Antimicrobial Resistance: Do We Know Everything? - Dr. Sid Thakur, Assistant Professor, North Carolina State University, from the 2013 NIAA Merging Values and Technology conference, April 15-17, 2013, Louisville, KY, USA. More presentations at http://www.trufflemedia.com/agmedia/conference/2013-niaa-merging-values-and-technology

1. Antimicrobial Resistance:
Do we know everything?
Dr. Sid Thakur
Assistant Professor
Swine Health & Production
CVM, NCSU

2. Research Focus
Antimicrobial Resistance
Pathogen detection, Phenotypic &
Genotypic Characterization
On
farm, Slaughter, Retail, Human
Sample & Environment
Molecular Characterization:
Mechanisms of resistance
 Transfer of resistance
 Virulence genes
Develop Sensitive and Rapid
Detection Tests
 Microarray
 Real-time PCR
Global Epidemiology

4. Antimicrobial Resistance:
Lost in Translation !!!
Are bacterial pathogens smarter than humans ?

5. Pathogen Evolution:
How good bacteria go bad
 Difference between good and bad pathogen
 Few genes encoded on plasmids or chromosome
 E. coli (commensal) : E. coli O157: H7 (HUS)
Stephens & Murray, Current Biology, 2001 Raskin et al., Cell, 2006

6. EID Global Trends1940-2004
 EID are dominated by zoonoses (60.3 %)
Jones et al.,
Nature, 2008
Drug
Resistant
Zoonotic
Wildlife

7.  NDM-1 (New Delhi metallo- β- Lactamase)
 Escherichia coli and Klebsiella pneumoniae
 Dec 2009 in India; Pakistan, US, UK, Japan, Brazil
 International Travel
Emergence of a new AR
mechanism in India
Number of cases by Country
Sweden (Dec 2009): 1st report
India (March 2010): 1st report
USA (June 2010): 3 cases
India (June 2010): More cases
UK (Aug 2010): 37 cases
Canada (Aug 2010): 1st report
Japan (Sept 2010): 1 report
All the patients had a travel history to
India within the past one year

8. Antimicrobials: Farm to Table
Sanitizers Disinfectants
Food Tissue Treatments
Aquaculture
Animal
Agriculture
Humans
Plant
Agriculture Food
retail
Food
Service
Food
Processing
Home
Antibiotics
Biocides Food Grade
Preservatives
Antimicrobial soaps/lotions
Personal hygiene
products
Food Tissue Treatments
Sanitizers Disinfectants
Food Tissue Treatments
Sanitizers Disinfectants
Sanitizers Disinfectants
Fungicides

9. Back to susceptible population:
Is it so simple?
Antimicrobial
Resistant
Antimicrobial
Susceptible
Antimicrobial
Exposure
Treatment
Environment
Treatment
Treatment
Antimicrobial
Resistant
Antimicrobial
Resistant

10. Reservoirs of Antimicrobial
Resistance determinants
 Focus is on the commodity
 Food animals, produce, humans
 Role of Environment
• Limited research conducted
• Need to focus more on the environment
• Study environmental reservoirs
• Food animals: Farm environment (lagoon, truck, lairage)
• Produce: Water for irrigation, birds, manure
• Humans: Hospital environment, community

11. Soil Antibiotic Resistosome
Vanessa et al.,
Science, 2006

12. Bacteria subsisting on antibiotics
Dantas et al., Science, 2008

13. Role of trucks in
Pathogen dissemination
 All in all out swine production system
 Four truck wash systems were selected that
service one integrated system.
– Samples obtained from each wash station
• 10 trucks pre-wash, 5 swabs from each
• 10 trucks post-wash, 5 swabs from each
 Fecal samples were also collected on farm, at
slaughter (Cecal content & MLN) & lairage
Applied Environmental Microbiology, 2009
Breeding/Farrowing Nursery Finishing

14. Role of Truck Wash
in Pathogen Dissemination
0
10
20
30
40
50
60
70
80
90
100
%Positive
Truck Wash A Truck Wash B Truck Wash C Truck Wash D
Truck Wash Salmonella Prevalence - Pre and Post Wash
Pre Wash
Post Wash
A = recycled water and Virkon-S B = recycled water and phenol
C = fresh water, soap, and phenol D = recycled water and phenol
0
10
20
30
40
50
60
70
80
90
100
%Positive
Truck Wash A Truck Wash B Truck Wash C Truck Wash D
Truck Wash Campylobacter Prevalence - Pre and Post Wash
Pre Wash
Post Wash
 Salmonella and Campylobacter isolates
at farm were different from those isolated
at the slaughter plant (Cecal content and MLN)
 Isolates from the Cecum and MLN were
similar to those isolated from the truck floor

15. Plasmid exchange in
Salmonella positive pigs
S. Typhimurium S. Muenchen
Ch. Pl Ch. Pl
1 2 3 4
- 23 kb
- 9.4 kb
-6.5 kb
-4.3 kb
-2.3 kb
-2.0 kb
Conjugation Analysis
Southern Hybridization
aphA1-Iab probe
AKSSuT ACKSSuT
Gebreyes and Thakur, AEM, 2005

16. DANMAP Data-2011
DANMAP, 2011

17. S. Typhimurium Antimicrobial
Resistance profile: Pigs
DANMAP, 2011

18. S. Typhimurium AR:
Pigs, pork and human cases
DANMAP, 2011

19. Campylobacter jejuni:
Antimicrobial Resistance
DANMAP, 2011

20. FQ consumption: Humans
DANMAP, 2011

21. DANMAP 2011 Report:
Key Highlights
DANMAP, 2011

22. NARMS Retail meat (Poultry)
Antimicrobial Resistance Data
C. jejuni
CIP/NAL

23. Are resistant pathogens are
better fit to survive in vivo ?
Luo, et al., PNAS, 2005
 No Fluoroquinolone (FQ) selection pressure in this model
FQ sensitive
C. jejuni
FQ resistant
C. jejuni
FQ sensitive
and FQ resistant
C. jejuni
FQ sensitive
C. jejuni
FQ resistant
C. jejuni
FQ resistant
C. jejuni

24. Antibiotic Exposed Antibiotic-free (ABF)
 Use of antimicrobials
 Intensive
 Indoor
 No use of antimicrobials
 Extensive
 Outdoor
USDA-NIFA Study
Salmonella, Campylobacter & Clostridium
VS

25. Sampling Scheme
Conventional Farm ( All-in -All-out)
10 cohorts (35 pigs/cohort)
Farrowing Nursery FinishingTruckTruck
8 days old 6,10 weeks 16,26 weeks
Lairage
Slaughter
Truck Kill Floor
Pre-Evis ChillingPost-Evis
MLN, Carcass Swab Carcass Swab
Environment samples ( n=5 each) at farm
Feed Water Swab Lagoon Soil Truck
Environment samples ( n=5 each) at Slaughter
Lairage Truck

26. Antimicrobial Free System
water, feed, swabs,
soil (n=5 each)
Lariage
Truck (n=5 each)
Environment at Farm and Slaughter
water, feed, swabs,
soil (n=5 each)
water, feed, swabs,
soil (n=5 each)
Farrowing—Nursery--
Finishing
Rotate on
new land
SlaughterTruck
• Post evis
• MLN
• Post chill
8 cohorts
35 pigs/cohort

27. Antimicrobials
AZI CIP ERY GEN TET FFN NAL TEL CLI MDR
Resistance%
0
20
40
60
80
100
ABF
Conventional
Pig v/s Environment
Antimicrobials
AZI CIP ERY GEN TET FFN NAL TEL CLI MDRResistance%
0
20
40
60
80
100
ABF
Conventional
EnvironmentSwine
AZI = azithromycin, ERY = erythromycin, CIP = ciprofloxacin , GEN = gentamicin, TET =
tetracycline, FFN = florfenicol, NAL = nalidixic acid, TEL = telithromycin, CLI = clindamycin;
Macarena and Thakur, Applied Environmental Microbiology, 2012

28. Key Outcomes
Campylobacter and C. difficile
Pig
Environment
Antimicrobial
Exposed
Antimicrobial
free (ABF)
Antimicrobial
Resistant
Strains
Antimicrobial
Resistant
Strains

29. C. coli Population Tree
ABF pigs and carcass
ABF env at farm and slg.
Conv pigs and carcass
Conv env at farm and slg.
ST-5776
ST-854
ST-1068
ST-1186

30. Phylogenetics
Clustering of STs
Conventional system
ABF system
Prod. System Unique STs
ABF 14
Conventional 32
ST-5764
ST-5770, ST-
1827, ST-
1109, ST-5772
ST-5248
Macarena and Thakur, PLOS ONE, 2012

31. Clostridium difficile AR Profile
Antimicrobials
TET AMP MET ERY CIP VAN MDR
Resistance%
0
20
40
60
80
100
ABF
CONVENTIONALFarm
Antimicrobials
TET AMP MET ERY CIP VAN MDRResistance%
0
20
40
60
80
100
ABF
CONVENTIONALEnvironment
AMP: Ampicillin; CIP: Ciprofloxacin; ERY: Erythromycin; MET: Metronidazole;
TET: Tetracycline; VAN: Vancomycin; MDR: Multidrug Resistance
Susick and Thakur, Veterinary Microbiology, 2012

32. Salmonella Serotypes
 A total of 1090 Salmonella isolates
 24 different Salmonella serotypes

33. Salmonella Campylobacter
A; Ampicillin
C: Chloramphenicol
S: Streptomycin
Su: Sulfamethoxazole
T: Tetracycline
S. Typhimurium
AR Pattern: ACSSuT
S. Typhimurium
AR Pattern: SSuT
Slaughter
Carcass
Ecological Niche
FarmFarm

34. Salmonella Genotypic Similarity
PFGE pattern Antimicrobials Serotype Farm Stage
Sample type
A-Antimicrobial Free System; C- Conventional System

35. Lagoon
Sprayer/
effluent
5 plots
Day 0, Before
Application
• 10 Lagoon
• 5 Effluent
• 25* Soil
Day 0, After
Application
• 25 Soil
samples
Day 7
• 25 Soil
samples
Day 14
• 25 Soil
samples
*5 replicates for the five 1m2 plots, 20m apart
Salmonella environmental
transmission from swine manure
SAMPLE COLLECTION

36. 0
20
40
60
80
100
Before D0 D7 D14 Before D0 D7 D14
Salmonella
Prevalence
%
Farm1 Farm2
Preliminary Results

37. Summary
• Move away from a generalized discussion on
antimicrobial resistance solution
• Pathogen ecological niche: specific
environment
• Renew focus on the role of environment
• Antimicrobial resistance is a global problem

38. Questions
(www.tmelab.org)