemia (UK)
10% cases hospitalised, 5 days
Meningitis, Cholecystitis, Pancreatitis, Hepatitis,
Peritonitis, Myocarditis, Abcess ……
There’s a lot of it about…
BUT…
Common source outbreaks are rarely identified and
the source of the majority of cases reported in the UK
is unknown. In one case control study, epidemiology
of infection for over 60% of cases remained unknown.
Transmission pathways for
Campylobacter INFECTION
Campylobacter world
The world of Salmonella Enterica
Selective culture
Enrichment culture
S.typhimurium
DT104
S.ealing
S.goldcoast
S.virchow
Serotyping
Serotype specific phage typing
Plasmid profiling
Antibiograms
PFGE
S.typhi
S.enteritidis PT4
Campylobacter world
Selective culture
Enrichment culture (foods)
Typing methods
Campylobacter spp.
Phenotyping
• Serotyping
– Penner serotyping (HS)
• 65 serotypes
– Lior serotyping (HL)
• >100 serotypes
– Colindale (modified HS)
• Phagetyping
– Preston/Krakhria-Lior/Grajewski/Colindale
• Biotyping
– Preston
Problems with phenotyping
•
•
•
•
•
Isolates which fail to react
Need to constantly expand reagent panel
Limited availability of reagents
Lack of standardisation
Variable expression
Molecular sub-typing for C.jejuni
•
•
•
•
•
•
•
•
Restriction endonuclease analysis
RFLPs
Ribotyping
PFGE
But problems remain,
fla typing
Ambiguities need combinations
Standardisation
PCR RFLP
Roll out
RAPD
ERICs
Crossroads
control and prevention
sporadic infection
epidemiology
Ambiguous
typing
population
genetics
Campylobacter infection
Campylobacter isolate
characterisation
• Is central to disease surveillance and
epidemiology, requiring methods that
are
– accurate
– comprehensive
– reproducible
Multilocus sequence typing
(MLST)
Amplify  450-bp
fragments of seven
house-keeping genes
Chromosomal
DNA
Sequence the seven gene fragments on both strands
Compare sequences of each gene fragment with the known alleles at the locus
Assign alleles at the seven loci to give the allelic profile
Compare the allelic profile with those of isolates within a central database via the
internet and assign a sequence type (ST)
C. jejuni sequence types
Name
aspA
glnA
gltA
glyA
pgm
tkt
uncA
ST-21
ST-45
ST-206
ST-61
ST-48
ST-257
ST-353
ST-42
ST-403
ST-52
ST-177
ST-354
ST-22
ST-433
ST-362
ST-179
ST-49
2
4
2
1
2
9
7
1
10
9
17
8
1
2
1
1
3
1
7
21
4
4
2
17
2
27
25
2
10
3
59
2
6
1
1
10
5
2
1
4
5
3
16
2
8
2
6
4
49
7
5
3
4
37
2
2
62
2
4
19
10
5
2
4
38
4
2
17
2
1
2
6
7
4
10
5
10
22
8
11
3
17
11
40
11
1
7
1
3
1
5
3
9
5
3
2
12
3
12
66
32
11
5
1
5
17
5
6
6
3
7
6
4
6
3
35
8
3
6
Genetic diversity within Penner
serotypes
Penner serotype
1
2
4
5
6
Number of STs
32
33
12
8
8
Population diversity of C. jejuni
Serotype and phagetype
Diversity Index
0.989
Serotype, phage and biotype
0.997
Combined genotype
MLST
0.930
0.99
Do we need yet another typing
scheme for Campylobacter?
• A nucleotide sequence based approach
capitalises on technology which is largely
automated and increasingly applied to the
characterisation of pathogens
Advantages of MLST
• The technique is portable, reproducible and
relatively quick, easy and cheap to perform
• Unlike antigen and antibiotic resistance genes,
housekeeping genes are selectively neutral
• Freedom from reliance on serological typing
reagents which are becoming more difficult to
produce (recent changes in Animal Procedures
Act)
Frequency of clonal complexes in
different sample sources (n=160)
Source
ST-21
ST-45
ST-61
Ruminant
35 (41%)
11 (13%)
17 (20%)
Avian
3 (11%)
10 (35%)
_
Wild mammal
5 (21%)
9 (37%)
2 (9%)
Environment
1 (5%)
10 (48%)
1 (5%)
_
Frequency (%)
Comparison of the frequency of STcomplexes from animal and environmental
sources with human infections
50
45
40
35
30
25
20
15
10
5
0
Ruminant
Avian
Wild mammal
Environment
Human infection
ST-21
ST-45
ST-61
Preston 2000 isolates
Number of Clonal
Complexes
17
Number of Sequence
Types
58
Number of HS
serotypes
28
Preston 2000 isolates ST clusters versus
HS serotype
ST
No isolates
HS serotype
ST-104
5
HS 5(1); HS 16(1);
HS 50(2);HS NT(1)
ST-45
4
HS 12(3);HS NT(1)
ST-262
2
HS NT
ST-19
2
HS NT
Clonal Complexes for C.jejuni isolated
from GI infections in Preston area NW
England
25
20
15
10
5
0
ST-21
ST-45
ST-205
ST-47
ST-61
ST-19
ST-53
ST-311
ST-17
Comparison of clonal complexes for UK
human infections: 1991-2000
100%
UA
ST-61
ST-48
ST-45
ST-257
ST-21
ST-206
80%
60%
40%
20%
0%
1991
2000
Glastonbury outbreak
Investigation of outbreak isolates with MLST
Outbreak
Kettering 93
Kettering 93
Kettering 93
Kettering 93
Kettering 93
Kettering 93
Kettering 93
Kettering 93
Kettering 93
Kettering 93
Kettering 93
Kettering 93
glnA
21
21
21
7
21
21
21
7
21
21
21
21
gltA
5
3
5
3
5
3
5
3
5
3
5
3
glyA
37
37
37
4
37
37
37
4
37
37
37
37
pgm
2
2
2
8
2
2
2
8
2
2
2
2
tkt uncA ST
1
8
206
1
5
206
1
8
206
9
5
42
1
8
206
1
5
206
1
8
206
9
5
42
1
8
206
1
5
206
1
8
206
1
5
206
3
3
3
3
3
3
3
1
1
1
1
1
1
1
5
5
5
5
5
5
5
17
17
17
17
17
17
17
11
11
11
11
11
11
11
11
11
11
11
11
11
11
6
6
6
6
6
6
6
49
49
49
49
49
49
49
11
11
11
11
11
11
11
Glastonbury 93 1
Glastonbury 93 1
4
4
2
2
2
2
6
6
3
3
17
17
61
61
14
14
France
France
France
France
France
France
France
aspA
2
2
2
1
2
2
2
1
2
2
2
2
FlaSVR
11
11
11
9
11
11
11
9
11
11
11
11
Multilocus sequence typing of C.jejuni
ST 21 Complex
HS1
PT76
HS2
PT52
ST-61
HS4
PT121
HS4
PT55
HS11
PT90
HS19
PT90
ST 17
ST 22
Campylobacter detection and typing from
faeces or foods - future prospects
Detection and report of Camp spp.
by EIA or PCR
24 - 48 hours
DNA Purification and typing
24 - 48 hours
Further report to GP, EHO, CCDC, RE
Cluster analysis and investigation
24 hours
3 - 5 days
C. jejuni sequence types
Name
aspA
glnA
gltA
glyA
pgm
tkt
uncA
ST-21
ST-45
ST-206
ST-61
ST-48
ST-257
ST-353
ST-42
ST-403
ST-52
ST-177
ST-354
ST-22
ST-433
ST-362
ST-179
ST-49
2
4
2
1
2
9
7
1
10
9
17
8
1
2
1
1
3
1
7
21
4
4
2
17
2
27
25
2
10
3
59
2
6
1
1
10
5
2
1
4
5
3
16
2
8
2
6
4
49
7
5
3
4
37
2
2
62
2
4
19
10
5
2
4
38
4
2
17
2
1
2
6
7
4
10
5
10
22
8
11
3
17
11
40
11
1
7
1
3
1
5
3
9
5
3
2
12
3
12
66
32
11
5
1
5
17
5
6
6
3
7
6
4
6
3
35
8
3
6
AACTGGGTCCAGGCAACATCATTATCCGG
AACTGGGTCGAGGCAACATCATTATCCGG
Single Nucleotide Polymorphisms
Place and Time and Type
C.jejuni
HS11 PT1
7 cases in
20 days
21 cases all year
in NW and S
Lancs HA
The Iceland Experiment
1.Public awareness campaign
2.Frozen chicken
USDA Intervention studies
Norman Stern
• Novel biological agents to reduce or eliminate
Campylobacter from chicken intestinal flora
– Competitive exclusion
• Bacteriocins
– 15 trials
– Administer bacteriocin 5 days before slaughter
– 5-fold or total reduction in Campylobacter from
chicken at slaughter
Acknowledgements
Preston PHL
Eric Bolton
Roisin Ure
David Wareing (Dynal Biotech)
University of Staffordshire
Mishele Barrigas
Pete Gowland
DEFRA Epidemiology Unit,
University of Liverpool
Nigel French
Richard Kemp
Howard Leatherbarrow
WCIED, Oxford
Frances Colles
Kate Dingle
Martin Maiden
Rachel Urwin