statens serum institut spread of resistant e.coli and resistance genes from animals to humans anette...

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


Agenda• Danmap- brief history

• Olaquindox Resistance in Indicator E. colifrom 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


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

• Since 1996, DANMAP reported annually on the occurrence of antimicrobial resistance in pathogenic and indicator bacteria from animals, foods and humans in Denmark

Statens S

erum Institut


DANMAPDanish IntegratedAntimicrobialMonitoring andResearch Programme

DANMAP pdf-files: www.danmap.org


http://www.strama.se/news.asp

http://www.danmap.org

http://www.vetinst.no/Arkiv/Zoonosesenteret/NORM_NORM-VET_2003.pdf

http://www.swab.nl


Consumers are increasingly concernedabout food safety

Animal to human transfer of bacteria Animal to human transfer of bacteria (or resistance (or resistance determinants) determinants) carrying resistance to antimicrobials considered carrying resistance to antimicrobials considered critically important in human medicinecritically important in human medicine


Transfer of antimicrobial resistance between animals and humans

Statens S

erum Institut

National Center for Antimicrobials and Infection Control

EnvironmentEnvironment

Direct contactDirect contact


Public Health Concerns

Salmonella & E. coli

Fluroquinolones

3rd Gen. Cephalosporins

Aminoglycosides

Sulphonamides

Campylobacter

Fluroquinolones

Macrolides

Most G- Concerns relate to the THERAPEUTIC use of antibiotics in food animals

Enterococci,

Streptococci,

Staphylococci

Vancomycin

Streptogramins

Most G+ concerns relate to the GROWTH PROMOTER use of antibiotics


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-)


Olaquindox Resistance in Indicator E. colifrom Animals and Humans

• It has recently been shown that olaquindox resistance is encoded by oqxA and oqxB (efflux pump system)

• This system has been found in E. coli isolates from pigs.

Hansen et al. 2005 AAC


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


Trends in consumption of antimicrobials in food animals in Denmark

0

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To

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Therapeutic AGPs

AGP for Finisher pigsand broilersterminated

AGP for Weaner pigsterminated

Profit ontherapeuticsreduced


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


Veterinary practiceVeterinary practice

Private LaboratoriesPrivate Laboratories

Regional Food Regional Food Control LaboratoriesControl Laboratories

Regional hospital Regional hospital

laboratorieslaboratories

General practiceGeneral practice

DanishDanish InstituteInstitute for for Food and Food and VeterinaryVeterinary

researchresearch

Danish Medicines AgencyDanish Medicines Agency

SamplesSamples

IsolatesIsolates

DiagnosticDiagnosticsubmissionsubmission

IsolatesIsolates

SamplesSamples

SamplesSamples

Danish Zoonosis Danish Zoonosis CentreCentre

Slaughter plantsSlaughter plantsSamplesSamples

Hu

man

sH

um

ans

DataData

DataData

Foo

ds

Foo

ds

Foo

d a

nim

als

Foo

d a

nim

als

DataData

DataData

IsolatesIsolates

DataData

DataData

DataData

DANMAP - data flow

VetStatVetStat

DataData

Statens Serum InstitutStatens Serum Institut

DataData

DataData


Frederiksberg Municipality

RingkoebingFrederiksborg

Funen

Storstroem

Bornholm

Viborg

NorthJutland

Aarhus

Ribe

South Jutland Roskilde

Copenhagen MunicipalityWestZealand

Vejle Copenhagen County

-16 counties in DK

-15/16 clinical microbiology laboratories delivered data in 2004


Data Included in the DANMAP Integrated

surveillance

Origin

Indicator bacteria

Pathogens Consumption of antimicrobials

Humans + + +

Pigs (and pork) + + +

Poultry (and poultry meat) + + +

Cattle (and beef) + + +

Fish - - +


Indicator bacteria

• 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

Gram+ : E. faecium / E. faecalis

Gram - : E. coli


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 non-hospitalized persons was initiated in 2002


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


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 E-mail to DZC with code

Telephone interview / webDatabase (DZC)

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. enterococciPresence of enteric pathogens

Identification and testing


Respons rate

Year Invitered Answars Respons rate

2002 (from Marts)

760 111 14.6 %

2003 1055 130 12.3 %

2004 988 119 12.0 %


• 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

NOR-MAT: Results, 2002-2003


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


Comparison of Indicator E. coli from Pigs, Pork and Humans


Trends in Ampicillin Resistance among Indicator E. coli


Trends in Sulphomanide Resistance among Indicator E. coli


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)


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)


• 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.


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


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 :

Number of strains with more

than one sulR gene

sul1 sul2 sul3

Humans

35 40% 80% 0 20%

Pork 39 23% 74% 5% 5%

Pigs 162 53% 50% 11% 14%


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).


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


Fluoroquinolone consumption in DK – Primary health care, hospital and veterinary use

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

2001

Hospital

Primary health care

Veterinary use

Source: VetStat and The Danish Medicines Agency, 2004

Statens Serum Institut


Use of fluoroquinolones in animals in Denmark 2001 and 2002

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2001 2002

Source: VETSTAT 2001 & 2002


Companion animalsInvestigation 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.)


Consumption of ciprofloxacin and other fluoroquinolones in human therapy

Source: The Danish Medicines Agency, 2004

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Ciprofloxacin

Andrefluorquinoloner



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


0.5

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2.5

2002 (n=12.788) 2003 (n=13.081)

Cip

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DANMAP 2003: Ciprofloxacin resistance in Escherichia coli urine isolates from primary health care and hospital

Primary health care Hospital

Source: DANMAP report 2003

0.5

1.0

1.5

2.0

2.5

2002 (n=20.406) 2003 (n=22.933)

% C

ipro

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Data: The average number of ciprofloxacin resistant E. coli isolates from 7 clinical microbiology laberatories (with 95 % c.i.)



0 1 2 3 40

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Fluorquinolone useDDD/1,000 inhab.-days

% n

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Source: Kahlmeter et al. 2003. JAC vol. 52, p. 1005-1010 and ESAC

Fluoroquinolone use and nalidixan-R Escherichia coli, Europe (year 2000)



WWW.TRAINAU.dk


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


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.


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


P5-Animal and human models for studying the consequences of antimicrobial resistance on human health

P6-Mathematical models for assessing the impact of non-human antimicrobial resistance

P7-the effect of antimicrobial drug metabolites on selection of resistant bacteria


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

• 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


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


http://www.danmap.org

statens serum institut spread of resistant e.coli and resistance genes from animals to humans anette...

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