Presented as Invited Paper on 11-02-2017 at International Conference on Reproductive Biology and Comparative Endocrinology (ICRBCE) 9-11 February 2017, organized by Department of Animal biology, University of Hyderabad, India.Abstracted as IL-35

Antimicrobial Drug Resistance Pattern of Bacteria Isolated from Cases of Abortion and Metritis in

Animals

Bhoj Raj Singh, DK Sinha, Vinodh Kumar OR, KP Singh, AM Pawde, Prasanna Vadhana, M. Bhardwaj, A Saraf, S. Dubey

ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, UP, 243122

Abortions in animals• Mostly noticed in 3rd trimester of pregnancy. Important non-specific

causes of abortions in dairy animals are:– Drug-induced (prostaglandins)– Insemination/intra-uterine infusion– Hypothyroidism– Trauma/stress (transport, noise, veterinary treatment etc.)– High fever and endotoxins (toxic plants, nitrate/nitrite, fungal toxins, other

disease)– Nutritional (malnutrition, vitamin A/selenium/vitamin E deficiency, goitre)– Twin pregnancy– Genetic (malformation)Specific causes: Infectious Causes (Bacterial, viral, ricketssial, parasitic, mycotic etc.)

• Abortion in 3-5% of pregnant animals are considered in normal range in a herd and need for identification of cause is not considered by vets.

• However, occurrence of >5% abortions necessitate to find the specific cause.

Specific causes or infectious causes of abortions

• Except for some endemic areas most the infective abortions are due to one or other bacteria. However, abortion may be caused by many types of infective agents. Definitive causes include: – Parasitic causes: Neospora caninum, a dog parasites, causing neosporosis

in cattle, common in some areas only.– Protozoon infections: Tichomonas fetus– Mycotic causes: Aspergillus infection.– Viral causes: Bovine viral dirrhoea (BVD), Infectious bovine rhinotracheitis

(IBR), Blue tongue virus (BTV), Parainfluenza-3 virus, Schmallenberg virus– Bacterial causes: Brucella abortus, Listeria monocytogenes, Laptospira

spp., Mycoplasma spp., Trueperella (Arcanobacterium) pyogenes, Campylobacter fetus venerealis and rarely other Campylobacter spp. too, Chlamydia abortus, Ureaplasma diversu, Salmonella, Histophilus somni etc.

• Opportunistic bacteria are often causes more abortions than definitive causes of abortions.

Metritis & Pyometra

• Accumulation of pus in uterus is pyometra and swelling in endometrium, and myometrium preventing conception or occurring after dystokia, faulty insemination or trauma is usually the cause of the condition.

• In dairy animals, it occurs most commonly after calving complicated by dystokia, retained fetal membranes, twins or stillbirths.

• In most of the cases one or other pyogenic bacteria is often associated with the disorder.

• Some common bacteria that cause enteritis include:– Salmonella enterica ssp. enterica serovars– Escherichia coli– Staphylococcus aureus and other staphylococci.– Streptococcus species– Campylobacter jejuni– Yersinia enterocolitica– Bacillus species

Abortion and still birth scenario in Brucellosis positive herd

2010-11 2011-12 2012-13 2013-14 2014-15 2015-160.00

5.00

10.00

15.00

20.00

25.00

30.00

12.20

5.26

7.14

5.084.55 4.84

11.48 11.90

6.06

1.96

18.60

25.00

11.6311.11

9.70

4.74

11.18

6.23

Murra buffalo, n~230 Tharparkar herd n~200 Crossbred herd n~600

Abortion and stillbirths rates in Indian Veterinary Research Institute, Iz-atnagar, Dairy Farm over last six years (2010-2016)

AS Rate=100*numer of abortions & still births/Total pregnancies

Perc

ent a

bort

ion+

still

birt

hs

About 60% of the abortions were in the first time pregnant animals

Materials and Methods• Samples collected: Aborted foetuses from diaries in Bareilly Region

from April 2011- December 2016). From 246 foetuses samples of stomach contents, liver, spleen and kidneys were collected.

• Uterine swab from 21 clinical cases of pyometra/ metritis were processed.

• Samples were processed for isolation and characterization of Bacteria (Aerobic and microaerobic).

• Isolated bacteria were tested for antibiotic sensitivity using disc diffusion assay.

• For detecting ESBL, Carbapenemase, NDM and other MBLs using E-strips and PCR.

• Serum from dams was collected and tested for antibodies for the isolated bacteria from foetuses to confirm association of infection with abortion.

PCR for detection of NDM (621 bp), KPC (438 bp) &OXA (389 bp) specific products

Results of our StudyThe Most Common Bacteria identified as cause of abortion in dairy animals (shown

as percent of cases) in Bareilly (Rohilkhand) Region

Escheric

hia

Aeromonas

Enterobacte

r

Strepto

coccu

s

Bruce

lla

Klebsiella

Staphylococcu

s

Alcalig

enes

Moraxella

Acineto

bacter

Raoultella

terri

gena

Pseudomonas

Salmonella

Proteus

0.0

5.0

10.0

15.0

20.0

25.0 23.5

10.1 9.37.2 6.4 5.2 4.4 4.0 3.8 3.4 2.0 1.8 1.8 1.6

Bacteria belonging to 37 genera were identified from aborted foetal tissues or membranes and were associated with abortion.

0.0

5.0

10.0

15.0

20.0

25.023.5

10.19.3

7.26.4

5.24.4 4.0 3.8 3.4

2.0 1.8 1.8 1.6 1.4 1.4 1.2 1.2 1.0 1.0 1.0 1.0 0.8 0.8 0.8 0.6 0.4 0.4 0.4 0.4 0.2 0.2 0.2 0.2 0.2 0.2 0.2

% o

f cas

es

Bacteria associated with abortions in animals

Bacteria associated with metritis in dogs and buffaloes (in two cases no bacteria could be detected)

0

2

4

6

8

10

1210

4

21 1 1

Num

ber

of c

ases

Herbal Antimicrobial Drug Sensitivity of Bacterial Strains Associated with Reproductive Tract

Infections in Animals

Agar wood Oil

Patchouli o

il

Guggul oil

Sandal

wood oilZR

EO

Lemon grass

oilCitr

al

Beetel leaf o

il

Holy basil o

il

Cinnamledehyde

Cinnamon oil

Thyme oil

Ajowan oil

Carvacro

l0.0

20.0

40.0

60.0

80.0

100.0

120.0

18.8

37.132.9

56.1

24.4

34.2

60.0

47.254.0

86.1 86.5

96.8 94.698.7

7.8 9.4 9.7 11.419.1

33.440.6

64.7

87.393.7 96.7 98.3 98.8 99.5

G+ve (91) G-ve (425) Total (516)

Perc

ent s

ensi

tive

stra

ins

Antibiotic Sensitivity of Bacterial Strains Associated with Reproductive Tract Infections in Animals

Novobiocin

Vancomycin

Penicillin

Clindamycin

Ampicillin

Erythro

mycin

Amoxicilli

n

Cefoxitin

Nalidixi

c acid

Azithro

mycin

Piperacillin

Aztreonam

Ceftazidim

e

Nitrofuranto

in

Cotrimoxa

zole

Cefepime

Strepto

mycin

Colistin

Cefotaxime

Imipenem

Ciprofloxa

cin

Polymyxin B su

lphate

Moxalacta

m

Ceftriaxo

ne

Chloramphenicol

Gentamicin

Tigecycli

ne0.0

20.0

40.0

60.0

80.0

100.0

120.0

57.7

84.7

48.7

76.1

57.4

82.9

74.7

49.2

16.3

90.4

81.5

45.850.0

83.1

74.4

64.6 62.259.4

81.3

88.0

78.775.5 77.8

89.2 87.191.1

100.0

5.40.0

9.7 11.9

19.6

31.6

39.0

53.5

64.8 65.1 65.669.1

73.6 74.0 75.880.8 82.7 83.3 84.4 84.7 86.1 87.1 87.1

90.7 92.1 93.2 95.5

G+ve (91) G-ve (425) Total (516)

The Most Effective antibiotics against bacteria causing RTI

• The most effective drugs on G+ve bacteria (in increasing order of effectivity): Clindamycin, Moxalactam, Ciprofloxacin, Cefotaxime, Piperacillin, Erythromycin, Nitrofurantoin, Vancomycin, Chloramphenicol, Imipenem, Ceftriaxone, Azithromycin, Gentamicin and Tigecycline

• The most effective antimicrobials on G-ve bacteria: Ceftazidime, Nitrofurantoin, Cotrimoxazole, Cefepime, Streptomycin, Colistin, Cefotaxime, Imipenem, Ciprofloxacin, Polymyxin B sulphate, Moxalactam, Ceftriaxone, Chloramphenicol, Gentamicin and Tigecycline.

• The myth prevalied: – Imipenem is reported to be more active against G+ve bacteria and meropenem om

G-ve bacteria (Zhanel et al., 2007, Drug). However, this study revealed that Imipenem was better than Meropenem in vitro both on G+ and G-ve Bacteria (p ≤0.03). For meropenem, 26.9% G+ve and 24.8% G-ve bacteria were resistant, while for imipenem, 12% G+ve and 15.3% G-ve bacteria were resistant.

– Aztreonam agar can be used as selective agar for Staphylococci (Wood et al., 1993): Our observation revealed that >25% staphylococci were sensitive to aztreonam and may not grow on aztreonam agar.

ESBL, MBL and NDM production by bacteria associated with abortion and metritis

Bacteria Percent of strains positive for

Type ESBL NDM MBL MDR

G+ve (91) 19.8 1.0 0.0 50.5

G-ve (425) 34.8 1.6 2.8 47.8

Total (516) 32.2 1.5 2.3 48.3

Bacteria Drug resistance trait

Associated with

Animal

Acinetobacter lwoffii KPC Abortion CattleAeromonas bestiarum OXA Abortion CattleAeromonas popoffii (2) NDM 1 Abortion CattleAeromonas trota NDM 1 Abortion CattleEnterobacter agglomerans OXA Abortion CattleEscherichia coli (2) NDM 1 and OXA

Abortion Cattle

Escherichia coli (2) NDM1 Abortion (1), pyometra (1)

Cattle, dog

Raoultella terrigena OXA Abortion Cattle

NDM-1 (New Delhi metallo-β-lactamase) (Class B)OXA (oxacillinase) group of β-lactamases (Class D)KPC (K. pneumoniae carbapenemase) (Class A)

Carbapenemase producing bacteria associated with RTI detected in animals

Multiple Drug Resistance (MDR) detected in bacteria associated with RTI in animals

0 1 2 3 4 5 6 7 8 9 100.0

5.0

10.0

15.0

20.0

25.0

30.0

14.3

25.3

9.9

16.5

9.9

6.6

9.9

4.42.2

0.01.1

10.4

21.620.2

15.113.6

9.2

5.6

2.41.2 0.7 0.0

Gram Positive bacteria (91)

Gram Negative bacteria (425)

Number of drugs for which resistance was detected

Perc

ent o

f str

ains

res

istan

t to

drug

s

To determine MDR, resistance to the following antimicrobials was counted.For GNBs: Ampicillin, gentamicin, nitrofurantoin, nalidixic acid, cotrimoxazole, ciprofloxacin, chloramphenicol, carbapenem, azithromycin, colistin, cefotaxime, moxalactam, aztreonam.For GPBs: Ampicillin, gentamicin, nitrofurantoin, cotrimoxazole, ciprofloxacin, chloramphenicol, carbapenem, azithromycin, clindamycin, erythromycin, vancomycin, novobiocin, cefotaxime, moxalactam.

Important Facts Revealed• Sensitivity of bacteria to one of the less effective (Sandal wood oil, 18.5%) and one

of the most effective (Carvacrol, >99%) herbal antimicrobials had strong positive correlation with sensitivity to the most of the antibiotics.

• Nalidixic acid inhibition zone had negative correlation with zone of inhibition of SWO and

• Meropenem inhibition zone had negative correlation with zone of inhibition of agar-wood oil. But, agar wood oil inhibition zone was positively correlated with those of azithromycin, erythromycin, novobiocin ceftriaxone and moxalactam.

• Tigecycline inhibition zone had negative correlation with zone of inhibition of betel leaf oil and

• Ceftriaxone inhibition zone had negative correlation with zone of inhibition of thyme oil.

• MDR in strains was positively correlated with detection of ESBL, NDM and carbapenemase.

• MDR had negative correlation with zone of inhibition by citral and thyme oil.• MHDR had no correlation with MDR, and production of ESBL, NDM and

carbapenemase.• MHDR was positively correlated to zone of inhibition by meropenem and MDR

with zone of inhibition by ceftriaxone.

Conclusions

• Besides the known causes of abortions in animals, opportunistic pathogens appears to be more important cause of sporadic abortions.

• Some of the herbal antimicrobial are as good as antibiotics or even better, the question is how we can use these to alleviate the infections of reproductive tract.

• In vitro studies are just indicative and real picture may be lucid after in vivo studies for which a concerted one health study is the need of the day.

Thanking you all and the organizers for proving this

opportunity to interact with learned peoples.