This document discusses antimicrobial resistance (AMR) in dairy animals and alternative management strategies. It provides an introduction on the current status of AMR in dairy animals and the factors responsible for AMR development. It then covers the mechanisms of bacterial resistance and various diagnostic methods for AMR. The document discusses the animal-human interface in the transmission of resistant bacteria and strategies to prevent and contain AMR. Finally, it presents various alternative management strategies that can be used instead of antibiotics, including vaccines, probiotics, bacteriophages, phytocompounds, antimicrobial peptides, and ribosomally synthesized bacterial peptides.
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Antimicrobial Resistance in Dairy Animals and it's Alternative Management
1. Prospective of Antimicrobial Resistance
in
Dairy Animals and it’s Alternative Management
Strategies
By
Dr. Anupama Verma
M.V. Sc. Scholar (V-1693/16)
DEPARTMENT OF VETERINARY CLINICAL MEDICINE
College of veterinary Sciences and Animal Husbandry S -1
2. CONTENTS
Total slide : 49
1- Introduction: Current status of Antimicrobial
Resistance in Dairy Animals ( S: 3-9)
2-Factors responsible for Antimicrobial Resistance
in Dairy Animals ( S: 10-12)
3- Mechanisms of Antimicrobial Resistance (S: 13-16)
4-AMR Diagnostics Methods (S:17-18)
5- Animal- Human Interface (S:19-22)
6- Strategies for prevention and Containment of AMR
(S:23-24)
7- Alternative Management Strategies (S:25-46)
8- Conclusion (S:48) S -2
4. After discovery of first antibiotic (Penicillin, 1928) :
- Retarded the prevalence of infectious diseases
- Saved millions of life ( till second world war)
(Abraham EP, Chain E,1940)
In 1945, Sir Alexander Fleming, during his Nobel Prize speech stated
that
‘‘ Bacteria could develop resistance against antibacterial therapies,,
WHO report in 2014, the discovery of each and every new antibiotic
has been followed by reports of emerging resistance against it
INTRODUCTION: AMR
S -4
5. INTRO..
‘‘ AMR in microbes is defined as their
unresponsiveness to standard doses of clinically
relevant antimicrobial drugs”
(Ganguly NK et al.,2011)
S-5
7. Development of Antimicrobial Resistance against
Various Antibiotics Source : CDC ;2020
1942
1967
1976
Staphylococcus
aureus
Streptococcus
pneumonia
Pencillinase
producing
Neisseria
gonorrhoea
AMR identified in bacteria
against penicillin
Methicillin Methicillin resistant Staphylococcus aureus 1960
Extended spectrum
cephalosporins (cefotaxime)
Extended spectrum beta-lactamase producing
Escherichia coli
1983
Azithromycin Neisseria gonorrhea 1983
Imipenem
Carbapenemase producing Klebsiella
pneumonia(KPC)
1996
Ciprofloxacin Neisseria gonorrhea 2007
Daptomycin Staphylococcus aureus 2004
Ceftazidime-Avibactum
Carbapenemase producing Klebsiella
pneumonia( KPC)
2015
S -7
8. Scenario In India
India – ‘Epicentre of AMR catastrophe’
Analysis of Milk sample revealed that,
- 48 % of Gram-negative bacilli detected in cow and buffalo
milk were extended-spectrum β-lactamases (ESBL)
producers (West Bengal)
- 47.5 % were resistant to Oxytetracycline (Gujarat)
(Das et al., 2017)
-2.4 % of S. aureus were Vancomycin resistant(West Bengal)
(Bhattacharya et. al., 2017)
- Vancomycin resistant S. aureus (VRSA) was reported for
the first time in bovine and goat milk by Bhattacharyya et
al., 2016
S-8
9. Emerging AMR
Extended spectrum beta-lactamase producers
AmpC type beta-lactamase producers
Fluroquinolone resistant enterobacteriaceae
Methicillin resistant S. aureus and CoNS
Mettalo-lactamase producing enterobacteriaceae
(EC & KP)
Vancomycin resistant S. aureus (VRSA)
Colistin resistant enterobacteriaceae
(Bhoj R et al.,2021) S-9
12. Other associated factors
AMR Consultation when
animal is sick
(Garg et al.,2019)
Unrestricted access
to antibiotics
(Bhushan et al.,2016)
Farm practices
(Chauhan et al.,2018)
Selling of milk from cows treated with antibiotics
Avoidance of withdrawal periods
Residue levels of antibiotics
Misuse/ unplanned used of antibiotic at
field level
Lack of operational laboratory facilities
(lack of microbiologists, equipment etc)
S-12
18. CONVENTIONAL METHODS NON CONVENTIONAL
METHODS
PHENOTYPIC METHODS
(Evaluating the bacterial growth in the
presence of antibiotics)
Manual
oAgar dilution
o Gradient test
oDisk diffusion
oBroth micro dilution- use ready-made
cartridges or plates including positive controls and
gradient concentrations of antibiotics
Automated
oVITEK® 2 COMPACT
o Sensititre™ ARIS™ 2X
oAlfred 60AST system
MOLECULAR BASED METHODS
(Detect the AMR genes)
oPCR based methods
oIsothermal Amplification methods
oDNA Microarrays
Fourier Transform
Infrared (FTIR)
Spectroscopy
MALDI-TOF Mass
Spectrometry
Genome sequencing and
Meta genomics
S-18
20. Conceptual representation of possible movement of
antibiotic-resistant bacterial strains/genes between
different ecosystems
S-20
21. Cont..
• Recently, several reports have portrayed the presence of large number of resistant
bacteria and corresponding genes in a variety of animal food products.
(Coetzee J et al.,201
• Whole-genome sequencing and phylogenetic studies proposed that the methicillin-
resistant Staphylococcus aureus (MRSA) in livestock has evolved from methicillin-
susceptible S. aureus strains of human origin.
• Identification of similar or clonally related bacterial strains of animal origin in
human populations without any direct exposure to animals, linking them to the
consumption and/or handling of food.
( Founou et al.,2016)
• Livestock animals transmit livestock-associated MRSA and ESBL producing E. coli
to human. (Horigana et al.,2016 Kock et al.,2016)
S-21
22. KEY STEPS IN REGULATION OF
AMR IN INDIA
YEAR IMPLEMENTATION DETAILS
2011 “Jaipur Declaration on Antimicrobial Resistance” WHO South-East Asia
Region.
2012 The “National Programme on Containment of Antimicrobial Resistance”
was launched under the 12th five-year plan (2012–2017).
2017 National network of veterinary laboratories for AMR established
Adoption of National action plan on AMR
Antibiotic Residue limits in meat was released by FSSAI
Enrolment in WHO Global Antimicrobial Resistance Surveillance System
(GLASS).
2019 Manufacture, sale and distribution of colistin for food-producing animals,
dairy and animal feed supplements prohibited.
SOURCE- Antimicrobial Resistance and Infection Control (2020) S-22
24. RESPONSIBILITY
FOR
SECURED
FUTURE
GOVERNMENT/
POLICY MAKERS
FARMERS SCIENTISTS
INDUSTRIES
VETERINARIANS
•Assess trends of resistance
at National level
• Conduct research to
develop devices for rapid
and reliable diagnosis
1- Hygiene
/Disinfection and
surveillance system
Implementation of
current strategies at
ground level.
•Control self
medication practices
•Ensure completion
of applied drug
therapies
Data of antimicrobial
production, its sale and usage
should be shared with
government regulatory bodies
and veterinarians.
S-24
27. A- VACCINES
Comparative resistant phenomenon with respect to antibiotics and vaccines
(M, mutation; HGT, horizontal gene transfer; AV, antigenic variation; antibiotic-
resistant bacteria; antibiotic-sensitive bacteria).
S-27
28. Cont..
Recombinant vaccines (subunit antigens or genetically
engineered organisms) generally possess multiple
immunogenic epitopes, thereby requiring multiple mutations
for allowing resistance development
Eg : Gavac and Tick GUARD- Cattle (against Ripcephalus
microplus)
HIDATIL EG95- Sheep and Goat (against Echinococcus
granulosus (Riosa et al.,2016)
Eri silkworm larvae were explored for production of VLPs of
FMD O/IND/R2/75 using recombinant baculovirus encoding
polyprotein of FMD virus (Kumar et al.,2016 )
S-28
29. B:CHICKEN EGG YOLK ANTIBODIES
Role of IgY in prophylaxis and treatment of rotavirus diarrhea in animal neonates has
been recently reviewed in Thu et al.,(2017)
S-29
30. Lactobacillus sp.
Bacillus sp.
Enterococcus sp.,
Bifidobacterium sp.,
Lactococcus sp.,
Pediococcus sp.,
Saccharomyces
(Cheng et al.,2016) S-30
C- Probiotics, Prebiotics and
Synbiotics
1- Bovamine™
2- BovamineDefend™
This is Lactobacillus sp. based DFMs,
currently marketed for reducing E. coli
O157:H7 in cattle.
(Tellez et al.,2015)
31. Cont..
Prebiotics are mainly carbohydrate substrates
1- Oligosaccharides polysaccharides
2-Polyols,
3- Protein hydrolyzates, etc.
(Cheng et al.,2014, Uyeno et al.,2015)
4- Fructooligosaccharide along with spray-dried bovine serum
minimized the incidence and severity of enteric disease in calves.
(Quigley et al.,2002)
Synbiotic supplementation to animal feed augments the lactate
and antibody production, lowers intestinal pH, which alters the intestinal
microflora, and minimizes harmful bacteria in the gut.
(Hamasalim .,2016)
S-31
32. D:PHAGE THERAPY
• Targets a narrow group of bacteria and prevents dysbiosis as
the autochthonous bacterial flora remains unharmed.
S-32
STRATEGIES
33. In 2006, a phage cocktail of six types of pure bacteriophages, designated
as LMP-102™ was approved by US-FDA for application as food
additives for preventing Listeria spp. contamination of meat.
(Cheng et al.,2014)
Endolysins isolated from phage φ3626 were also shown to treat Clostridium spp. infections.
(Courchesne et al.,2009)
Protein HydH5, a virion associated peptidoglycan hydrolase from phage phiIPLA88 showed
antagonistic activity against S. aureus. (Rodriguez et al.,2011)
Lytic phage-producing depolymerase and ciprofloxacin are united to treat the biofilm
of Klebsiella pneumoniae than when ciprofloxacin was used alone.
(Verma et al.,2016)
S-33
The clustered regularly interspaced short palindromic repeats-associated (CRISPR-Cas) system in
bacteria provides sequence-based adaptive immunity against mobile genetic elements such as
viruses and plasmids. (Beisel et al.,2014)
35. Plants extracts explored against various diseases
causing bacterial pathogen in dairy animals
Scientific name
(common name)
Plant part
used
Type of extract
Antibacterial
activity against
Reference
Senna macranthera
(Large flower Cassia)
Roots Dichloromethane Staphylococcus aureus
Andrade
et al.,2015
Allium sativum
(garlic)
Cloves Juice Cryptosporidium sp.
Mageed
et al.,2015
Psidium guajava
(guava)
T.Foecum-graceum
(fenugreek)
leaves,
seeds
Methanol
S.aureus, E.coli,
P.aeruginosa,
Salmonella sp.
Balkrishna
et al.,2016
Cinnamon
casia oil
seed -
S.aureus, S.epidermicus,
S.hyicus, E.coli
Bhoj et al.,2016
Curcuma longa
( Aloe vera) leaf
Aqueous,
ethanol,
ethyl acetate
E.coli, S.aureus,
P.aeruginosa
Punniamurthy
et al.,2017
Panicum turgidum
(Desert Grass)
- aqueous
Streptococccus aureus,
Candida albicans
El-Desoukey
et al.,2017
S-35
36. F:ANTIMICROBIAL PEPTIDES
Host defense peptides possessing structural and
functional diversity.
(Source: Shai et al.,1999) S-36
37. Strub et al.,1996, studied bovine AMP, Chromacin, isolated from bovine
chromaffin granules, which inhibits the growth of both Gram-positive
and Gram-negative bacteria.
Bacitracin methylene salicylic acid and Bacitracin zinc had been
approved in USA and China as feed additives. (Cheng et al.,2014)
Milk proteins acts as precursors for antimicrobial peptides and have
been considered for application both as dietary supplements in functional
foods and as drugs.
In an interesting study, a modified peptide, L10 (WFRKQLKW)
obtained from bovine lactoferrin displayed potent antibacterial and
antifungal activity against ESBL producing Gram-negative bacteria and
Candida isolates. (Mishra et al.,2014)
S-37
38. It is ribosomally synthesized bacterial antimicrobial peptides (AMPs)
that can kill or inhibit closely related bacterial strains
(Tagg et al.,1976)
E.g – Colicins, Microcins, Nisin and Mutacin etc.
Three new variants of nisin A M17Q, nisin A HTK, and nisin A T2L showed
antibacterial activity against S.aureus strains associated with bovine mastitis.
(Field et al.,2021)
Microcin C7 and Colicins 1b and E1 from E. coli as well as enterocin RM6
inhibits pathogenic enterobacteria, i.e., Enterobacter agglomerans, E. coli,
K. pneumoniae, Morganella morganii, Salmonella enterica, Shigella flexneri
and Yersinia enterocolitica (Huang et al.,2013)
S-38
G:BACTERIOCINS
39. Commercially available products
Teat sanitizer : Amibicin N® (Nisin based)
Dairy Wipes :Wipe-Out® (Nisin based )
Teat dip: containing lantibiotic and lacticin 3147
(Pieterse R et al.,2010)
Teat seal :
1- Orbeseal (Nisin based )
(Pfizer Animal Health.,2004
2- Osmonds Teat Seal 2(®(Bismuth subnitrate-based)
(Ryan et al.,1994)
S-39
40. Stimulating factor
Act on feed components in Animal GIT
(
Assist Digestion and Nutrient Bioavailability
Improve General Health and Immunity
Reduces Practices of Drug Abuse
H: FEED ENZYMES
S-40
41. TRIVIAL NAME CLASSIFICATION GENERAL FUNCTION
α-Amylase,β-Amylase
α-Galactosidase
β-Glucanase
Carbohydrase Hydrolyzes starch,
oligosaccharides and
β-glucans
Lipase Lipase Hydrolyze glycerides
Bromelain, Ficain,
Papain,Pepsin
Protease Hydrolyzes proteins
Phytase Phosphatase Hydrolyzes phytate
Source :Regul Toxicol Pharmacol (2010)
In a study by Tewoldebrhan et al.,2017, feeding of β-mannanase enzyme (commercially
available as CTCZYME),reduce the somatic cell counts in milk samples of cows.
Phytase has been reported to have some characteristics effects on mineral
(i.e., calcium, phosphorus) digestibility along with production and secretion of mucin,
which influence the organization of intestinal epithelial surface and eventually
microbial composition of the gut. (Knowlton et al.,2007)
S-41
43. ZnO NPs possess antibacterial, antineoplastic, angiogenic, and wound-
healing properties, and has been proposed as a feed additive for mastitis
management.
(Raguvaran R et al.,2015)
S-43
TYPE Effective against Reference
Amoxicillin NPs E.coli,
S.aureus,
S.agalactiae
Xuefeng et al.,2009
Violecein NPs S.aureus Berni et al.,2013
AgNPs
Capsaicin
MDR-ESBL
producing E.coli
Kar et al.,2016
44. J: CYTOKINES
Cytokines plays an important role regulating the innate immune response
and are promising candidates for therapeutic interference in infectious
and autoimmune diseases (Dhama et al.,2013)
Application of cytokines alone or as conjunct therapy to antibiotics
improves the cure rate of bovine mastitis. (Hossain MK et al.,2017)
In a study , Recombinant cytokines, mammay glands Infusion of
cytokines (IL-1, IL-2) that led to increased polymorphonuclear cells,
with enhanced inducible oxygen radical formation in the milk and
thereby effectively preventing S. aureus infection. (Daley M et al.,1993)
S-44
45. K: IMMUNOSTIMULANTS
Imms, directly enhance innate immune responses through the
activation of phagocytes, neutrophils, alternative
complement system, and increased lysozyme activity.
S-45
Zelnate®, an innovative cytosine-
phosphate-guanine (CpG) motif-based
immunostimulant for animal health. It
effectively reduces bovine respiratory
disease caused by Mannheimia
haemolytica.
46. Immunostimulant Examples
Mineral substances Selenium, zinc
Amino acids Arginine, leucine, ubenimex
Vitamins A, E, C
Herbals Astragalus, Echinacea
Plant polysaccrides Algal polysaccharides, Astragalus
polysaccharide, chitosan, ganoderan
Microbial preprations BCG vaccine, cholera toxin B subunit,
Corynebacterium seedlings, muroetasin
Bacterial extracts β-glucan, peptidoglycan
Immunologic adjuvents Freund’s adjuvant, propolis, liposome,
Hormones and hormone like substances Growth hormone, metallothionein,
thymopentin, thymosin
Chemical synthetics Cimetidine, imiquimod, levomisole, pidotimod,
polyinosinic acid
Biological cytokines Interferon, transfer factor, interleukin, immune
globulin
SOURCE: Cheng et al.,2014, Song et al.,2014, Masihi et al.,2000 S-46
47. 8-CONCLUSION
• Although a new ray of hope arises with alternative
strategies but there is still a huge void between the
activity spectrum of antibiotic alternatives and
antibiotics itself.
• Interventions that address AMR from the animal health
perspective need to be encouraged and should be
designed in a manner that allows for their monitoring
and evaluation.
• So, it will be healthier approach if we will explore
alternatives as complementary strategy not as
replacement policy. S-47
48. References
• Abraham EP, Chain E. An enzyme from bacteria able to destroy penicillin Rev Infect Dis (1940) 10:677–
8.
• World Health Organization. WHO’s First Global Report on Antibiotic Resistance Reveals Serious,
Worldwide Threat to Public Health. (2014). Available from :
http://www.who.int/mediacentre/news/releases/2014/amr-report/en/
• Ganguly NK, Arora NK, Chandy SJ, Fairoze MN, Gill JP, Gupta U, et al. Rationalizing antibiotic use to
limit antibiotic resistance in India. Indian J Med Res (2011) 134(3):281–94
• McManus MC. Mechanisms of bacterial resistance to antimicrobial agents. Am J Health Syst Pharm
(1997) 54(12):1420–33.
• Centers for disease control and prevention, National centre for emerging and zoonotic infectious diseases
(NCEZID), Division of healthcare quality promotion (DHQP), Available at
cdc.gov/drugresistance/about.html, Accessed on 09.04.2020.
• Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiology and Molecular Biology
Reviews 2010;74(3):417-433.
• Pariza MW, Cook M. Determining the safety of enzymes used in animal feed. Regul Toxicol Pharmacol
(2010) 56:332–42. doi:10.1016/j.yrtph.2009.10.005
• Shai, Y. Mechanism of the binding, insertion and destabilization of phospholipid bilayer membranes by
α-helical antimicrobial and cell non-selective membrane-lytic peptides. Biochim. Biophys. Acta
Biomembr. 1999
• Brodzki P, Kostro K, Krakowski L, Marczuk J. Inflammatory cytokine and acute phase protein
concentrations in the peripheral blood and uterine washings of cows with subclinical endometritis in the
late postpartum period. Vet Res Commun (2015) 39(2):143–9. doi:10.1007/s11259-015-9635-4
S-48
