Emergence of antibiotic resistance in captive wildlife

Bhoj R SinghDivision 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).

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).

BackgroundStudies 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).

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).

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

• 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

Tigecyc

line

Chloramphenico

l

Cefepime

Cefoxitin

Imipenem

Gentamicin

Cefotaxim

e

Piperacilli

n Tazto

bactam

Ceftriaxo

ne

Moxalac

tam

Nitrofuran

toin

Meropenem

Ciprofloxacin

0.0

5.0

10.0

15.0

20.0

25.0

30.0

5.9

12.2 12.7 14.2 15.2 15.517.7

21.5 21.7 22.2 22.725.2 25.8

Perc

ent r

esis

tant

isol

ates

Thirteen most effective in vitro antimicrobials on veterinary clinical isolates (2225 tested)

Penicillin

Erythromyci

n

Ampicillin

Amoxycillin

Nalidixic

acid

Aztreonam

Ceftazidim

e

Ceftazidim

e+Clav

Amoxy+sulbact

am

Piperacillin

Cotrimoxaz

ole

Tetra

cyclin

e

Amoxyclav

Azithromyci

n0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.072.5

61.6 60.555.8 55.7

43.639.3 38.4 38.0 37.5

35.3 34.8 34.1

28.9

Perc

ent r

esis

tant

Thirteen least effective in vitro antimicrobials on veterinary clinical isolates (2225 tested)

2011 (n, 248) 2012 (n, 441) 2013 (n, 402) 2014 (n, 286) 2015 (n, 489)0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

9.7

12.7

21.7

27.3

30.7

7.7

9.8

20.9

32.9

37.0

3.2 2.74.2

5.2

7.4

ESBL+ve Carbapenemase+ve MBL+ve

Increase in ESBL, CR and MBL bacteria in animals

Perc

ent P

ositi

veAMR Trends in veterinary clinical isolates

2012 (n, 48) 2013 (n, 340) 2014 (n, 192) 2015 (n, 390) 2016 (n, 440)0

5

10

15

20

25

30

35

40

8.3

18.8

24.0

34.1

29.5

Colistin resistance (%) in bacteria (1410) isolated from sick animals over 5 years (2012-2016)at IVRI, Izatnagar

2011 (58) 2012 (79) 2013 (63) 2014 (10) 2015 (42) 2016 (29)0.0

20.0

40.0

60.0

80.0

100.0

120.0

31.8

15.720.6 20.0

45.2

27.627.3

8.66.3

0.0

7.1

13.8

40.9

0.0 1.6 0.04.8

0.0

13.8

20.3

54.0

90.0

54.8

44.843.1

25.3

61.9

20.0

64.3

44.8

61.1

50.0

100.0

90.093.1

68.4

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)

Carbapenem resistance

Metallo-B-lacatmase (MBL) positive

New Delhi MBL (NDM) producers

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0

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

5.9Aquatic 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

0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

2.3

58.0

55.4

27.0

3.91.7

2.9

40.7 40.7

15.9

1.30.0

2.3

29.732.0

17.0

5.7

0.73.0

58.0

53.9

28.1

5.4

1.50.0

71.2

45.5

51.5

22.7

16.7

0.0

71.2

44.2

14.0

2.34.7

9.5

58.1

51.6

48.3

17.2

3.41.9

62.0

51.9

38.7

8.0

5.14.9

71.1

54.7

31.1

2.8

0.0

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

• 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).

AME MEO GO Carvacrol PEO MME AO LGO SWO0.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

100.0

90.7

68.9

91.8

2.3

94.2

81.0

94.3

72.4

84.9

66.7

90.086.2

9.5

92.0

33.3

85.0

67.7

90.3

Wild Herbivores (175) Wild Carnivores (66) Wild birds (40) Aquatic environment (31)

Perc

ent r

esis

tant

stra

ins

Herbal drug resistance pattern in bacteria isolated from Captive wildlife and aquatic environment

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.

• 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.

Tigecy

cline

Chloramphen

icol

Gentam

icin

Cefotax

ime

Ciprofloxacin

Imipen

em

Piperacill

in Tazto

bactam

Nitrofuran

toin

Meropen

em

Ceftria

xone

0.0

5.0

10.0

15.0

20.0

25.0

30.0

4.45.3

6.9

16.7 17.3

22.721.8

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

G-ve bacteria G+ve bacteria

The ten most effective antimicrobials on bacteria of captive wildlife origin (281)

0

10

20

30

40

50

60

70

80

90

100

29.827.7

34.632.6

44.4

37.4 37.5

66.7

79.3

91.6

33.3

53.3

21.1

56.7

0

32.4 32.436.7

17.07

31.03

G-ve Bacteria G+ve Bacteria

Perc

ent R

esist

ant s

train

s

The ten least effective antimicrobials on bacterial strains from captive wildlife (281)

2011 2012 2013 2014 2015 20160.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

10.5

2.66.3

9.011.9

24.126.1 25.6

23.820.0 21.4

31.0

4.2

9.0 7.6 6.02.4

6.9

30.0

35.1

72.5

80.0

60.0

55.6

36.4

16.7

25.4

10.0

22.024.1

Tetracycline Nalidixic acid Ciprofloxacin AzithromycinCefotaxime Series6

Increasing Resistance trends for some selected antibiotics in captive wildlife bacteria

2011 2012 2013 2014 2015 20160.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

90.0

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

69.871.6

77.8

70.0

64.3

34.5

Chloramphenicol Amoxi+clavulanic acid Ceftriaxone Penicillin

Drugs showing decreased AMR trend in bacteria of captive wildlife

2011 2012 2013 2014 2015 20160.0

20.0

40.0

60.0

80.0

100.0

120.0

0.0 1.7 0.0 0.0 2.4 3.4

82.979.2

57.8

50.0

71.4 72.4

80.083.6

87.5

80.0

71.4

86.289.1

96.291.7

80.0 78.6

89.786.1

90.493.7

90.0

81.0 82.8

Carvacrol Lemon grass oil Sandal Wood Oil Patchouli oil Gguggul oil

Trend of herbal AMR in bacteria isolated from wild life

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.

• 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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