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STATENS SERUM INSTITUT
Spread of Resistant E.coli and Resistance
Genes from Animals to Humans
Anette M. Hammerum
National Center for Antimicrobials and Infection Control
Statens Serum Institut, Copenhagen, Denmark
STATENS SERUM INSTITUT
Agenda
• Danmap- brief history
• Olaquindox Resistance in Indicator E. coli
from Animals and Humans
• Normat-study
• Comparison of Indicator E. coli from Pigs, Pork and Humans
• Detection of sul genes in pig, pork and humans
• Flouroquinolones in animals and humans
• TRAINAU
STATENS SERUM INSTITUT
DANMAP – brief history
• The Danish Integrated Antimicrobial Resistance
Monitoring and Research Programme (DANMAP)
was established by the Danish Ministry of Food,
Agriculture and Fisheries and the Danish Ministry of
Health in 1995
Statens Serum Institut
• Since 1996, DANMAP reported annually on the
occurrence of antimicrobial resistance in pathogenic
and indicator bacteria from animals, foods and
humans in Denmark
STATENS SERUM INSTITUT
DANMAP
Danish Integrated
Antimicrobial
Monitoring and
Research Programme
DANMAP pdf-files:
www.danmap.org
STATENS SERUM INSTITUT
http://www.danmap.org
http://www.strama.se/news.asp
http://www.vetinst.no/Arkiv/Zoonosesenteret/
NORM_NORM-VET_2003.pdf
http://www.swab.nl
STATENS SERUM INSTITUT
Consumers are increasingly concerned
about food safety
Animal to human transfer of bacteria (or resistance
determinants) carrying resistance to antimicrobials considered
critically important in human medicine
National
Center
for Antimicrobials
STATENS
SERUM
INSTITUTand Infection Control
Transfer of antimicrobial resistance
between animals and humans
Statens Serum Institut
Environment
Direct contact
STATENS SERUM INSTITUT
Public Health Concerns
Salmonella & E. coli
Enterococci,
Fluroquinolones
Streptococci,
3rd Gen. Cephalosporins
Staphylococci
Aminoglycosides
Vancomycin
Sulphonamides
Streptogramins
Campylobacter
Fluroquinolones
Macrolides
Most G- Concerns relate to the
THERAPEUTIC use of antibiotics in
food animals
Most G+ concerns relate to the
GROWTH PROMOTER use of
antibiotics
STATENS SERUM INSTITUT
Antimicrobial Growth Promoters (AGP)
Antimicrobial substances used as a supplement in
animal feed in sub-therapeutic concentrations
Avoparcin (G+)
Spiramycin (G+)
Bacitracin (G+)
Avilamycin (G+)
Virginiamycin (G+)
Flavomycin (G+)
Tylosin (G+)
Carbadox (G-)
Olaquindox (G-)
STATENS SERUM INSTITUT
Olaquindox Resistance in Indicator E. coli
from Animals and Humans
• It has recently been shown that olaquindox resistance is
encoded by oqxA and oqxB (efflux pump system)
Hansen et al. 2005 AAC
• This system has been found in E. coli isolates from pigs.
STATENS SERUM INSTITUT
Olaquindox resistence in E. coli isolates from
humans
• Olaquindox resistance can been found in persons with
close contact to animals and in person with no contact to
pigs
STATENS SERUM INSTITUT
AGP for
Finisher pigs
and broilers
terminated
250
AGP for
Weaner pigs
terminated
200
150
100
50
Therapeutic
AGPs
20
04
20
03
20
02
20
01
20
00
19
99
19
98
19
97
19
96
19
94
Profit on
therapeutics
reduced
19
92
0
19
90
Tonnes (active component)
Trends in consumption of antimicrobials
in food animals in Denmark
STATENS SERUM INSTITUT
Effect of the AGP Stop on Antimicrobial Consumption
and Occurrence of Resistance in Food Animals in
Denmark
• Total antimicrobial consumption in animals reduced by >50% from 1994
to 2002
• Slow increase in consumption of antimicrobials for therapy in pigs - No
increase in broilers
• Substantial quantitative reduction of antimicrobial resistance in bacteria
from animals and food
STATENS SERUM INSTITUT
DANMAP - data flow
Diagnostic
submission
Veterinary practice
Data
Samples
Private Laboratories
Isolates
Data
Danish Institute for
Food and Veterinary
research
Data
Data
Slaughter plants
Samples
Isolates
Regional Food
Control Laboratories
Regional hospital
laboratories
Danish Zoonosis
Centre
Data
Isolates
Data
Data
Statens Serum Institut
Data
Samples
General practice
Data
Samples
Danish Medicines Agency
VetStat
Data
STATENS SERUM INSTITUT
-16 counties in DK
-15/16 clinical
microbiology laboratories
delivered data in 2004
North
Jutland
Bornholm
Viborg
Aarhus
Frederiksborg
Ringkoebing
Copenhagen County
Vejle
Ribe
West
Zealand
Copenhagen Municipality
Frederiksberg Municipality
Funen
South
Jutland
Roskilde
Storstroem
STATENS SERUM INSTITUT
Data Included in the DANMAP Integrated
surveillance
Origin
Humans
Indicator Pathogens Consumption of
bacteria
antimicrobials
+
+
+
Pigs (and pork)
+
+
+
Poultry (and poultry meat)
+
+
+
Cattle (and beef)
+
+
+
Fish
-
-
+
STATENS SERUM INSTITUT
Indicator bacteria
Gram+ : E. faecium / E. faecalis
Gram - : E. coli
• Easily obtained from various animal species as
well as from humans
• Good ability to acquire resistance to various
antimicrobial substances
• Easily obtained from healthy individuals
STATENS SERUM INSTITUT
Indicator Bacteria in DANMAP
• E. coli (and Enterococcus faecalis og Enterococcus faecium)
from meat and healthy animals has been sampled since
1996, whereas a continuously sampling of faces from nonhospitalized persons was initiated in 2002
STATENS SERUM INSTITUT
Normal Flora Study at SSI
Methods:
• Volunteers were found through the Danish Civil Registry system, which
is a continuously updated register of all residents in DK.
• The scientific ethics committee for Copenhagen and Frederiksberg
municipalities approved the protocol
• Age and gender profile of the invited volunteers were in compliance
with the total Danish population
STATENS SERUM INSTITUT
Selection of persons via CPR
(DZC)
1st Letter:
Invitation + Declaration of consent
Declaration of consent to DZC
2nd Letter: Material for faeces sample
+ questionnaire (with code)
Faeces sample sent to SSI
NOR-MAT, 2002-ongoing
 One random E. coli, E. faecium and E.
faecalis isolate / sample
 MIC determination (Sensititre) for growth
promoters and antibiotics used for therapy
 A selective method to detect vancomycin res.
enterococci
Presence of enteric pathogens
E-mail to DZC with code
Identification and testing
Database (DZC)
Telephone interview / web
STATENS SERUM INSTITUT
Respons rate
Year
Invitered
Answars
Respons rate
2002
(from Marts)
760
111
14.6 %
2003
1055
130
12.3 %
2004
988
119
12.0 %
STATENS SERUM INSTITUT
NOR-MAT: Results, 2002-2003
• From March 2002 to December 31, 2003, 1815 individuals were invited to
participate in the study
• 248 (13.7%) responded to the letter
• In total, 93 E. faecium isolates, 119 E. faecalis isolates, and 195 E. coli
isolates were obtained
STATENS SERUM INSTITUT
NOR-MAT: Results, 2002-2003
• A single E. faecalis isolate was gentamicin-resistant and one
vancomycin resistant E. faecium isolate was detected (selective
method)
• 10% E. coli isolates were multi-resistant
• The carrier-rate of Salmonella and Campylobacter in the normal
population is lower than 1%, however several pathogenic E. coli
was identified
STATENS SERUM INSTITUT
Comparison of Indicator E. coli from
Pigs, Pork and Humans
STATENS SERUM INSTITUT
Trends in Ampicillin Resistance among Indicator
E. coli
STATENS SERUM INSTITUT
Trends in Sulphomanide Resistance among
Indicator E. coli
STATENS SERUM INSTITUT
Detection of sul1, sul2 and sul3 in sulphonamide
resistant Escherichia coli isolates obtained from
healthy humans, pork and pigs in Denmark
(Hammerum et al. accepted for publication)
STATENS SERUM INSTITUT
Background
• In 2003, antibiotic treatment of pigs accounted for almost 80% of the total
amount of antibiotics administered to food animals in Denmark.
• The combination of sulphonamide and trimethroprim was the second
most used antimicrobial therapy for sows and piglets in 2003 (approx. 4
tonnes)
STATENS SERUM INSTITUT
• Sulphonamide or trimethroprim are used for treatment of the majority
uncomplicated urinary tract infections of humans in Denmark
(approx. 3 tonnes)
• Potential transfer of sulphonamide resistant (sulR) Escherichia coli
from animals, directly or via handling of raw meat, to humans is
therefore undesirable.
• SulR is often encoded by sul1 and sul2 in Enterobacteriaceae.
Recently a third gene, sul3, was found to encode for sulR.
STATENS SERUM INSTITUT
Materials:
• 998 E. coli isolates, obtained from pig faeces collected at slaughter,
Danish pork collected at retail outlets and from faeces from healthy
persons in Denmark
• All sulphonamide resistant E. coli isolates were investigated for the
presence of sul1, sul2, and sul3 genes by PCR
STATENS SERUM INSTITUT
Prevalence of sul1, sul2 and sul3 in 236
Sulphonamide Resistant (sulR) E. coli isolates
Origin
No. of isolates
tested
No. of isolates PCR-positive
for :
sul1
sul2
sul3
Number of strains with
more
than one sulR gene
Humans
35
40%
80%
0
20%
Pork
39
23%
74%
5%
5%
Pigs
162
53%
50%
11%
14%
STATENS SERUM INSTITUT
Discussion and Conclusion:
• The proportion of sulR E. coli isolates obtained from faeces of pigs
increased significantly from 23% to 31% from 2002 to 2003 (DANMAP
2003). This coincided with an increased consumption of sulphonamide
and trimethroprim by pigs.
• Urinary tract infections are generally thought of as infections caused by
bacteria from the host’s own faecal flora (Murray et al. 2004).
• A recent study has demonstrated a close resemblance between certain
food borne and human isolates of extra intestinal pathogenic E. coli, which
suggests a possible transmission of E. coli causing urinary tract infections
through food products (Johnson et al. 2005).
STATENS SERUM INSTITUT
Discussion and Conclusion:
• The presence of sulR E. coli in faeces from pigs, in pork and in the gut
flora of healthy humans is therefore of concern
• The origin of the sulphonamide resistance genes found in isolates
from healthy human E. coli isolates is speculative, but spread via the
food chain seems very likely
Statens
SerumSERUM
Institut INSTITUT
STATENS
Fluoroquinolone consumption in DK –
Primary health care, hospital and veterinary use
2001
Veterinary
use
Primary
health care
Veterinary
use
2002
Veterinary
use
Primary
health care
Hospital
2003
Hospital
Hospital
Primary
health care
Year
Veterinary use
(kg)
Primary health care
(kg)
Hospital (kg)
Total
2001
185
220
115
520
2002
97
238
143
478
2003
53
348
154
555
Source: VetStat and The Danish Medicines Agency, 2004
STATENS SERUM INSTITUT
Use of fluoroquinolones in animals in
Denmark 2001 and 2002
20
18
14
12
10
8
6
4
December
November
October
August
July
2002
September
2001
May
April
March
February
0
June
2
January
kg active compound
16
Source: VETSTAT 2001 & 2002
STATENS SERUM INSTITUT
Companion animals
Investigation of previously neglected reservoirs:
•
Companion animals (dogs, cats, etc.): VETSTAT data indicates large
consumption of fluoroquinolones and cephalosporins in companion
animals
•
•
In 2003 danes had in total 550,000 dogs, 650,00 cats
24 milllion slaughter pigs, 130 million broiler chickens, 1.2 million
cattle and dairy cows were produced during 2003
Antimicrobials
Consumption in
companion animals
(kg)
Consumption in food
animals and
companion animals
(kg)*
Cephalosporins
254
461
Fluoroquinolones
24
53
(Heuer, Jensen and Hammerum 2005) EID, vol.11, no 2.)
Statens
SerumSERUM
Institut INSTITUT
STATENS
Consumption of ciprofloxacin and other
fluoroquinolones in human therapy
Consumption
DDD/1,000 inhab.-days
0,35
0,3
0,25
Ciprofloxacin
0,2
0,15
0,1
0,05
jan
-1
se 994
p-1
9
m a 94
j -1
9
jan 95
-1
se 996
p-1
9
m a 96
j -1
9
jan 97
-19
9
se
p-1 8
m a 998
j -1
9
jan 99
-2
se 000
p-2
0
m a 00
j -2
0
jan 01
-20
0
se
p-2 2
0
m a 02
j -2
0
jan 03
-20
04
0
Source: The Danish Medicines Agency, 2004
Andre
fluorquinoloner
STATENS SERUM INSTITUT
Ciprofloxacin consumption in primary health care
- Price versus consumption
Source: Muscat M, et al. EPI-NEWS 2004, week 41. Available at: URL:
http://www.ssi.dk/graphics/en/news/epinews/2004/pdf/2004_41.pdf
Statens
SerumSERUM
Institut INSTITUT
STATENS
DANMAP 2003: Ciprofloxacin resistance in
Escherichia coli urine isolates from primary health
care and hospital
Data: The average number of ciprofloxacin resistant E. coli isolates from 7
clinical microbiology laberatories (with 95 % c.i.)
Hospital
Primary health care
2.5
% Cipro-R E.coli
Cipro-R E.coli
2.5
2.0
1.5
1.0
0.5
2.0
1.5
1.0
0.5
2002 (n=12.788)
2003 (n=13.081)
Source: DANMAP report 2003
2002 (n=20.406)
2003 (n=22.933)
Statens
SerumSERUM
Institut INSTITUT
STATENS
% nalidixan resistant
E. coli from uncomplicated
urinary tract infections
Fluoroquinolone use and nalidixan-R Escherichia
coli, Europe (year 2000)
30
25
20
15
10
5
0
0
1
2
3
4
Fluorquinolone use
DDD/1,000 inhab.-days
Source: Kahlmeter et al. 2003. JAC vol. 52, p. 1005-1010 and ESAC
STATENS SERUM INSTITUT
WWW.TRAINAU.dk
STATENS SERUM INSTITUT
What is TRAINAU?
• TRAINAU is a multidisciplinary early-stage research training (EST)
site on identification, characterisation and assessment of public
health risks associated with non-human antimicrobial usage, i.e.
antimicrobial use in animals
• The project will bring together young researchers with different
backgrounds (veterinary and medical microbiology, epidemiology,
statistics, pharmacology, and analytical chemistry) and provide them
with the scientific and technological competences necessary to
obtain substantial progress in this field
STATENS SERUM INSTITUT
STATENS SERUM INSTITUT
Seven PhD Fellowships in Denmark
Open for Applications
Location: Students will primarily be enrolled at the Royal
Veterinary and Agricultural University, but the research activities
will also be carried out at one or more of the TRAINAU
institutions: The Royal Veterinary and Agricultural University,
Statens Serum Institut, The Danish University of Pharmaceutical
Sciences & Danish Institute for Food and Veterinary Research all
situated in Copenhagen, Denmark.
STATENS SERUM INSTITUT
List of proposed PhD projects:
P1-Transmission of antimicrobial resistant bacteria and resistance
genes through the food chain
P2-Transmission of antimicrobial resistant bacteria and resistance
genes by contact with animals and by environmental exposure
P3- In vivo and in vitro models for investigatigating the impact of
different veterinary treatment strategies on development of
antimicrobial resistance
P4-Associations between non-human antimicrobial resistance usage
and occurence of resistant bacteria in animals and food
STATENS SERUM INSTITUT
P5-Animal and human models for studying the consequences
of antimicrobial resistance on human health
P6-Mathematical models for assessing the impact of nonhuman antimicrobial resistance
P7-the effect of antimicrobial drug metabolites on selection of
resistant bacteria
STATENS SERUM INSTITUT
Is it relevant to consider animals in relation
to antimicrobial resistance in humans?…
Yes!
• Antimicrobial resistance in bacterial populations comes from
exposure to selective pressure
• Substantial amounts of antimicrobials are used in animals
worldwide (Therapy, Prophylaxis and Growth Promotion) creating
large resevoirs of antimicrobial resistance
STATENS SERUM INSTITUT
Thanks to:
Karin Sixhøj Pedersen, Frank Hansen, Stine Frese-Madsen, Aviaja Maya
Sicka, Rikke Kubert and Inge Hansen for technical assistance
Lone Jannok Porsbo, Dorthe Sandvang, Ole E. Heuer, Line Bagger-Skjøt, Christian Brandt, Lotte Jakobsen, Sigrid R.
Andersen, Anne Mette Seyfarth,Eleni Galanis, Katharina E. P. Olsen, Jens S. Andersen, Vibeke F. Jensen, Jakob Neimann,
Lars Bogø Jensen, Lars Hestbjerg Hansen, Dominique L. Monnet, Henrik C. Wegener and Niels Frimodt-Møller for help
with data handling and scientific input
STATENS SERUM INSTITUT
http://www.danmap.org