In this presentation antimicrobial drug resistance (AMR) in captive wildlife has been compared with AMR in bacteria isolated from veterinary clinical cases. In captive wildlife bacteria resistant to carbapenems, all generations of cephalosporins, producing ESBL, MBL, and NDM were prevalent. In this study 36.7% bacterial isolates from captive wildlife were ESBL producers and about 45% were MDR type. In recent past not much increase in AMR in bacteria of captive wildlife was observed. Carvacrol was found to be the most effective herbal antimicrobial. About 67.5% bacteria from birds kept in zoo and >71% those from zoo carnivores had MDR. This was much more than in strains of wild herbivore origin (<30%). Herbal drugs resistance was more common in bacteria from herbivore captive wildlife in contrast to AMR for conventional antimicrobials in bacteria from carnivores. Bacteria from carnivores had higher levels of multidrug resistance than those from omnivores or herbivores. No such difference was evident in bacterial isolates from domesticated or pet herbivores and carnivores. Omnivore animals (pigs) and human isolates had almost similar levels of AMR but much higher than herbivores. Position in food chain and Food of the host play an important role in occurrence of AMR bacteria. The study revealed that members of Enterobacteriaceae are the major players in the propagation of AMR. And for maintenance or propagation of AMR Enterobacteriaceae members, wild carnivores are the major abode. The study indicated that AMR was common in bacteria of captive wildlife too as in other biotic and abiotic components of the environment. However, the level of AMR was much more aggravated than in domestic animals. It can be concluded that if we need to monitor the AMR in any locality it will be more informative to look for the AMR strains in wildlife and aquatic environment than in livestock. This might be due to the concentration of the AMR strains in wild carnivores having a specific niche in food chain. More long term studies on large number of isolates from wide variety of captive wildlife living in different geographical and climatological conditions are required for better understanding of AMR trends.

1. Emergence of antibiotic
resistance in captive wildlife
Bhoj R Singh
Division of Epidemiology
ICAR-Indian Veterinary Research Institute,
Izatnagar-243122, India, +91-8449033222
brs1762@ivri.res.in; brs1762@gmail.com
Sumedha Gandharava
Presented on 6th January 2017 as lead paper in National Congress on Wildlife Health (NCWH) at ICAR-Indian
Veterinary Research Institute, Izatnagar (6-7 January 2017).

2. Ubiquitous AMR
• Presently AMR in microbes is not limited to livestock and the human
population it has spread ubiquitously all over the globe.
• AMR has invaded widely into wildlife populations.
• AMR is more prominent in wildlife than in the human and domestic
animals (Jobbins and Alexander, 2015).
• Captive wildlife in zoo is a huge reservoir of AMR bacteria (Ahmed et al.,
2007; Power et al., 2013; Sala et al., 2016).
• The food may be an important source of AMR bacteria in wildlife and
captive wildlife (Li et al., 2011)
• Though the mechanisms of its spread are unclear (Vittecoq et al., 2016).

3. Background
Studies in past few years have revealed that anti-microbial drug resistance (AMR)
in microbes has spread ubiquitously to all over the globe and is not limited
only to livestock and the human population. The AMR has invaded into
wildlife populations, though the mechanisms of its spread are unclear
(Vittecoq et al., 2016), it is more prominent than in the human or domestic
animal populations (Jobbins and Alexander, 2015). Studies abroad have
indicated that captive wildlife in zoo are reservoirs of AMR bacteria (Ahmed et
al., 2007; Power et al., 2013; Sala et al., 2016) and their food may be an
important source of acquiring AMR bacteria (Li et al., 2011). However, AMR
status in captive wildlife in India is a little explored field. The present
investigation dealt with AMR bacteria in captive wildlife. Attempts have been
made to understand trends of AMR in bacteria in captive wildlife over 2011 to
2016 through comparison of AMR in microbes isolated from captive or
contained wildlife with those from livestock, pet animals, poultry birds,
humans and aquatic environment.
In epidemiology of AMR, wildlife plays an important role and fact is known since
long (Linton, 1977), however little attention has been paid towards
understanding the exact mechanism. Though it is not lucid how it happens but
wildlife plays important role in spread of AMR through horizontal gene
transfer (Nordman and Poirel, 2005).

4. Basis of the Information
• In last 6 years more than 2225 bacterial strains isolated from
human (128), pig (324), aquatic environment (31), domestic
herbivores (820), herbivore pets (216), pet carnivores (219),
domestic birds (52) & captive wild carnivores (66), captive
wild herbivores (175), and captive wild birds (40) were
tested.
• Strains were tested against 9 herbal and 30 conventional
antimicrobials (antibiotics).
• Strains were tested using conventional (disc diffusion assay,
E-test, DDD assay) and molecular methods (specific PCR) to
determine ESBL, CR, MBL and NDM production (CLSI, 2015).

5. Bacteria belonging to more than
64 species of 29 genera were isolated from captive wild life
and tested for antimicrobial resistance.
Acinetobacter boumanni, Acinetobacter haemolyticus, Aeromonas bestiarum,
Aeromonas caviae, Aeromonas hydrophila, Aeromonas jandaei , Aeromonas
media, Aeromonas schubertii, Aeromonas veronii, Agrobacterium tumefaciens,
Alcaligenes denitrificans, Alcaligenes faecalis, Arsenophonus nasoniae, Bacillus
cereus, Bacillus pentothenticus, Bacillus polymyxa, Bacillus spp., Brucella abortus,
Citobacter amalonaticus, Citrobacter freundii, Edwardsiella ictaluri, Enterobacter
agglomerans, Enterobacter gregoviae, Enterobacter spp., Enterococcus faecalis,
Enterococcus spp., Erwinia amylovora, Erwinia ananas, Erwinia spp., Escherichia
coli, Escherichia fergusonii, Gallibacterium anatis haemolytica, Geobacillus
steariothermophilus, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae ssp.
pneumoniae, Kluyvera ascorbate, Kluyvera cryocrescens, Micrococcus spp.,
Pasteurella multocida, Pragia fontium, Proteus mirabilis, Proteus penneri, Proteus
vulgaris, Pseudomonas aeruginosa, Pseudomonas paucibacillus, Pseudomonas
pseudoalcaligenes, Pseudomonas testosteronii, Pseudomonas spp., Raoultella
terrigena, Salmonella enterica 6,8 (UI), Salmonella Kentucky, Serratia odorifera,
Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis,
Staphylococcus spp., Streptococcus canis, Streptococcus macacae, Streptococcus
porcinus, Streptococcus pyogenes, Streptococcus spp., Streptococcus suis, Vibrio
anguillarum

6. • Results indicated that all kinds of drug resistance were prevalent in
bacteria isolated from wildlife/ captive wildlife. Though trends of
AMR were undulating, it was evident that bacteria resistant to even
the carbapenems, all generations of cephalosporins, producing
ESBL, MBL, and NDM were prevalent in zoo as well as wild animals.
The important bacteria isolated from wild animals having MDR and
resistance to new generation antimicrobials included members of
Acinetobacter (2), Aeromonas (10), Agrobacterium (1), Alcaligenes
(2), Bacillus (7), Brucella (1), Edwardsiella (2), Enterobacter (22),
Erwinia (1), Escherichia (32), Gallibacterium (1), Hafnia (2),
Klebsiella (7), Micrococcus (1), Pragia (1), Proteus (7), Pseudomonas
(12), Raoultella (2), Salmonella (2), Staphylococcus (5),
Streptococcus (5) and Vibrio (1) species. However, the majority
(~62%) of the isolates belonged to Enterobacteriaceae family
known for frequent horizontal gene transfer for AMR (Nordman and
Poirel, 2005). The strains of ubiquitous Escherichia coli and
Enterobacter agglomerans dominated the scene. However, it was
evident that in recent past there is no much increase in trends of
AMR in bacteria of captive wildlife.
Observations

7. 5.9
12.2 12.7
14.2 15.2 15.5
17.7
21.5 21.7 22.2 22.7
25.2 25.8
0.0
5.0
10.0
15.0
20.0
25.0
30.0
Percentresistantisolates
Thirteen most effective in vitro antimicrobials on
veterinary clinical isolates (2225 tested)

8. 72.5
61.6 60.5
55.8 55.7
43.6
39.3 38.4 38.0 37.5
35.3 34.8 34.1
28.9
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0Percentresistant
Thirteen least effective in vitro antimicrobials on
veterinary clinical isolates (2225 tested)

9. 9.7
12.7
21.7
27.3
30.7
7.7
9.8
20.9
32.9
37.0
3.2 2.7
4.2
5.2
7.4
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
2011 (n, 248) 2012 (n, 441) 2013 (n, 402) 2014 (n, 286) 2015 (n, 489)
ESBL+ve Carbapenemase+ve MBL+ve
Increase in ESBL, CR and MBL bacteria in animals
PercentPositive
AMR Trends in veterinary clinical isolates

10. 8.3
18.8
24.0
34.1
29.5
0.0
5.0
10.0
15.0
20.0
25.0
30.0
35.0
40.0
2012 (n, 48) 2013 (n, 340) 2014 (n, 192) 2015 (n, 390) 2016 (n, 440)
Colistin resistance (%) in bacteria (1410) isolated from
sick animals over 5 years (2012-2016)at IVRI, Izatnagar

11. 31.8
15.7
20.6 20.0
45.2
27.627.3
8.6
6.3
0.0
7.1
13.8
40.9
0.0
1.6
0.0
4.8
0.0
13.8
20.3
54.0
90.0
54.8
44.8
43.1
25.3
61.9
20.0
64.3
44.8
61.1
50.0
100.0
90.0
93.1
68.4
0.0
20.0
40.0
60.0
80.0
100.0
120.0
2011 (58) 2012 (79) 2013 (63) 2014 (10) 2015 (42) 2016 (29)
Carbapenem resistance Metallo-B-lacatmase (MBL) positive
New Delhi MBL (NDM) producers ESBL positive
Multi-drug resistance Multi-herbal -drug resistance
Trends of different types of AMR in microbes of captive
wildlife origin (2011 to 2016)

12. 27.1
3.9
1.6
39.7
15.5
5.2
23.5
12.1
8.1
16.1
8.0
1.1
17.2
3.4
3.4
11.8
0.0
0.0
14.8
3.3
0.0
30.6
6.1
0.0
82.4
29.4
5.9
0.0 20.0 40.0 60.0 80.0 100.0
Carbapenem resistance
Metallo-B-lacatmase (MBL)
positive
New Delhi MBL (NDM)
producers
Aquatic environment
Human
Equids
Wild birds
Poutry
Wild carnivores
Wild Herbivores
Pigs
Pet carnivores
Domestic herbivores
NDM, MBL and Carbapenem resistance bacteria from
veterinary clinical isolates

13. 2.3
58.0
55.4
27.0
3.9
1.7
2.9
40.7 40.7
15.9
1.3
0.0
2.3
29.7
32.0
17.0
5.7
0.7
3.0
58.0
53.9
28.1
5.4
1.5
0.0
71.2
45.5
51.5
22.7
16.7
0.0
71.2
44.2
14.0
2.3
4.7
9.5
58.1
51.6
48.3
17.2
3.4
1.9
62.0
51.9
38.7
8.0
5.14.9
71.1
54.7
31.1
2.8
0.0
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
Carvacrol resistant Multi-drug resistance extended spectrum
B-lactamase
production
Carbapenem
resistant
Metallo B-Lactamase
prositive
New Delhi B-
Lactamase positive
Domestic Herbivores (820)
Herbivore Pets (216)
Wild Herbivores (175)
Pet Carnivores (219)
Wild Carnivores (66)
Domestic birds (52)
Wild birds (40)
Aquatic environment (31)
Pig (324)
Human (128)
Comparison of different drug-resistance types in bacteria
isolated from captive wildlife and other sources

14. • The most effective herbal antimicrobial in the study was
carvacrol.
• Except for resistance to Zanthoxylum rhetsa essential oil
(MEO) which was more common among bacterial isolates
from wild carnivores, for all other herbs, drug resistance can
be monitored on bacteria isolated from herbivore wildlife or
from aquatic environment.
• The observation is quite in contrast to resistance to
conventional antimicrobials including antibiotics.
• Resistance to conventional antimicrobials including all kinds
of antibiotics was much more common in bacteria isolated
from carnivores.
• The observation is in concurrence with earlier observations
indicating that microbes from wild carnivores are the best
targets to monitoring emergence and the existence of drug
resistance in any geographical area (Vittecoq et al., 2016).

15. 90.7
68.9
91.8
2.3
94.2
81.0
94.3
72.4
84.9
66.7
90.0
86.2
9.5
92.0
33.3
85.0
67.7
90.3
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
100.0
AME MEO GO Carvacrol PEO MME AO LGO SWO
Wild Herbivores (175) Wild Carnivores (66) Wild birds (40) Aquatic environment (31)
Percentresistantstrains
Herbal drug resistance pattern in bacteria isolated from
Captive wildlife and aquatic environment

16. Earlier studies (Vittecoq et al., 2016; Jobbins and Alexander, 2015;
CristóbalAzkarate et al., 2014) clearly indicated that it is not easy to understand
from where the AMR reached and spreading in wild animals and the food is said to
be the main source (Li et al., 2011). However, it was clear that carnivores have
higher levels of multidrug resistance than omnivores or herbivores (Vittecoq et al.,
2016; Jobbins and Alexander, 2015; Cristóbal Azkarate et al., 2014) as observed in
the present study.
In the study 36.7% bacterial isolates from captive wildlife were ESBL
producers and about 45% were MDR type. In earlier studies in Japan (Ahmed et al.,
2007) it was less than 25%, it might be the effect of geography, temporality, and
types of animals screened.
The major reason, however, appears to be the fact that in the study in
Japan healthy animals were screened while in our study all isolates were from sick
or dead animals. In a recent study in Australia (Power et al., 2013) more than 45%
wallaby faecal samples had ESBL producing bacteria.
The results revealed that about 67.5% isolates from birds kept in zoos
and > 71% from zoo carnivores had MDR which was much more than in strains of
wild herbivore origin (<30%).
In the study bacteria isolated from wild carnivores had higher levels of
MDR, ESBL, NDM, and MBL production, higher levels of MDR and carbapenems
and carvacrol resistance similar to isolates from the aquatic environment.

17. • In a recent study on birds of prey (raptors) in Italy (Sala et al.,
2016) all the isolates were MDR type. In earlier studies too
(Jobbins and Alexander, 2015) herbivore-wild-animals are
reported to carry fewer MDR strains than those in carnivores
and aquatic environment.
• However, in earlier studies (Vittecoq et al., 2016; Jobbins and
Alexander, 2015; CristóbalAzkarate et al., 2014) it was the
scenario against early antibiotics, in this study it was evident
that not only for MDR strains but also for NDM, MBL strains
carnivore wild animals were the better reservoir or source
than herbivores either in wild or under domestication.
• However, in this study, no such difference was evident among
domesticated or pet herbivores and carnivores. Omnivore
animals (pigs) and human isolates had almost similar levels of
AMR but much higher than herbivore animals.
• Three of the ten most effective antimicrobials on bacteria
from wild animals were those used in therapeutics since five
decades or more including chloramphenicol, gentamicin and
nitrofurantoin.
• Among the 10 least effective antimicrobials on bacterial
strains of wildlife origin a few were those considered to be
the last resort drugs as pipercillin, monobactams and
ceftazidime clavulanic acid.

18. 4.4
5.3
6.9
16.7
17.3
22.7
21.8
23.8
26.7
24.7
0.0
2.4
16.3
0.0
18.6
0.0 0.0
7.1
10.5
22.6
0.0
5.0
10.0
15.0
20.0
25.0
30.0
G-ve bacteria G+ve bacteria
The ten most effective antimicrobials on bacteria of
captive wildlife origin (281)

19. 29.8
27.7
34.6
32.6
44.4
37.4 37.5
66.7
79.3
91.6
33.3
53.3
21.1
56.7
0
32.4 32.4
36.7
17.07
31.03
0
10
20
30
40
50
60
70
80
90
100
G-ve Bacteria G+ve Bacteria
PercentResistantstrains
The ten least effective antimicrobials on bacterial strains
from captive wildlife (281)

20. 10.5
2.6
6.3
9.0
11.9
24.1
26.1 25.6
23.8
20.0
21.4
31.0
4.2
9.0
7.6
6.0
2.4
6.9
30.0
35.1
72.5
80.0
60.0
55.6
36.4
16.7
25.4
10.0
22.0
24.1
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
2011 2012 2013 2014 2015 2016
Tetracycline Nalidixic acid Ciprofloxacin Azithromycin Cefotaxime
Increasing Resistance trends for some selected antibiotics
in captive wildlife bacteria

21. 31.8
47.6
51.7
20.0
26.8
15.8
50.0
16.2
8.6
40.0
64.3
12.5
40.9 40.0 38.9
20.0
28.6
17.2
69.8
71.6
77.8
70.0
64.3
34.5
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
80.0
90.0
2011 2012 2013 2014 2015 2016
Chloramphenicol Amoxi+clavulanic acid Ceftriaxone Penicillin
Drugs showing decreased AMR trend in bacteria of
captive wildlife

22. 0.0 1.7 0.0 0.0
2.4 3.4
82.9
79.2
57.8
50.0
71.4 72.4
80.0
83.6
87.5
80.0
71.4
86.2
89.1
96.2
91.7
80.0 78.6
89.7
86.1
90.4
93.7
90.0
81.0 82.8
0.0
20.0
40.0
60.0
80.0
100.0
120.0
2011 2012 2013 2014 2015 2016
Carvacrol Lemon grass oil Sandal Wood Oil Patchouli oil Gguggul oil
Trend of herbal AMR in bacteria isolated from wild life

23. Conclusions
• In captive wildlife bacteria resistant to carbapenems, all generations of
cephalosporins, producing ESBL, MBL, and NDM were prevalent.
• In this study 36.7% bacterial isolates from captive wildlife were ESBL producers and
about 45% were MDR type.
• In recent past not much increase in AMR in bacteria of captive wildlife was
observed.
• Carvacrol was found to be the most effective herbal antimicrobial.
• About 67.5% bacteria from birds kept in zoo and >71% those from zoo carnivores
had MDR. This was much more than in strains of wild herbivore origin (<30%).
• Herbal drugs resistance was more common in bacteria from herbivore captive
wildlife in contrast to AMR for conventional antimicrobials in bacteria from
carnivores.
• Bacteria from carnivores had higher levels of multidrug resistance than those from
omnivores or herbivores.
• No such difference was evident in bacterial isolates from domesticated or pet
herbivores and carnivores.

24. • Omnivore animals (pigs) and human isolates had almost similar levels of
AMR but much higher than herbivores.
• Position in food chain and Food of the host play an important role in
occurrence of AMR bacteria.
• The study revealed that members of Enterobacteriaceae are the major
players in the propagation of AMR. And for maintenance or propagation of
AMR Enterobacteriaceae members, wild carnivores are the major abode. The
observations are similar to earlier observations (Eze et al., 2015).
• The study indicated that AMR was common in bacteria of captive wildlife too
as in other biotic and abiotic components of the environment.
• However, the level of AMR was much more aggravated than in domestic
animals. It can be concluded that if we need to monitor the AMR in any
locality it will be more informative to look for the AMR strains in wildlife and
aquatic environment than in livestock. This might be due to the
concentration of the AMR strains in wild carnivores having a specific niche in
food chain.
• More long term studies on large number of isolates from wide variety of
captive wild life living in different geographical and climatological conditions
are required for better understanding of AMR trends.

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