1. Use and misuse of antimicrobials on farms: Ecological and health impact
Juan J Carrique-Mas
Oxford University Clinical Research Unit
Ho Chi Minh City, Vietnam
Biodiversity and Health Symposium
Phnom Penh, 17 November 2014
Good morning. It is a pleasure to be in PP introducing this very relevant topic.

2. Agenda AMR: A complex ‘ecohealth’ problem
Economic development and animal production
The ‘ecological web’ of AMR
Other AMR co-selecting environmental pollutants
Data from current projects (OUCRU-VN):
Antimicrobial consumption
Transmission of AMR between humans and animals
The way forward
Future projects
In my presentation I will introduce the problem of AMR – emphasizing its complexity.
Use of Ams are interlinked with animal production and economic development.
I will present the ecological web of AMR. I will briefly introduce to the issue of other co-selecting pollutants.
Then some results from on-going projects in VN that hopefully will help document the issues, both on AM consumption on farms and transmission between humans and animals
Finally some suggestions as to where to move on from here.

3. The ‘Confusogram’
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This diagramme comes from a book by Dr Prescott of Guelph University in Canada. In another publication it was referred as a confusogram.
It highlights the complex webs of circulation of AMR. It is a One Health issue, probably more than any other.
The overwhelming majority of research on the right side, hospitals and communities. Over the last 15 years or so emphasis on the food producing animals.
Clearly the greatest research gaps on research relate to the environment.
J. F. Prescott et al. (2000) Antimicrobial Therapy in Veterinary Medicine

4. The VAC system in Vietnam
We can see an example of this web in some producting systems in Asia. For example in Vietnam, the VAC system, stands for garden, pond, and animal pen which is an efficient way of recycling byproducts and manure, but also for facilitating AMR.
Source: FAO (2004)

5. Human and farm density in SE Asia*
Animal protein consumption: Vietnam
Fish and shrimp production in Vietnam (2004 to 2011)
Human and farm density in SE Asia*
Pig farms
Chicken farms
Humans
These maps are now a little bit old, but show how the densities of humans, chicken and pigs are inter-related.
Of the countries in the Indochina Peninsula, probably VN has the highest population densities, mostly in the Deltas and the cities of Ha Noi and HCMC. An important characteristic is the rapid observed increase in animal protein consumption, fuelled by fish (now VN is one of the first fish consuming countries in Asia), followed by pigs and poultry
* Sources: LEAD and FAO (2000, 2006)

6. Antimicrobial use in animal production
Fundamental differences in the use of AM in humans and animals. Use in animal production is almost always related to groups of animals,
except in the case of injectable drugs (most common in pigs only, but rare in aquaculture and poultry).
Growth promotion, prophylactic and methaphylactic use, treatment. In human medicine treatment is the most common use.
Some travellers have the habit of having doxacycline in developing countries.
S. Page and P. Gautier (2012), Rev. sci. tech. Off. int. Epiz., 2012, 31 (1),

7. Post antimicrobial effects
Fate of antimicrobials
Fate of antimicrobial resistant bacteria
Fate of resistant genes
Interaction with environmental bacteria?
Fate of antimicrobials
Distribution at sub-inhibitory concentrations
Fate of antimicrobial resistant bacteria
Pathogens and commensals
Direct transfer from host to host
Indirect transfer (food, water, environmental pathways)
This slide summarises the main impacts of the use of AM:
It concerns that availability of the antimicrobial that may keep promoting AMR after its intended aid. As a result of AMU you generate AMR bacteria that may reach humans or other individuals not treated.
Now the most recent concern is that a lot of the AMR promoted during the use of AM is in the form of AMR genes that can cross beyond bacterial species.
Fate of resistant genes
Vertical transfer
Horizontal transfer between unrelated bacteria
*Adapted from Da Costa et al. (2013) Int. J. Environ. Res. Public Health 2013, 10:

8. AMR and environmental bacteria
Environmental bacteria >95% of the earth’s microbiome
Naturally produce antimicrobials as signalling molecules at low concentrations
Naturally multi-drug resistant
Most plasmid-mediated ARG originate from the environment
i.e. qnr from waterborne bacteria
Since 1950’s humans have manufactured antimicrobials and used them at industrial levels, this results in unprecedented levels of exposure of the earth’s microbiome to antimicrobials

9. Ecological concerns
Commensal, pathogenic flora, and ARG interact with environmental organisms in certain hotspots
Farm, hospital
ARG integrated in gene-transfer elements (plasmids, transposons, integrons) the highest risk
ARG can be maintained in the absence of antimicrobial selective pressure and co-selected due to other stressors (siderophores, toxins, heavy metals, biocides)
Do changes in the natural ecosystems as a consequence of human activities accelerate evolution towards AMR?
ARG detected in bacteria from environmentally pristine locations, indicating dissemination
qnr encoded in plasmids detected in Aeromonas spp.
† J. Martinez (2009). Proc. R. Soc. B.

10. Confirmed hospital-acquired Acinetobacter baumannii infections, respiratory patients, ICU (HTD, HCMC (Vietnam) ( )
Source: James I. Campbell, OUCRU-HCMC

11. AMR in E. coli in humans and chickens in the Mekong Delta
Tien Giang
I will be presenting some studies we have been carried out to investigate AMU in poultry farms, and AMR in chickens and humans

12. VIBRE Project Rural areas (3 districts) Urban areas (1 city)
Chicken farm
Use of antimicrobials
Food habits
Use of antimicrobials
Food habits
Knowledge and use of antimicrobials:
Bio-security
Non-farmers (N=204)
Not involved in poultry farming
Matched by commune, age/sex to farmer
Urban inhabitants (N=102)
Not involved in poultry farming
Living in urban centre of My Tho
Chicken farms
(N=204)
Development of AMR E. coli ?
Transfer of AMR?
Use of antimicrobials
Contact with chickens
Food habits
Chicken farmers
(N=204)
Transfer of AMR?

13. Antimicrobial agents used in chicken farming, TG
Class of antimicrobial
Name of antimicrobial
No. (%) formulations containing the product (N=157)
No. (%) farms using (N=208)
Tetracyclines
Doxycycline, oxytetracycline, tetracycline
57 (36.3%)
53 (25.5%)
Polypeptides
Colistin
48 (30.6%)
41 (19.7%)
Macrolides
Tylosin, tilmicosin, erythromycin, spiramycin
40 (25.5%)
40 (19.2%)
Penicillins
Ampicillin, amoxicillin
41 (26.1%)
34 (16.3%)
Quinolones
Flumequine, oxolinic acid, norfloxacin, enrofloxacin
22 (14.0%)
19 (9.1%)
Aminoglycosides
Spectinomycin, neomycine, gentamicin, apramycin, streptomycin
19 (12.1%)
Phenicols
Florfenicol, thiamphenicol
14 (8.9%)
12 (5.8%)
Sulphonamides/
trimethoprim
Sulfamethoxazole, sulphadimidine, sulphadimetoxine, sulphadimerazine, trimethoprim
12 (7.6%)
Lincosamides
Lincomycin
4 (2.5%)
Pleuromutilin
Tiamulin
1 (0.6%)
1 (0.5%)
Studies show that over the last 90 days a total of 28 formulations had been used. Some of them include
critically important antimicrobials such as colistin, quinolones and aminoglycosides.

14. Antimicrobial formulations, TG
Penicillin
Polypeptide
Macrolides
Tetracyclines
Quinolones
Phenicols
Aminoglycosides
Sulphonamides
Lincosamides
Pleuromutilins
No. administrations
Penicillins 6 33 1 41
Polipeptides 2 5 3 48 25 4 40 13 7 57 20 22 8 14 19 12
Pleuromutilin
tylosin tartrate,
trimethoprim,
sulphadiazine, sulphamethoxazole
100/157 (64% products were ‘mixes’ of 2 or more antimicrobials)

15. AMU on chicken farms, Tien Giang
Variable
Level
All farms (N=208)
Number of antimicrobial products used over study period
40.9% 1
>1
18.3%
Method of administration
Water
82%
Feed 9%
Feed and water 4%
Type of administration
Prophylaxis
84%
Treatment
12%
Both
Timing
On arrival
34%
Continuously
18%
Periodic
29%
Other
19%
Advice given by
Drug seller
56%
District veterinarian
Friend/neighbour
Sales person 2%
We collect data on AMU on these farms

16. Antimicrobial consumption in relation to chicken production
Stratum
No. farms sampled
No. chickens sampled
No. chickens (census)
Fraction sampled
(%)
Sampling weight
Milligrams of active compound used per week per chicken (±SE)
Grams of active compound per 1,000 chickens produced
CG, hh 34
2,890
409,850
0.007
141.8
30.4 (±15.6)
901.2 (±622.8)
CG, sm
47,970
128,250
0.374
2.7
5.3 (±1.5)
167.5 (±63.9)
CT, hh 36
4,505
268,295
0.017
59.5
5.6 (±1.4)
327.8 (±122.4)
CT, sm
50,230
56,700
0.886
1.1
18.6 (±7.2)
193.1 (±57.3)
MT, hh
2,290
58,310
0.039
25.5
26.4 (±17.2)
413.8 (±256.4)
MT, sm
52,500
73,300
0.716
1.4
4.7 (±1.9)
156.6 (±63.7)
All
208
160,385
994,705
0.161
15.1 (±4.0)
358.1 (±113.5)

17. What’s on the menu? Colistin: 120 mg/Kg Neomycin: 400 mg/Kg
Florfenicol: 60mg/Kg
Kitasamycin: 300 mg/Kg
In Vietnam, most commercial animal feed rations are medicated
How easy it is to calculate the amount of AM necessary to produce pork

18. AMR testing in E. coli Disc diffusion test 1 ampicillin (10µg) 2
ceftriaxone (30µg) 3
ceftazidime (30µg) 4
chloramphenicol (30µg) 5
ciprofloxacin (1µg) 6
ciprofloxacin (5µg) 7
trimethoprim-sulphamethoxazole (1.25 µg /23.75µg) 8
gentamicin (10µg) 9
amikacin (30µg) 10
meropenem (10µg) 11
amoxicillin-clavulanic acid (20/10µg) 12
tetracycline (30µg)
Disc diffusion test

19. Antimicrobial resistance in E
Antimicrobial resistance in E. coli (non-selective plates) VIBRE Project
These can be taken as ‘randomly selected colonies from farmers and chickens’.

20. Diversity among chicken and human E. coli
N=5 colonies per study subject
Shannon-Weaver diversity index (H)
Shannon-Weaver index (H)

21. Human and chicken E. coli AMR patterns (I)1 2 3 4 5
---C--TE-STX----AMP-
AMC---C AMP-
AMP-
---C--TE-STX-----
-----TE------
---C--TE-----AMP- X
Matching: 3/50 (6%)

22. Comparisons of E. coli patterns from humans and chickens
Distances
No. calculations
Median [75% IQR]
Chicken vs. farmer a
201
2.76 [ ]
Chicken vs. rural control b
199
3.12 [ ]
Chicken vs. urban control c 97
3.47 [ ]
Farmer vs. Rural control d
196
3.02 [ ]
Farmer vs. Urban control e 95
3.20 [ ]
Rural vs. Urban control f
3.25 [ ]

23. Antimicrobial resistance in pig pathogens

24. AMR among pig pathogens (n=53)
Source: Severe disease in piglets in southern Vietnam: main bacterial aetiologies and antimicrobial resistance. J. Campbell et al, 2014 (in preparation)

25. Longitudinal studies on AMR in pig and chicken farms
Differences in the dynamics of AMR depending on the type of farming. Here we see results of AMR testing against a number of antimicrobials for one pig and poultry farm at three time points.
E. coli
Enterococcus spp.
White bar: day-old, grey bar: mid production, black bar: end of production

26. Environmental stressors and AMR: Quaternary ammonium compounds (QAC)

27. Use of QAC in chicken farms
Product
Disinfectant class
Percent farms using (%)
(N=208) 1
QAC
68.2 2
Peroxygen-based
3.4 3
Halogen-Releasing agent 1 4
Halogen-Releasing agent 2
1.9 5
Other
23.0
(in the cleaning process and in occupied animal houses)
QAC products represented 68.2% of all disinfectants used in Tien Giang province
Benkocid represented 56.7%
The cross-resistance of QAC and antibiotics could be achieved by linkage of different resistance mechanisms on the same plasmid, transposon or integron or any combination of these [4]. Many bacteria acquire resistance to QACs by changing the composition of their membrane proteins, LPS, fatty acids [6, 9], cell surface hydrophobicity

28. Adaptation experiments to QAC
Determine MIC for each isolate
Expose 105 cfu/ml of bacteria to 0.5MIC (37oC, 18-20h)
Stabilise bacteria in MHB without disinfectant (37oC, 5h)
Calibrate suspension to 105cfu/ml and transfer to new MHB supplemented with 0.75 MIC QAC disinfectant
Stabilise bacteria in MHB without disinfectant for 5h at 37oC
Expose bacteria to step-wise increase concentration of disinfectant
Until no growth observed, the highest concentration of disinfectant that bacteria can survive were used

29. Cross-resistance QAC and antimicrobials
Study ID TE CN
AMP
CIP CT
SXT C
AD01-c S I
AD01-p R
AD01-ef
AD02-c
AD02-p
AD02-ef
AD03-c
AD03-p
AD03-ef
AD04-c
AD04-p
AD04-ef
AD09-c
AD09-p
AD09-ef
AD10-c
AD10-p
AD10-ef
c = control; p = post adaptation; ef= after treating with PAβN

30. The link between AMU and AMR is uncontroversial
Chantziaras et al (2014) Journal of Antimicrobial Chemotherapy, 69:

31. A vociferous skeptical minority
WoK quoted >350 times!!!

32. Priorities for research….and for policy
Carry out farming with less antimicrobials
If antimicrobials are used, limit their impact on animals and their environment
Understand why and where antimicrobials are used in agriculture
Increase awareness
Change attitudes and behaviours
Surveillance of AMU and AMR:
Monitoring of antimicrobial use and ARG
Applied research
Understand impact of antimicrobial use on the environmental (AM, ARG, bacteria)
Intervention studies to reduce AMU
Technical solutions to limit impact.. and test them!

33. Acknowledgements Zoonoses Group ITU, CTU, Microbiology, Enterics Group
Mr. Trung Nguyen Vinh
Dr. Ngo T. Hoa
Mr. James Campbell
Dr. Constance Schultsz
Sub-Department of Animal Health Tien Giang, Dong Thap
Hospital Tropical Diseases HCMC
Funding:
ZoNMW / WOTRO (The Netherlands) (VIBRE Project)
The Wellcome Trust
Add pictures

34. Thanks for your attention!