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Antibiotic usage, residues and resistance genes from food animals to human and environment: An Indian scenario
Accepted Manuscript Title: Antibiotic Usage, Residues and Resistance Genes from Food Animals to human and environment: An Indian scenario Authors: Krishnasamy Sivagami, Vijayan Jaya Vignesh, Ramya Srinivasan Govindaraj Divyapriya, Indumathi M. Nambi PII: DOI: Reference:
S2213-3437(18)30100-3 https://doi.org/10.1016/j.jece.2018.02.029 JECE 2221
To appear in: Received date: Revised date: Accepted date:
15-11-2017 7-2-2018 16-2-2018
Please cite this article as: Krishnasamy Sivagami, Vijayan Jaya Vignesh, Ramya Srinivasan Govindaraj Divyapriya, Indumathi M.Nambi, Antibiotic Usage, Residues and Resistance Genes from Food Animals to human and environment: An Indian scenario, Journal of Environmental Chemical Engineering https://doi.org/10.1016/j.jece.2018.02.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antibiotic Usage, Residues and Resistance Genes from Food Animals to human and environment: An Indian scenario
Krishnasamy Sivagami,Vijayan Jaya Vignesh, Ramya Srinivasan
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Govindaraj Divyapriya, Indumathi M. Nambi*
Environmental and Water Resources Engineering Division, Department of Civil Engineering, Indian Institute of Technology Madras, Chennai, India – 600036 *
Corresponding author: Tel: +91-44-2257 4289
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Email: [email protected]
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Graphical abstract
Abstract
Antibiotics are majorly used in food animals for growth promotion and prophylactic purposes in public health and environment. The aim of this review is mainly to discuss about the antibiotics
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usage in poultry, livestock and aquaculture sectors, particularly in India, and the identification of antibiotic resistance in animals, their corresponding antibiotic resistance genes (ARGs) (if investigated) and their dissemination among environmental compartments in various parts of the country. It also discusses about the classification and mechanism of action of different antibiotics. It reports the risks, benefits, ARGs development mechanisms from food animals to humans and to
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the environment. Most of the current studies are done in the medical field on regulating/restricting
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antibiotic usage and tracking the propagation of ARGs in bacterial samples of sick patients and
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also report the identification of antibiotics and ARGs in wastewater treatment plant effluents
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across various countries. This study mainly reviews the evolution and transportation of ARGs in
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detail for Indian scenario.
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1. Introduction
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Keywords: Antibiotics; Resistance genes; Food animals; Indian scenario; Super bugs
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In recent times, the occurrence and detection of pharmaceutically active compounds in the aquatic environment is a serious health concern. Rampant use of antibiotics in hospitals, livestock, poultry
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and aquaculture farms, indiscriminate disposal of waste and wastewater from municipalities, animal farms and pharmaceutical industries into rivers, lakes and other water bodies over the years, have contributed to the development of the mammoth problem of antibiotic resistance. Further, animal protein intake grew in Asia, it increased from seven to twenty-five grams per
capita in five decades (CDDEP, 2017). Consequently, the quantity of diet obtained from rice and wheat progressively decreased. To meet the growing demand, developing countries have moved towards highly cost-efficient and vertically integrated intense poultry, livestock and aquaculture
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production systems. Moreover, a study conducted by Ramanan Laxminarayanan states that, the antibiotic use in food animals will increase by 82 per cent in India by 2030 (Van Boeckel et al., 2015), which would be quite an alarming increase.
Most of the antibiotic dose (30-80%) given to food animals are excreted because of partial metabolization of antibiotics. In addition, animal feed-containing antibiotics that are not consumed
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by animals will reach the soil sediments directly. Another source of antibiotics entering the
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environment is through manure. Solid waste generated from animal farms is mostly used as
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manure (called as farm yard manure) to fertilize the soil (Ramesh, 2016). However, most of the
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antibiotics leach during run off and end up in aquatic systems such as rivers and lakes. In spite of
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knowing the fact that antibiotics have been entering the environment through various sources, only
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a very few research investigations have been done on the fate and transport of antibiotics present in storage-manure or animal excreta. It is also known that the natural soil microbial communities get
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affected severely due to the presence of broad spectrum antibiotics in soil. It results in the
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emergence of ARGs in the soil bacteria and creates "super bugs" which severely impact the human and environmental health (Washer and Joffe, 2006).
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Aquaculture sector uses antibiotics for prophylactic purposes, as therapeutic measures and as feed additives (Aly and Albutti, 2014). Very few drugs like oxytetracycline hydro chloride, sulfamerazine and a combined preparation that contains sulfadimethozine and ormetoprim have been approved by FDA for use in aquaculture in developed countries. On the other hand, less
stringent guidelines in developing countries like India have led to rampant and reckless use of unapproved drugs in aquaculture (CIBA, 2016). It is a fact that 70-80% of the antibiotics used in aquatic farming end up in the environment. The reason for these antibiotics to end up in
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environmental compartments is partial/incomplete metabolism, which leads to development of antimicrobial resistance (AMR) in the exposed bacteria (Hernandez, 2012). Hence, there is a need to track the fate and transformation of antibiotics during their metabolism and transfer into the food chain and to identify forms of antibiotics in animal products and waste.
Antibiotic resistant bacteria (ARB) thrive in soil all over the world because soil contains a lot of
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antibiotic-producing strains. For instance, Actinomycetes is the most ubiquitous soil bacteria, and
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has the potential to synthesize more than half of the world’s antibiotics (such as erythromycin,
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streptomycin and tetracycline) (Berdy, 2012). If the soil is laden with antibiotics, then that is the
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best possible route for spreading antibiotic resistance through manure and other agricultural
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practices. Especially, opportunistic pathogens like Acinetobacter and Pseudomonas when present
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in the soil, due to their intrinsic capability to develop resistance, acquire ARGs from the natural
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environment (soil) and they become ARBs (Argudin et al., 2017). D’Costa et al.,(2006) stated that 480 Streptomyces strains cultured from soil were tested for 21
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antimicrobials and it was found that the strains were multi-drug resistant, on an average, to 8 antimicrobials. The most interesting revelation was that 2 strains were resistant to about 15 drugs.
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Thus, in nature, soil presents a single high potential reservoir of ARGs; if the resistance can be passed on to pathogens, there will be a linkage between the environment and human health to a great extent (Allen et al., 2010). ARGs are often positioned on mobile genetic elements like plasmids and transposons, which ensure the spread by horizontal gene transfer. Mobile elements
like plasmids harbor a lot of ARGs which have been found in clinically relevant pathogens found in soil and water like Salmonella, Shigella, Escherichia, Aeromonas, Pseudomonas and Klebsiella
2. Classification and mechanism of action of antibiotics
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(Stokes and Gillings, 2011).
Food animals are injected with antibiotics to take care of the clinical disease, to avoid general disease events and to promote growth. The use of antibiotics in food animals are classified as prophylactic and sub-therapeutic use. Following are the twelve classes of antibiotics that are used
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during the life cycle of food animals, (1) arsenicals, (2) polypeptides, (3) glycolipids, (4)
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tetracyclines, (5) elfamycins, (6) macrolides, (7) lincosamides, (8) polyethers, (9) beta-lactams,
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(10) quinoxalines, (11) streptogramins, and (12) sulfonamides (Landers et al., 2012). World Health
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Organization (WHO), World Organization for Animal Health (OIE) and the Food and Drug Administration (FDA) stated that fluoroquinolones, 3rd and 4th generation cephalosporins and
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macrolides as “critically important” antimicrobial agents (WHO/OIE/FDA, 2006). Antimicrobial
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agents are frequently used to treat various epidemic events and used for various purposes in
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livestock populations. Each family or subgroup of antimicrobials has its own purpose and mode of action to target species. Hence, each microbial species counters the antimicrobial agents with
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specific defense/resistance mechanism(s) for its survival. The main antimicrobial families, their mode of action and their general resistance development mechanisms are summarized in Table 1
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(Burch, 2012).
3. Antibiotics and ARGs
Residues of antibiotics are released into the environment due to incomplete absorption and metabolic activities of food animals. Antibiotics from food supplement may be lost either through
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leaching or elimination by urine and feces. The possibility of antibiotics reaching the environment depends on their properties, bio-availability and surrounding environmental characteristics (Kumararaj et.al., 2016). Development of antibiotic resistance is considered to be a major risk factor in addition to possible toxicity, allergy or carcinogenicity to humans (CIBA report, 2016). The bacteria which have acquired resistance against antibiotics are called Antibiotic Resistant
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Bacteria (ARB) (Magiorakos et al., 2012).
Sulfadiazine
Mode of action Purine synthesis of DNA which interferes folic synthesis rRNA binds with 50S subunit which prevents protein production
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Drugs Classification Sulfonamides
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Table 1. Classification of antibiotics used in food animals (Burch 2012)
Resistance mechanism Chromosome mutation, plasmid and integron mediated resistance
Methylation of 23S sub-unit of rRNA prevents binding and drug inactivation Inducible efflux in E.coli (Tet A, Tet Tetracyclines B) Binding site changes (Tet O, Tet M) Inhibits cell wall production β lactamase production primarily Penicillin, and binds enzymes which changes cell wall proteins so that they Beta lactamase sensitive, help form peptidoglycans cannot bind to penicillin binding Other penicillins, proteins. cephalosporins Interfere in DNA breakage - Target modification - DNA gyrase Quinolones, reunion step by binding Decreased permeabilityouter Flouro quinolones DNA-gyrase membrane porins mutation Binding of rRNA to 50S Methylation of rRNA in gram Macrolides/
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Lincosamide Macrolides,Tialmulin
Azalides
sub unit which inhibits positive organisms and inhibits transpeptidation and binding production of protein Phosphorylation and adenylation of amino glucoside stops the binding rRNA binds to sub units Resistance mechanism varies which inhibits protein and depending upon the type of cell wall production antibiotics
Aminoglycosides
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Others
ARBs are capable of transferring their resistance gene to other bacteria through plasmid DNAs by Horizontal Gene Transfer (HGT) by disseminating the ARG among other bacteria. ARBs also use genetic elements such as integrons, transposons and bacteriophages to disseminate the ARG.
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ARGs are considered to be emerging environmental contaminants because once they are into the
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system; they are capable of spreading the antibiotic resistance to other bacterial cells in the system
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as well. This dissemination of antibiotic resistance may happen by one or more of the following
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ways (Pruden et al., 2006, Burch 2012). (i). Horizontal gene transfer (HGT) (occurs between pathogens, non-pathogens and also between distantly related bacterial species). (ii). Multiple
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antibiotic resistant (MAR) integron - one antibiotic co-selecting for resistance to other antibiotics
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(iii). Persistent nature of DNA - on the death of ARG-containing cells, there occurs the release of DNA into the surrounding environment. The DNA which has been released is quite persistent and
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protected due to certain soil compositions. Thus, the DNA is transformed into other bacterial cells
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which are alive and hence resistance is further passed on. iv) Another way of transmission of resistance is, DNA sending messages to the ribosome (rRNA 50S and 30S sub units) to produce
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poly peptides or proteins for growth. When the bacterium is ready to divide, the DNA uncoils, and the rapid prolific bacteria has more chance to develop mutants which also increases the antibiotic resistance.
3.1. Antibiotic resistance - occurrence and dissemination
The contamination issue does not stop at the point of occurrence of antibiotics; it includes the presence of ARGs as well in the environment which is becoming a major global threat in recent
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years. The antibiotics widely used in livestock, poultry and aquaculture have been studied, are listed in the table below (Table. 2), to understand the possible direct impacts of improper use and/or disposal of antibiotics. It is important to study the link between the illegitimate use of antibiotics, exposure to antibiotics-containing water and the gain of resistance.
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3.2. Antibiotic resistance studies in India
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As mentioned earlier, pharmaceuticals such as antibiotics pose a great global threat due to build-up
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of antibiotic resistance in the environment, leading to untreatable infections which used to be
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treatable previously, due to the formation of super bugs. Antibiotic resistance is an emerging
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environmental and health crisis and requires immediate research attention and understanding of its
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occurrence and dissemination. The dissemination of antibiotic resistance involves three compartments, namely, (i) various sources, (ii) environmental pathways and exposure routes, and
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(iii) receptors. Figure 1 gives a representation of the compartments and how the sub-compartments
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are inter-related. Due to the excessive usage of antibiotics as growth promoters in livestock and other animals, animal excreta have significant concentrations of antibiotics, which when washed
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off with water and trickles down into the aquifers along with rainwater, results in contaminating the environment (Figure 1a). Similarly, excessive and inappropriate use of antibiotics by humans can lead to contamination of sewage treatment plants (Figure 1b). Hospital and pharmaceutical industry-wastes are also illegally let into sewage systems, further contaminating the wastewater treatment plants (WWTPs) (Figures 1c and 1d). Improper disposal of unused and/or expired
antibiotic pills by flushing down the drain and overuse of unprescribed antibiotics due to over-thecounter sales, can also result in polluting the WWTPs (Figures 1e and 1f). Eventually, by the above-mentioned ways, various components of the environment are contaminated by antibiotics
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and finally lead to ARBs and ARGs in the sewage. These result in further contaminating the water bodies due to discharge of treated sewage. Subsequently, humans encounter a similar circumstance (as that of animals) through various modes like oral ingestion of contaminated drinking water,
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inhalation of microbial aerosol and become a major vector for dissemination.
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Fig.1 Sources of antibiotic usage, its spread and transfer of resistance genes to humans
Table 2. Representative studies on antibiotic resistance in India
Animal Livestock -
Microorganisms E.coli
Antibiotics tested
Details
Nitrofurantoin, cotrimoxazole, tetracycline,ampicillin
10 out of the total (14) tested isolates were resistant to either one or more of
Location West Bengal
Ref. Manna et al., 2006
Salmonella
Bovine
S. aureus
Bovine
Shiga toxin producing E.coli (STEC) and nonSTEC isolates
Ducks
35 Enterococc us isolates
Chloramphenicol, gentamycin sulphate. Lincosamide and macrolide-based antibiotics were also tested
Poultry
Non-typhal salmonella isolates E. coli
Oxytetracycline
Piglets
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Out of 111 isolates, about 20-30% were resistant
-
Kumar et al., 2011
Out of the STEC strains, 17% were found to have eaeA gene and among nonSTEC strains, 14.28% had eaeA. All STEC were resistant to 3 or more of the antibiotics tested. All of them were non-sensitive to cephalexin and kanamycin Lincosamide and macrolide antibiotics were not effective. Enterococcus isolates were found to be susceptible to gentamycin sulphate and chloramphenicol All the strains were resistant to oxytetracycline
Gujarat
Arya et al, 2008
Assam
Saikia et al., 1995
South India
Saravana n et al.,2015 Singh et al., 1992
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Cloxacillin, cephaloridine, oxytetracycline, ampicillin, amoxicillin, chloramphenicol, trimethoprimsulphamethaxazole, doxycycline, chloramphenicol Ampicillin, erythromycin, nalidixic acid, oxytetracycline, sulfadiazine, cefixine, lincomycin,
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Samanta et al., 2014
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Poultry, humans, bovine, equine, sheep
West Bengal
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Levoflaxin, chloramphenicol, norfloxacin, ciprofloxacin, gentamycin, oxytetracycline Erythromycin, tetracycline, gentamycin, lincomycin Tetracycline,cephalexi n, enrofloxacin, kanamysin, cephaloridine, ampicillin, amikacin
these antibiotics 100% of the isolates were resistant to the antibiotics of concern
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Bovine Poultry
63.2% of the isolates Lucknow displayed resistance towards atleast one drug and out of which multi-resistant were 41%
Greater than 80% resistant to the antibiotics tested
Mizoram
Dutta et al., 2011
34 isolates Salmonella
16 antibiotics were tested
770 isolates of Vibrio cholerae
Polymyxin-B, cephalothin, chloramphenicol, streptomycin, tetracycline, oxytetrcycline, sulfadiazene
Fin fish
82 Vibrio parahaemol yticus isolates
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Cuttlefish and prawn Fin fish, shell fish and crustacea ns
Ampicillin, cefpodoxime, streptomycin, carbenicillin, cephalothin, amoxicillin, colistin, tetracycline, naxidic acid
In addition, referred to CDDEP, 2017
Sethi et al., 1976
25% of the drugs were multidrug resistant and 67.5 % were resistant to 2 or more of the tested antibiotics All the salmonella strains were susceptible
Mangalor Deekshit e et al., 2012
Hatha and Lakshma naperuma lsamy, 1995
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40 salmonella isolates
Various locations in India Tamil Nadu
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Bacitracin (98.7%), chloramphenicol (6.7%), gentamycin (16.2%), nalixidic acid (11.7%), neomycin (53.3%), novobiocin (90%), oxytetracycline (46.2%), penicillin G (92.9%), polymixin B (18.8%), streptomycin (19.6%) Multiple antibiotic resistance genes were tested for their presence
95.4% of the isolates were found to be resistant to the antibiotics tested 98.7% of the strains were resistant to Bactracin, Novobiocin and penicillin G
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704 salmonella strains 240 salmonella isolates
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SeafoodF ish and crustace
roxythromycin, penicillin chloramphenicol
Polymyxin-B, cephalothin Parangip andchloramphenicol were ettai found to be sensitive. Streptomycin, tetracycline, oxytetrcycline, sulfadiazine had greater than 25% resistant strains.Significant number of tested finfish samples had ARB. Greater than 80% of the Cochin isolates showed resistance against ampicillin, cefpodoxime, streptomycin, carbenicillin, cephalothin. 63% and 77% of the isolates displayed resistance against amoxicillin, colistin. All the isolates were sensitive to tetracycline and naxidic acid
Kamatet al., 2005 Sathiyam urthy et al., 1997
Sudha et al., 2012
Some of the major works since the seventies on the occurrence of antibiotic resistance in cattle, other livestock forms and poultry have been tabulated in Table 2 (CDDEP Report, 2017). Besides
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the works mentioned in the table, several research works have been reported as listed below. Resistance has been identified against many antibiotics in several of the E.coli (Kawoosa et al., 2007; Ghatak et al., 2013; Sharma et al., 2015) and S.aureus (Kumar et al., 2011; Tiwari et al., 2011; Dutta et al., 2011; Kumar et al., 2012; Preethirani et al., 2015) isolates obtained from bovine animals. Similarly, antibiotic resistance present in isolates from poultry had also been reported.
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Shivachandra et al. (2004) examined 123 isolates from chickens in various parts of India and found
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that all isolates exhibited complete resistance to sulfadiazine and larger part of the isolates
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displayed resistance against penicillin, erythromycin, amikacin and carbenicillin. On the other
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hand, 74% isolates were susceptible to chloramphenicol. Similarly, Mir et al. (2015) examined
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isolates from Salmonella enterica and found that majority of the isolates was resistant to numerous
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antibiotics such as β-lactam. Besides these, numerous studies have been reported on antibiotic resistance based on isolates from poultry (Kar et al., 2015; Rasheed et al., 2014, CDDEP, 2017).
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CDDEP, 2017 has also dealt with antibiotic resistance among food animals in detail. Antibiotic
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resistance can be transferred from animals/plants to humans by food chain, direct/indirect link with animal health workers/livestock industry and in agriculture through manure application. The
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spread of resistance between different ecosystems rely on the concentrations of antibiotic resistance genes (ARGs) and their responsible host bacteria present in an ecosystem and also largely vary on the rate of their exchange between ecosystems.
4. Massive use of antibiotics in veterinary medicine
Antibiotics used in veterinary and human medicine are from the same class and sometimes similar in structure. A study reported in the Proceedings of the National Academy of Sciences, found that
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the antibiotic consumption between 2010 – 2030 is expected to rise by a staggering 67% (Van Boeckel et al., 2015). More than 66% of the worldwide rise in antimicrobial consumption can be attributed to the increasing quantity of animals being raised for food production. Thus, veterinary medicine demands a greater and wider share of the sectoral antibiotic consumption globally. The real problem in this area is that, antibiotics were administered not only to treat the animals but for
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growth promotion, and significantly in disease-prevention, making the animal houses a hotspot of
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antibiotic resistance dissemination. Despite a large number of bans in the United States
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(quinolones in poultry industry), some important antibiotics continue to be routinely used for
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prophylactic measures among crowded animals.
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4.1. Transfer from poultry farm environment
Poultry litter constitutes one of the key sources of ARBs. In commercial poultry farms, antibiotics
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are administered from a day-old chick to an adult bird for a period of 6 weeks. Usually, poultry
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farm floors are covered with bedding materials. The bedding is made of materials such as softwood, and during the growing period, this gets mixed with chicken feces, skin, feather and
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insects. This mix is called the poultry litter and it gets replaced with fresh wood shavings between different time intervals. This litter is usually rich in mineral content, and can be used as a fertilizer. Antibiotics are used in poultry for growth promotion and for disease prevention. As previously mentioned, the metabolic rate of antibiotics is low and hence 90% of the administered dose is excreted in feces. Avian intestines are one of the potential reservoirs of E. coli. These bacteria
have a huge potential for zoonotics, so there is a higher chance for AMR spread from birds to humans (Ewers et al., 2009). Feeding greatly influences the microbial ecosystem of broiler litter. The population of beneficial bacteria can be increased, and pathogenic E. coli populations can be
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decreased in the litter by the addition of mannan and purified lignin to broiler feed (Baurhoo et al., 2007). Resistant E. coli from contaminated litter are transferred to the environment which in turn contaminate the surface water and groundwater through run-off happening while storing litter for long time periods in an open source.
Possible link between livestock/poultry and aquaculture
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4.2.
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Van Boeckel (2015) has predicted that antibiotic consumption by chickens in India in chicken is
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rapidly increasing and area of high consumption is expected to grow by 312% by 2030. The major
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concern is that India has not come up with regulatory guidelines to limit the usage of antibiotics in livestock or poultry. There is a common practice existing in integrated farming in Asia, where the
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organic waste from livestock and poultry which is rich in ARB, are fed to farm fish. Thus, transfer
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of resistance happens in the aquaculture farms (Jayathilakan, 2012). This link can be established
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with different perspectives. Companion animals like dogs and cats are being more or less treated as family members by pet lovers. Thus, these pets receive the most advanced medical treatment with
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newer antibiotics. There is always a risk of zoonotic infections. Due to over-usage of antibiotics, increasing resistant strains are produced which impact human health. In recent times, there are
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reports
of
increasing
resistance
in
animals.
For
example,
carbapenemase-positive
Enterobacteriaceae, pig farm associated MRSA ST398, quinolone resistance in food products (plasmid mediated) have been reported recently (Argudin et al., 2017).
4.3.
Spread of AMR in the environment from wastewater
There is also a common practice in countries like India where animal manure is widely used in agriculture. Animal waste represents one of critical links in spreading AMR because they harbor
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AMR bacteria. Antimicrobials are never completely metabolized and finally find their way into sewage treatment plants. In countries like India, pharmaceuticals and personal care products are abundant in WWTP and thus the selection pressure for AMR is high in these environments (Marathe et al., 2013). It is a pity that our WWTP are not equipped for detecting or treating pharmaceutical or personal care products and this is a massive source of antimicrobial exposure to
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the environment. Even some personal care products like hand wash soaps which contain triclosan,
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serve as a potent antimicrobial selection source in the domestic wastewater (Jutkina et al., 2017).
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4.4. Spread of AMR from environmental sources like rivers/lakes to humans/animals/plants
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In India, many rivers and streams are polluted with industrial and domestic wastewater. Many
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occasions when wastewater is let into the rivers without treatment lead to massive inputs of AMRladen bacteria into the natural unpolluted environments. This water is being used to feed the
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livestock, and is also used for irrigation and thus, transfer is obvious in these aquatic environments.
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A recent research article in 2017 investigated water samples collected from the Musi River near a WWTP of a pharmaceutical industry in Hyderabad (Lubbert et al., 2017). Samples were analyzed
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for 25 antibiotics with liquid chromatography–tandem mass spectrometry. High concentrations of moxifloxacin, voriconazole, and fluconazole were detected in the sewers in an industrial area in Patancheru–Bollaram area. In a recent research study, Vibrio coralliilyticus, one of the most
prevalent marine pathogens responsible for coral disease in aquaculture farms was isolated from samples collected from ten different sites in Cochin and Kumarakom and four different shrimp
farm hatcheries. Thirty different strains of V. coralliilyticus were analyzed for antibiotic resistance towards 20 antibiotics using disk diffusion method. Resistance was observed against beta lactams (amoxicillin,
ampicillin
and
carbenicillin),
tetracycline
(oxytetracycline),
pyrimidine
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(trimethoprim), ntirofurans (nitrofurantoin and furazolidone), sulphonamides (sulphamethoxasole), quinolones (enrofloxacin) and macrolides (erythromycin) (Silvester et al., 2017). Vignesh et al., (2016) reported that the water and sediment samples obtained at the eco-regions of Chennai coast were detected with ARB. Most of the bacterial strains of a total of 960 isolates belong to the four major genera. Highest resistances were shown for vancomycin and penicillin with the frequency of
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53.6 % and 52.6% respectively. Chloramphenicol, ciprofloxacin, gentamicin and tetracycline were
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Risks and benefits of using antibiotics
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5.
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observed to be most effective on the bacteria with the frequency range of less than 7%.
The market value of antimicrobial drugs used to treat animal diseases stood at $20.1 billion in
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2010. It has been steadily increasing as $8.65 billion in 1992 and predicted to rise to $42.9 billion
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in 2018 (GIA, 2012). Best quality meat with high protein content and low fat is being produced
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from animals fed with antibiotics (Hughes and Heritage, 2002). Tetracycline and penicillin usage in poultry have considerably improved egg production and hatchability (Gustafson and
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Bowen, 1997). Besides poultry, antibiotic-supplemented feed had a marked improvement in the health of livestock. Sulfamethazine and chlortetracycline supplements, significantly reduced the
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rate of relapse and respiratory disease morbidity (Gallo and Berg, 1995). Apart from their antimicrobial effects, antibiotics (particularly macrolides) have an anti-inflammatory potential which rationalizes their beneficial effects (Buret, 2010). Livestock, poultry and aquaculture industries thus rely on the crucial role of antibiotics for efficient animal food production strategies.
It has been well documented that the diarrhoeagenic E. coli has been the underlying cause of acute diarrhea in Indian children. A study reported that rural child population living near poultry farms in Tamil Nadu are at high risk for E.coli resistance to fluoroquinolones, tetracycline, and
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gentamicin (Murthy et al., 2008). Emergence of global resistance of avian influenza strain H5N1 is due to unrestricted usage of amantadine in China. Amantadine, a potential life-saving drug rendered ineffective during the H5N1 outbreak. In India, amantadine drug resistant H5N1 avian influenza was reported in West Bengal in 2011.
One of the major diseases affecting poultry industry is Salmonellosis which has posed a severe
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threat resulting in substantial morbidity. High stocking density, high dust levels, low ventilation
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and stress in poultry farms have the potential to exacerbate the problem. Center for Disease
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Dynamics, Economics &Policy (CDDEP) and Centre for Science and Environment (2014) have
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reported increased levels of ARB in chickens in Punjab (CSE, 2014). These studies have proven
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that more than 66% of the farms use antibiotics for growth promotion, out of which, farm animals
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have 3 times more likeliness to develop antibiotic resistance. Huge demand for good quality meat has increased the antibiotic usage in animal food industry which results in wider selection of
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pathogens. The result of expansion of this sector is mainly due to the demand for the poultry. The extreme growth in consumption is expected to grow further by 312% by 2030 (Boeckel et. al.,
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2015).
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However, a major grey area in this field is that, even though several research works are conducted across the world on identifying the antibiotics spread and the resistance gene associated with it in various compartments of the environment such as water and wastewater, studies with respect to Indian scenario is minimal and needs to be explored to a great extent. Further, despite several research explorations happening globally to understand and fight this critical issue, other means
such as vaccines and bacteriophages may turn out to be an alternate method to treat resistant bacterial infections (Raghunathan, 2008). At the same time, experimentation of such alternate methods must be assessed of their pros and cons before commercializing them.
Implications on human health
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6.
A research study published in the Lancet in 2010 by Karthikeyan Kumarasamy et al. about the NDM1 superbug created a storm in the healthcare sector of India. Politicians and physicians felt exposed after this publication about multidrug resistance in India and medical tourism in the
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country was severely criticized (Kumarasamy et al., 2010). In 2016, Indian researchers have
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identified mcr-1, which has resistance to the last mile antibiotic (colistin) that humans have access
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to. Environmental reservoirs of AMR have demonstrated the potential to serve as the source of
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antibiotic resistance genes for clinically relevant microbes (Ghosh and Lapara, 2007; Perry and Wright, 2013). Further, antimicrobial compounds which end up in aquatic systems have the
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potential to interrupt native microbial species which is essential to maintaining the ecosystem
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(nitrification/denitrification), soil fertility and biological cycles (Costanzo et al., 2005; Kinney et
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al., 2006; Kummerer, 2004). As such, antimicrobials are widely recognized as emerging environmental contaminants (Pruden et al., 2006) and, given their alarming levels were classified
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as a priority risk group in recent years.
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Besides, several human pathogens have started gaining resistance. Among enteric pathogens, Vibrio cholera seems to have attained resistance towards nalidixic acid, furazolidone and cotrimoxazole and is susceptible to tetracycline (near Delhi region, India). However, in some areas of Bangladesh, tetracycline seems to be ineffective. This shows that resistance spectrum varies between regions and antibiotic consumption practices (Sharma et al., 2007; Saha et al., 2006).
Typhoid and paratyphoid causing micro-organisms had higher susceptibility to antibiotics, back in late 1980s, which made them treatable using chloramphenicol, ampicillin and cotrimaxazole. However, soon after acquirement of resistance to these drugs to a great extent, the usage of
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fluoroquinolones has been in the fore-front. Later, chloramphenicol lost sensitivity for a short period, following which due to the popularization of fluoroquinolone, sensitivity to chloramphenicol was restored (Raghunath and Narayanan1989; Anand et al., 1990; Raghunath, 2008). Ray et al. (2006) and Khaki et al. (2007) have studied the resistance of Neisseria gonorrhoeae (pathogen causing sexually transmitted diseases) towards penicillin and
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fluoroquinolone. This has resulted in a situation where third generation antibiotics need to be used.
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Furthermore, it is becoming more difficult to treat deadly diseases like tuberculosis by using basic
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level drugs. Mycobacterium tuberculosis has acquired multi-drug resistance (simultaneous
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resistance to rifampicin and isoniazid). In addition, it has lost sensitivity to fluoroquinoline and a few second line drugs which has made it an extensively drug resistant bacteria (Ramachandran and
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Narayanan 2008; Raghunath, 2008; Jesudasan et al., 2003). Further, Mycobacteria genus also
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causes leprosy, (the causative pathogen being Mycobacteria leprae). However, this pathogen has
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started acquiring resistance to rifampicin, clofazimine and dapsone. This is not a healthy sign for
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curing such dangerous diseases.
On the other hand, urinary infections are less dangerous and common among out-patient infection
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complaints, they have acquired resistance to antibiotics as well. Based on the research work conducted by Akram et al. (2007) in Aligarh, India, 100 samples out of 920 exhibited loss of susceptibility to the antibiotics tested. Out of 100 samples, 61% was E.coli and 22% was Klebsiella spp. that were resistant. In addition, ampicillin and co-trimoxazole were found to be ineffective against Gram negative bacilli. Kumar et al. (2017) have investigated AMR patterns of
654 enteric pathogens in India. They have conducted a detailed study on AMR traits on multi-drug enteric pathogens. The study included microbial species namely, P.stuartii, K.pneumoniae, E.coli, S. typhimurium, S.flexneri and P.aeruginosa. Their study indicates that these pathogens are
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resistant against 22 antibiotics which belong to 9 distinct classes of antibiotics. The major findings of their study are as follows. (i) About 97% of strains have lost susceptibility towards at least 2 antibiotics, (ii) 24% against at least 10 antibiotics and (iii) 3% of the isolates seem to exhibit resistance against at least 15 antibiotics.
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7. Conclusions
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This review mainly focuses on (i) the usage and prevalence of antibiotics in the environment, (ii)
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presence of antibiotic resistance in animals and (iii) dissemination of antibiotic resistance through
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various mechanisms in Indian scenario. Due to excessive and inappropriate human consumption of antibiotics, and administration of antibiotics to animals (not just for treatment of infections, but
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also for prophylactic purposes and as a form of growth promoters), it has resulted in the presence
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of antibiotics in the environment,in turn leading to dissemination of antibiotic resistance.
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The best way to control the spread of antibiotic resistance is to raise awareness to the general
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public by print and social media to increase the demand for antibiotic-free food products. Veterinarians, agricultural experts and legal authorities in India should address the issue of
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antibiotic resistance by regulating policies which can monitor or track the antibiotic usage, limiting its dosage, etc. Apart from these, people involved in waste management and municipal authorities should be educated about proper disposal of expired medications, poultry and aquaculture wastewater. Non-therapeutic use of antibiotics should be tracked and phased out.
A systematic nationwide rigorous surveillance system to monitor the antibiotic residues in veterinary, poultry and aquaculture environments is very much essential. Vertical surveillance cannot identify all the major drivers of AMR whereas a cross-sectional surveillance can ensure
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better understanding of underlying factors in spread of AMR to best identify the sectoral contribution. The other effective method is to control or prevent infection in humans and animal sources, which can be done by healthcare professionals. Wastewater treatment facilities built near the pharmaceutical manufacturing facilities must treat the effluents and remove the antibiotics and
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their metabolites before their release into the environment.
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S2213-3437(18)30100-3 https://doi.org/10.1016/j.jece.2018.02.029 JECE 2221
To appear in: Received date: Revised date: Accepted date:
15-11-2017 7-2-2018 16-2-2018
Please cite this article as: Krishnasamy Sivagami, Vijayan Jaya Vignesh, Ramya Srinivasan Govindaraj Divyapriya, Indumathi M.Nambi, Antibiotic Usage, Residues and Resistance Genes from Food Animals to human and environment: An Indian scenario, Journal of Environmental Chemical Engineering https://doi.org/10.1016/j.jece.2018.02.029 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Antibiotic Usage, Residues and Resistance Genes from Food Animals to human and environment: An Indian scenario
Krishnasamy Sivagami,Vijayan Jaya Vignesh, Ramya Srinivasan
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Govindaraj Divyapriya, Indumathi M. Nambi*
Environmental and Water Resources Engineering Division, Department of Civil Engineering, Indian Institute of Technology Madras, Chennai, India – 600036 *
Corresponding author: Tel: +91-44-2257 4289
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Email: [email protected]
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Graphical abstract
Abstract
Antibiotics are majorly used in food animals for growth promotion and prophylactic purposes in public health and environment. The aim of this review is mainly to discuss about the antibiotics
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usage in poultry, livestock and aquaculture sectors, particularly in India, and the identification of antibiotic resistance in animals, their corresponding antibiotic resistance genes (ARGs) (if investigated) and their dissemination among environmental compartments in various parts of the country. It also discusses about the classification and mechanism of action of different antibiotics. It reports the risks, benefits, ARGs development mechanisms from food animals to humans and to
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the environment. Most of the current studies are done in the medical field on regulating/restricting
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antibiotic usage and tracking the propagation of ARGs in bacterial samples of sick patients and
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also report the identification of antibiotics and ARGs in wastewater treatment plant effluents
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across various countries. This study mainly reviews the evolution and transportation of ARGs in
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detail for Indian scenario.
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1. Introduction
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Keywords: Antibiotics; Resistance genes; Food animals; Indian scenario; Super bugs
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In recent times, the occurrence and detection of pharmaceutically active compounds in the aquatic environment is a serious health concern. Rampant use of antibiotics in hospitals, livestock, poultry
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and aquaculture farms, indiscriminate disposal of waste and wastewater from municipalities, animal farms and pharmaceutical industries into rivers, lakes and other water bodies over the years, have contributed to the development of the mammoth problem of antibiotic resistance. Further, animal protein intake grew in Asia, it increased from seven to twenty-five grams per
capita in five decades (CDDEP, 2017). Consequently, the quantity of diet obtained from rice and wheat progressively decreased. To meet the growing demand, developing countries have moved towards highly cost-efficient and vertically integrated intense poultry, livestock and aquaculture
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production systems. Moreover, a study conducted by Ramanan Laxminarayanan states that, the antibiotic use in food animals will increase by 82 per cent in India by 2030 (Van Boeckel et al., 2015), which would be quite an alarming increase.
Most of the antibiotic dose (30-80%) given to food animals are excreted because of partial metabolization of antibiotics. In addition, animal feed-containing antibiotics that are not consumed
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by animals will reach the soil sediments directly. Another source of antibiotics entering the
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environment is through manure. Solid waste generated from animal farms is mostly used as
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manure (called as farm yard manure) to fertilize the soil (Ramesh, 2016). However, most of the
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antibiotics leach during run off and end up in aquatic systems such as rivers and lakes. In spite of
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knowing the fact that antibiotics have been entering the environment through various sources, only
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a very few research investigations have been done on the fate and transport of antibiotics present in storage-manure or animal excreta. It is also known that the natural soil microbial communities get
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affected severely due to the presence of broad spectrum antibiotics in soil. It results in the
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emergence of ARGs in the soil bacteria and creates "super bugs" which severely impact the human and environmental health (Washer and Joffe, 2006).
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Aquaculture sector uses antibiotics for prophylactic purposes, as therapeutic measures and as feed additives (Aly and Albutti, 2014). Very few drugs like oxytetracycline hydro chloride, sulfamerazine and a combined preparation that contains sulfadimethozine and ormetoprim have been approved by FDA for use in aquaculture in developed countries. On the other hand, less
stringent guidelines in developing countries like India have led to rampant and reckless use of unapproved drugs in aquaculture (CIBA, 2016). It is a fact that 70-80% of the antibiotics used in aquatic farming end up in the environment. The reason for these antibiotics to end up in
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environmental compartments is partial/incomplete metabolism, which leads to development of antimicrobial resistance (AMR) in the exposed bacteria (Hernandez, 2012). Hence, there is a need to track the fate and transformation of antibiotics during their metabolism and transfer into the food chain and to identify forms of antibiotics in animal products and waste.
Antibiotic resistant bacteria (ARB) thrive in soil all over the world because soil contains a lot of
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antibiotic-producing strains. For instance, Actinomycetes is the most ubiquitous soil bacteria, and
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has the potential to synthesize more than half of the world’s antibiotics (such as erythromycin,
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streptomycin and tetracycline) (Berdy, 2012). If the soil is laden with antibiotics, then that is the
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best possible route for spreading antibiotic resistance through manure and other agricultural
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practices. Especially, opportunistic pathogens like Acinetobacter and Pseudomonas when present
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in the soil, due to their intrinsic capability to develop resistance, acquire ARGs from the natural
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environment (soil) and they become ARBs (Argudin et al., 2017). D’Costa et al.,(2006) stated that 480 Streptomyces strains cultured from soil were tested for 21
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antimicrobials and it was found that the strains were multi-drug resistant, on an average, to 8 antimicrobials. The most interesting revelation was that 2 strains were resistant to about 15 drugs.
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Thus, in nature, soil presents a single high potential reservoir of ARGs; if the resistance can be passed on to pathogens, there will be a linkage between the environment and human health to a great extent (Allen et al., 2010). ARGs are often positioned on mobile genetic elements like plasmids and transposons, which ensure the spread by horizontal gene transfer. Mobile elements
like plasmids harbor a lot of ARGs which have been found in clinically relevant pathogens found in soil and water like Salmonella, Shigella, Escherichia, Aeromonas, Pseudomonas and Klebsiella
2. Classification and mechanism of action of antibiotics
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(Stokes and Gillings, 2011).
Food animals are injected with antibiotics to take care of the clinical disease, to avoid general disease events and to promote growth. The use of antibiotics in food animals are classified as prophylactic and sub-therapeutic use. Following are the twelve classes of antibiotics that are used
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during the life cycle of food animals, (1) arsenicals, (2) polypeptides, (3) glycolipids, (4)
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tetracyclines, (5) elfamycins, (6) macrolides, (7) lincosamides, (8) polyethers, (9) beta-lactams,
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(10) quinoxalines, (11) streptogramins, and (12) sulfonamides (Landers et al., 2012). World Health
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Organization (WHO), World Organization for Animal Health (OIE) and the Food and Drug Administration (FDA) stated that fluoroquinolones, 3rd and 4th generation cephalosporins and
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macrolides as “critically important” antimicrobial agents (WHO/OIE/FDA, 2006). Antimicrobial
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agents are frequently used to treat various epidemic events and used for various purposes in
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livestock populations. Each family or subgroup of antimicrobials has its own purpose and mode of action to target species. Hence, each microbial species counters the antimicrobial agents with
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specific defense/resistance mechanism(s) for its survival. The main antimicrobial families, their mode of action and their general resistance development mechanisms are summarized in Table 1
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(Burch, 2012).
3. Antibiotics and ARGs
Residues of antibiotics are released into the environment due to incomplete absorption and metabolic activities of food animals. Antibiotics from food supplement may be lost either through
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leaching or elimination by urine and feces. The possibility of antibiotics reaching the environment depends on their properties, bio-availability and surrounding environmental characteristics (Kumararaj et.al., 2016). Development of antibiotic resistance is considered to be a major risk factor in addition to possible toxicity, allergy or carcinogenicity to humans (CIBA report, 2016). The bacteria which have acquired resistance against antibiotics are called Antibiotic Resistant
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Bacteria (ARB) (Magiorakos et al., 2012).
Sulfadiazine
Mode of action Purine synthesis of DNA which interferes folic synthesis rRNA binds with 50S subunit which prevents protein production
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Drugs Classification Sulfonamides
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Table 1. Classification of antibiotics used in food animals (Burch 2012)
Resistance mechanism Chromosome mutation, plasmid and integron mediated resistance
Methylation of 23S sub-unit of rRNA prevents binding and drug inactivation Inducible efflux in E.coli (Tet A, Tet Tetracyclines B) Binding site changes (Tet O, Tet M) Inhibits cell wall production β lactamase production primarily Penicillin, and binds enzymes which changes cell wall proteins so that they Beta lactamase sensitive, help form peptidoglycans cannot bind to penicillin binding Other penicillins, proteins. cephalosporins Interfere in DNA breakage - Target modification - DNA gyrase Quinolones, reunion step by binding Decreased permeabilityouter Flouro quinolones DNA-gyrase membrane porins mutation Binding of rRNA to 50S Methylation of rRNA in gram Macrolides/
A
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Lincosamide Macrolides,Tialmulin
Azalides
sub unit which inhibits positive organisms and inhibits transpeptidation and binding production of protein Phosphorylation and adenylation of amino glucoside stops the binding rRNA binds to sub units Resistance mechanism varies which inhibits protein and depending upon the type of cell wall production antibiotics
Aminoglycosides
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Others
ARBs are capable of transferring their resistance gene to other bacteria through plasmid DNAs by Horizontal Gene Transfer (HGT) by disseminating the ARG among other bacteria. ARBs also use genetic elements such as integrons, transposons and bacteriophages to disseminate the ARG.
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ARGs are considered to be emerging environmental contaminants because once they are into the
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system; they are capable of spreading the antibiotic resistance to other bacterial cells in the system
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as well. This dissemination of antibiotic resistance may happen by one or more of the following
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ways (Pruden et al., 2006, Burch 2012). (i). Horizontal gene transfer (HGT) (occurs between pathogens, non-pathogens and also between distantly related bacterial species). (ii). Multiple
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antibiotic resistant (MAR) integron - one antibiotic co-selecting for resistance to other antibiotics
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(iii). Persistent nature of DNA - on the death of ARG-containing cells, there occurs the release of DNA into the surrounding environment. The DNA which has been released is quite persistent and
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protected due to certain soil compositions. Thus, the DNA is transformed into other bacterial cells
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which are alive and hence resistance is further passed on. iv) Another way of transmission of resistance is, DNA sending messages to the ribosome (rRNA 50S and 30S sub units) to produce
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poly peptides or proteins for growth. When the bacterium is ready to divide, the DNA uncoils, and the rapid prolific bacteria has more chance to develop mutants which also increases the antibiotic resistance.
3.1. Antibiotic resistance - occurrence and dissemination
The contamination issue does not stop at the point of occurrence of antibiotics; it includes the presence of ARGs as well in the environment which is becoming a major global threat in recent
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years. The antibiotics widely used in livestock, poultry and aquaculture have been studied, are listed in the table below (Table. 2), to understand the possible direct impacts of improper use and/or disposal of antibiotics. It is important to study the link between the illegitimate use of antibiotics, exposure to antibiotics-containing water and the gain of resistance.
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3.2. Antibiotic resistance studies in India
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As mentioned earlier, pharmaceuticals such as antibiotics pose a great global threat due to build-up
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of antibiotic resistance in the environment, leading to untreatable infections which used to be
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treatable previously, due to the formation of super bugs. Antibiotic resistance is an emerging
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environmental and health crisis and requires immediate research attention and understanding of its
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occurrence and dissemination. The dissemination of antibiotic resistance involves three compartments, namely, (i) various sources, (ii) environmental pathways and exposure routes, and
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(iii) receptors. Figure 1 gives a representation of the compartments and how the sub-compartments
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are inter-related. Due to the excessive usage of antibiotics as growth promoters in livestock and other animals, animal excreta have significant concentrations of antibiotics, which when washed
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off with water and trickles down into the aquifers along with rainwater, results in contaminating the environment (Figure 1a). Similarly, excessive and inappropriate use of antibiotics by humans can lead to contamination of sewage treatment plants (Figure 1b). Hospital and pharmaceutical industry-wastes are also illegally let into sewage systems, further contaminating the wastewater treatment plants (WWTPs) (Figures 1c and 1d). Improper disposal of unused and/or expired
antibiotic pills by flushing down the drain and overuse of unprescribed antibiotics due to over-thecounter sales, can also result in polluting the WWTPs (Figures 1e and 1f). Eventually, by the above-mentioned ways, various components of the environment are contaminated by antibiotics
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and finally lead to ARBs and ARGs in the sewage. These result in further contaminating the water bodies due to discharge of treated sewage. Subsequently, humans encounter a similar circumstance (as that of animals) through various modes like oral ingestion of contaminated drinking water,
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A
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inhalation of microbial aerosol and become a major vector for dissemination.
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Fig.1 Sources of antibiotic usage, its spread and transfer of resistance genes to humans
Table 2. Representative studies on antibiotic resistance in India
Animal Livestock -
Microorganisms E.coli
Antibiotics tested
Details
Nitrofurantoin, cotrimoxazole, tetracycline,ampicillin
10 out of the total (14) tested isolates were resistant to either one or more of
Location West Bengal
Ref. Manna et al., 2006
Salmonella
Bovine
S. aureus
Bovine
Shiga toxin producing E.coli (STEC) and nonSTEC isolates
Ducks
35 Enterococc us isolates
Chloramphenicol, gentamycin sulphate. Lincosamide and macrolide-based antibiotics were also tested
Poultry
Non-typhal salmonella isolates E. coli
Oxytetracycline
Piglets
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Out of 111 isolates, about 20-30% were resistant
-
Kumar et al., 2011
Out of the STEC strains, 17% were found to have eaeA gene and among nonSTEC strains, 14.28% had eaeA. All STEC were resistant to 3 or more of the antibiotics tested. All of them were non-sensitive to cephalexin and kanamycin Lincosamide and macrolide antibiotics were not effective. Enterococcus isolates were found to be susceptible to gentamycin sulphate and chloramphenicol All the strains were resistant to oxytetracycline
Gujarat
Arya et al, 2008
Assam
Saikia et al., 1995
South India
Saravana n et al.,2015 Singh et al., 1992
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Cloxacillin, cephaloridine, oxytetracycline, ampicillin, amoxicillin, chloramphenicol, trimethoprimsulphamethaxazole, doxycycline, chloramphenicol Ampicillin, erythromycin, nalidixic acid, oxytetracycline, sulfadiazine, cefixine, lincomycin,
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Samanta et al., 2014
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D
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A
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Poultry, humans, bovine, equine, sheep
West Bengal
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Levoflaxin, chloramphenicol, norfloxacin, ciprofloxacin, gentamycin, oxytetracycline Erythromycin, tetracycline, gentamycin, lincomycin Tetracycline,cephalexi n, enrofloxacin, kanamysin, cephaloridine, ampicillin, amikacin
these antibiotics 100% of the isolates were resistant to the antibiotics of concern
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Bovine Poultry
63.2% of the isolates Lucknow displayed resistance towards atleast one drug and out of which multi-resistant were 41%
Greater than 80% resistant to the antibiotics tested
Mizoram
Dutta et al., 2011
34 isolates Salmonella
16 antibiotics were tested
770 isolates of Vibrio cholerae
Polymyxin-B, cephalothin, chloramphenicol, streptomycin, tetracycline, oxytetrcycline, sulfadiazene
Fin fish
82 Vibrio parahaemol yticus isolates
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Cuttlefish and prawn Fin fish, shell fish and crustacea ns
Ampicillin, cefpodoxime, streptomycin, carbenicillin, cephalothin, amoxicillin, colistin, tetracycline, naxidic acid
In addition, referred to CDDEP, 2017
Sethi et al., 1976
25% of the drugs were multidrug resistant and 67.5 % were resistant to 2 or more of the tested antibiotics All the salmonella strains were susceptible
Mangalor Deekshit e et al., 2012
Hatha and Lakshma naperuma lsamy, 1995
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40 salmonella isolates
Various locations in India Tamil Nadu
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Bacitracin (98.7%), chloramphenicol (6.7%), gentamycin (16.2%), nalixidic acid (11.7%), neomycin (53.3%), novobiocin (90%), oxytetracycline (46.2%), penicillin G (92.9%), polymixin B (18.8%), streptomycin (19.6%) Multiple antibiotic resistance genes were tested for their presence
95.4% of the isolates were found to be resistant to the antibiotics tested 98.7% of the strains were resistant to Bactracin, Novobiocin and penicillin G
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704 salmonella strains 240 salmonella isolates
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SeafoodF ish and crustace
roxythromycin, penicillin chloramphenicol
Polymyxin-B, cephalothin Parangip andchloramphenicol were ettai found to be sensitive. Streptomycin, tetracycline, oxytetrcycline, sulfadiazine had greater than 25% resistant strains.Significant number of tested finfish samples had ARB. Greater than 80% of the Cochin isolates showed resistance against ampicillin, cefpodoxime, streptomycin, carbenicillin, cephalothin. 63% and 77% of the isolates displayed resistance against amoxicillin, colistin. All the isolates were sensitive to tetracycline and naxidic acid
Kamatet al., 2005 Sathiyam urthy et al., 1997
Sudha et al., 2012
Some of the major works since the seventies on the occurrence of antibiotic resistance in cattle, other livestock forms and poultry have been tabulated in Table 2 (CDDEP Report, 2017). Besides
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the works mentioned in the table, several research works have been reported as listed below. Resistance has been identified against many antibiotics in several of the E.coli (Kawoosa et al., 2007; Ghatak et al., 2013; Sharma et al., 2015) and S.aureus (Kumar et al., 2011; Tiwari et al., 2011; Dutta et al., 2011; Kumar et al., 2012; Preethirani et al., 2015) isolates obtained from bovine animals. Similarly, antibiotic resistance present in isolates from poultry had also been reported.
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Shivachandra et al. (2004) examined 123 isolates from chickens in various parts of India and found
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that all isolates exhibited complete resistance to sulfadiazine and larger part of the isolates
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displayed resistance against penicillin, erythromycin, amikacin and carbenicillin. On the other
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hand, 74% isolates were susceptible to chloramphenicol. Similarly, Mir et al. (2015) examined
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isolates from Salmonella enterica and found that majority of the isolates was resistant to numerous
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antibiotics such as β-lactam. Besides these, numerous studies have been reported on antibiotic resistance based on isolates from poultry (Kar et al., 2015; Rasheed et al., 2014, CDDEP, 2017).
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CDDEP, 2017 has also dealt with antibiotic resistance among food animals in detail. Antibiotic
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resistance can be transferred from animals/plants to humans by food chain, direct/indirect link with animal health workers/livestock industry and in agriculture through manure application. The
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spread of resistance between different ecosystems rely on the concentrations of antibiotic resistance genes (ARGs) and their responsible host bacteria present in an ecosystem and also largely vary on the rate of their exchange between ecosystems.
4. Massive use of antibiotics in veterinary medicine
Antibiotics used in veterinary and human medicine are from the same class and sometimes similar in structure. A study reported in the Proceedings of the National Academy of Sciences, found that
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the antibiotic consumption between 2010 – 2030 is expected to rise by a staggering 67% (Van Boeckel et al., 2015). More than 66% of the worldwide rise in antimicrobial consumption can be attributed to the increasing quantity of animals being raised for food production. Thus, veterinary medicine demands a greater and wider share of the sectoral antibiotic consumption globally. The real problem in this area is that, antibiotics were administered not only to treat the animals but for
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growth promotion, and significantly in disease-prevention, making the animal houses a hotspot of
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antibiotic resistance dissemination. Despite a large number of bans in the United States
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(quinolones in poultry industry), some important antibiotics continue to be routinely used for
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prophylactic measures among crowded animals.
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4.1. Transfer from poultry farm environment
Poultry litter constitutes one of the key sources of ARBs. In commercial poultry farms, antibiotics
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are administered from a day-old chick to an adult bird for a period of 6 weeks. Usually, poultry
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farm floors are covered with bedding materials. The bedding is made of materials such as softwood, and during the growing period, this gets mixed with chicken feces, skin, feather and
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insects. This mix is called the poultry litter and it gets replaced with fresh wood shavings between different time intervals. This litter is usually rich in mineral content, and can be used as a fertilizer. Antibiotics are used in poultry for growth promotion and for disease prevention. As previously mentioned, the metabolic rate of antibiotics is low and hence 90% of the administered dose is excreted in feces. Avian intestines are one of the potential reservoirs of E. coli. These bacteria
have a huge potential for zoonotics, so there is a higher chance for AMR spread from birds to humans (Ewers et al., 2009). Feeding greatly influences the microbial ecosystem of broiler litter. The population of beneficial bacteria can be increased, and pathogenic E. coli populations can be
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decreased in the litter by the addition of mannan and purified lignin to broiler feed (Baurhoo et al., 2007). Resistant E. coli from contaminated litter are transferred to the environment which in turn contaminate the surface water and groundwater through run-off happening while storing litter for long time periods in an open source.
Possible link between livestock/poultry and aquaculture
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4.2.
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Van Boeckel (2015) has predicted that antibiotic consumption by chickens in India in chicken is
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rapidly increasing and area of high consumption is expected to grow by 312% by 2030. The major
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concern is that India has not come up with regulatory guidelines to limit the usage of antibiotics in livestock or poultry. There is a common practice existing in integrated farming in Asia, where the
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organic waste from livestock and poultry which is rich in ARB, are fed to farm fish. Thus, transfer
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of resistance happens in the aquaculture farms (Jayathilakan, 2012). This link can be established
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with different perspectives. Companion animals like dogs and cats are being more or less treated as family members by pet lovers. Thus, these pets receive the most advanced medical treatment with
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newer antibiotics. There is always a risk of zoonotic infections. Due to over-usage of antibiotics, increasing resistant strains are produced which impact human health. In recent times, there are
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reports
of
increasing
resistance
in
animals.
For
example,
carbapenemase-positive
Enterobacteriaceae, pig farm associated MRSA ST398, quinolone resistance in food products (plasmid mediated) have been reported recently (Argudin et al., 2017).
4.3.
Spread of AMR in the environment from wastewater
There is also a common practice in countries like India where animal manure is widely used in agriculture. Animal waste represents one of critical links in spreading AMR because they harbor
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AMR bacteria. Antimicrobials are never completely metabolized and finally find their way into sewage treatment plants. In countries like India, pharmaceuticals and personal care products are abundant in WWTP and thus the selection pressure for AMR is high in these environments (Marathe et al., 2013). It is a pity that our WWTP are not equipped for detecting or treating pharmaceutical or personal care products and this is a massive source of antimicrobial exposure to
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the environment. Even some personal care products like hand wash soaps which contain triclosan,
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serve as a potent antimicrobial selection source in the domestic wastewater (Jutkina et al., 2017).
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4.4. Spread of AMR from environmental sources like rivers/lakes to humans/animals/plants
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In India, many rivers and streams are polluted with industrial and domestic wastewater. Many
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occasions when wastewater is let into the rivers without treatment lead to massive inputs of AMRladen bacteria into the natural unpolluted environments. This water is being used to feed the
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livestock, and is also used for irrigation and thus, transfer is obvious in these aquatic environments.
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A recent research article in 2017 investigated water samples collected from the Musi River near a WWTP of a pharmaceutical industry in Hyderabad (Lubbert et al., 2017). Samples were analyzed
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for 25 antibiotics with liquid chromatography–tandem mass spectrometry. High concentrations of moxifloxacin, voriconazole, and fluconazole were detected in the sewers in an industrial area in Patancheru–Bollaram area. In a recent research study, Vibrio coralliilyticus, one of the most
prevalent marine pathogens responsible for coral disease in aquaculture farms was isolated from samples collected from ten different sites in Cochin and Kumarakom and four different shrimp
farm hatcheries. Thirty different strains of V. coralliilyticus were analyzed for antibiotic resistance towards 20 antibiotics using disk diffusion method. Resistance was observed against beta lactams (amoxicillin,
ampicillin
and
carbenicillin),
tetracycline
(oxytetracycline),
pyrimidine
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(trimethoprim), ntirofurans (nitrofurantoin and furazolidone), sulphonamides (sulphamethoxasole), quinolones (enrofloxacin) and macrolides (erythromycin) (Silvester et al., 2017). Vignesh et al., (2016) reported that the water and sediment samples obtained at the eco-regions of Chennai coast were detected with ARB. Most of the bacterial strains of a total of 960 isolates belong to the four major genera. Highest resistances were shown for vancomycin and penicillin with the frequency of
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53.6 % and 52.6% respectively. Chloramphenicol, ciprofloxacin, gentamicin and tetracycline were
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Risks and benefits of using antibiotics
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5.
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observed to be most effective on the bacteria with the frequency range of less than 7%.
The market value of antimicrobial drugs used to treat animal diseases stood at $20.1 billion in
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2010. It has been steadily increasing as $8.65 billion in 1992 and predicted to rise to $42.9 billion
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in 2018 (GIA, 2012). Best quality meat with high protein content and low fat is being produced
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from animals fed with antibiotics (Hughes and Heritage, 2002). Tetracycline and penicillin usage in poultry have considerably improved egg production and hatchability (Gustafson and
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Bowen, 1997). Besides poultry, antibiotic-supplemented feed had a marked improvement in the health of livestock. Sulfamethazine and chlortetracycline supplements, significantly reduced the
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rate of relapse and respiratory disease morbidity (Gallo and Berg, 1995). Apart from their antimicrobial effects, antibiotics (particularly macrolides) have an anti-inflammatory potential which rationalizes their beneficial effects (Buret, 2010). Livestock, poultry and aquaculture industries thus rely on the crucial role of antibiotics for efficient animal food production strategies.
It has been well documented that the diarrhoeagenic E. coli has been the underlying cause of acute diarrhea in Indian children. A study reported that rural child population living near poultry farms in Tamil Nadu are at high risk for E.coli resistance to fluoroquinolones, tetracycline, and
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gentamicin (Murthy et al., 2008). Emergence of global resistance of avian influenza strain H5N1 is due to unrestricted usage of amantadine in China. Amantadine, a potential life-saving drug rendered ineffective during the H5N1 outbreak. In India, amantadine drug resistant H5N1 avian influenza was reported in West Bengal in 2011.
One of the major diseases affecting poultry industry is Salmonellosis which has posed a severe
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threat resulting in substantial morbidity. High stocking density, high dust levels, low ventilation
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and stress in poultry farms have the potential to exacerbate the problem. Center for Disease
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Dynamics, Economics &Policy (CDDEP) and Centre for Science and Environment (2014) have
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reported increased levels of ARB in chickens in Punjab (CSE, 2014). These studies have proven
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that more than 66% of the farms use antibiotics for growth promotion, out of which, farm animals
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have 3 times more likeliness to develop antibiotic resistance. Huge demand for good quality meat has increased the antibiotic usage in animal food industry which results in wider selection of
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pathogens. The result of expansion of this sector is mainly due to the demand for the poultry. The extreme growth in consumption is expected to grow further by 312% by 2030 (Boeckel et. al.,
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2015).
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However, a major grey area in this field is that, even though several research works are conducted across the world on identifying the antibiotics spread and the resistance gene associated with it in various compartments of the environment such as water and wastewater, studies with respect to Indian scenario is minimal and needs to be explored to a great extent. Further, despite several research explorations happening globally to understand and fight this critical issue, other means
such as vaccines and bacteriophages may turn out to be an alternate method to treat resistant bacterial infections (Raghunathan, 2008). At the same time, experimentation of such alternate methods must be assessed of their pros and cons before commercializing them.
Implications on human health
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6.
A research study published in the Lancet in 2010 by Karthikeyan Kumarasamy et al. about the NDM1 superbug created a storm in the healthcare sector of India. Politicians and physicians felt exposed after this publication about multidrug resistance in India and medical tourism in the
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country was severely criticized (Kumarasamy et al., 2010). In 2016, Indian researchers have
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identified mcr-1, which has resistance to the last mile antibiotic (colistin) that humans have access
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to. Environmental reservoirs of AMR have demonstrated the potential to serve as the source of
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antibiotic resistance genes for clinically relevant microbes (Ghosh and Lapara, 2007; Perry and Wright, 2013). Further, antimicrobial compounds which end up in aquatic systems have the
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potential to interrupt native microbial species which is essential to maintaining the ecosystem
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(nitrification/denitrification), soil fertility and biological cycles (Costanzo et al., 2005; Kinney et
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al., 2006; Kummerer, 2004). As such, antimicrobials are widely recognized as emerging environmental contaminants (Pruden et al., 2006) and, given their alarming levels were classified
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as a priority risk group in recent years.
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Besides, several human pathogens have started gaining resistance. Among enteric pathogens, Vibrio cholera seems to have attained resistance towards nalidixic acid, furazolidone and cotrimoxazole and is susceptible to tetracycline (near Delhi region, India). However, in some areas of Bangladesh, tetracycline seems to be ineffective. This shows that resistance spectrum varies between regions and antibiotic consumption practices (Sharma et al., 2007; Saha et al., 2006).
Typhoid and paratyphoid causing micro-organisms had higher susceptibility to antibiotics, back in late 1980s, which made them treatable using chloramphenicol, ampicillin and cotrimaxazole. However, soon after acquirement of resistance to these drugs to a great extent, the usage of
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fluoroquinolones has been in the fore-front. Later, chloramphenicol lost sensitivity for a short period, following which due to the popularization of fluoroquinolone, sensitivity to chloramphenicol was restored (Raghunath and Narayanan1989; Anand et al., 1990; Raghunath, 2008). Ray et al. (2006) and Khaki et al. (2007) have studied the resistance of Neisseria gonorrhoeae (pathogen causing sexually transmitted diseases) towards penicillin and
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fluoroquinolone. This has resulted in a situation where third generation antibiotics need to be used.
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Furthermore, it is becoming more difficult to treat deadly diseases like tuberculosis by using basic
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level drugs. Mycobacterium tuberculosis has acquired multi-drug resistance (simultaneous
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resistance to rifampicin and isoniazid). In addition, it has lost sensitivity to fluoroquinoline and a few second line drugs which has made it an extensively drug resistant bacteria (Ramachandran and
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Narayanan 2008; Raghunath, 2008; Jesudasan et al., 2003). Further, Mycobacteria genus also
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causes leprosy, (the causative pathogen being Mycobacteria leprae). However, this pathogen has
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started acquiring resistance to rifampicin, clofazimine and dapsone. This is not a healthy sign for
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curing such dangerous diseases.
On the other hand, urinary infections are less dangerous and common among out-patient infection
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complaints, they have acquired resistance to antibiotics as well. Based on the research work conducted by Akram et al. (2007) in Aligarh, India, 100 samples out of 920 exhibited loss of susceptibility to the antibiotics tested. Out of 100 samples, 61% was E.coli and 22% was Klebsiella spp. that were resistant. In addition, ampicillin and co-trimoxazole were found to be ineffective against Gram negative bacilli. Kumar et al. (2017) have investigated AMR patterns of
654 enteric pathogens in India. They have conducted a detailed study on AMR traits on multi-drug enteric pathogens. The study included microbial species namely, P.stuartii, K.pneumoniae, E.coli, S. typhimurium, S.flexneri and P.aeruginosa. Their study indicates that these pathogens are
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resistant against 22 antibiotics which belong to 9 distinct classes of antibiotics. The major findings of their study are as follows. (i) About 97% of strains have lost susceptibility towards at least 2 antibiotics, (ii) 24% against at least 10 antibiotics and (iii) 3% of the isolates seem to exhibit resistance against at least 15 antibiotics.
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7. Conclusions
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This review mainly focuses on (i) the usage and prevalence of antibiotics in the environment, (ii)
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presence of antibiotic resistance in animals and (iii) dissemination of antibiotic resistance through
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various mechanisms in Indian scenario. Due to excessive and inappropriate human consumption of antibiotics, and administration of antibiotics to animals (not just for treatment of infections, but
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also for prophylactic purposes and as a form of growth promoters), it has resulted in the presence
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of antibiotics in the environment,in turn leading to dissemination of antibiotic resistance.
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The best way to control the spread of antibiotic resistance is to raise awareness to the general
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public by print and social media to increase the demand for antibiotic-free food products. Veterinarians, agricultural experts and legal authorities in India should address the issue of
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antibiotic resistance by regulating policies which can monitor or track the antibiotic usage, limiting its dosage, etc. Apart from these, people involved in waste management and municipal authorities should be educated about proper disposal of expired medications, poultry and aquaculture wastewater. Non-therapeutic use of antibiotics should be tracked and phased out.
A systematic nationwide rigorous surveillance system to monitor the antibiotic residues in veterinary, poultry and aquaculture environments is very much essential. Vertical surveillance cannot identify all the major drivers of AMR whereas a cross-sectional surveillance can ensure
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better understanding of underlying factors in spread of AMR to best identify the sectoral contribution. The other effective method is to control or prevent infection in humans and animal sources, which can be done by healthcare professionals. Wastewater treatment facilities built near the pharmaceutical manufacturing facilities must treat the effluents and remove the antibiotics and
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their metabolites before their release into the environment.
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References
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1. Akram, M., Shahid, M., & Khan, A. U. (2007). Etiology and antibiotic resistance patterns of community-acquired urinary tract infections in JNMC Hospital Aligarh, India. Annals of clinical microbiology and antimicrobials, 6(1), 4.
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2. Allen, H. K., Donato, J., Wang, H. H., Cloud-Hansen, K. A., Davies, J., & Handelsman, J. (2010). Call of the wild: antibiotic resistance genes in natural environments. Nature Reviews Microbiology, 8(4), 251-259.
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3. Aly, S. M., & Albutti, A. (2014). Antimicrobials use in aquaculture and their public health impact. Journal of Aquaculture Research & Development, 5(4), 1. 4. Anand, A. C., Kataria, V. K., Singh, W., & Chatterjee, S. K. (1990). Epidemic multiresistant enteric fever in eastern India. The Lancet, 335(8685), 352.
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5. Antibiotic use and resistance in food animals: Current policy and recommendations, The Centre for Diseases Dynamics, Economics and Policy (CDDEP), 2017. https://www.cddep.org/publications/antibiotic_use_and_resistance_food_animals_current_ policy_and_recommendations/
6. Argudín, M. A., Deplano, A., Meghraoui, A., Dodémont, M., Heinrichs, A.,Denis, O., Nonhoff, C., & Roisin, S. (2017). Bacteria from Animals as a Pool of Antimicrobial Resistance Genes. Antibiotics, 6(2), 12.
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7. Arya, G., Roy, A., Choudhary, V., Yadav, M. M., & Joshi, C. G. (2008). Serogroups, Atypical Biochemical Characters, Colicinogeny and Antibiotic Resistance Pattern of Shiga Toxin‐producing Escherichia coli Isolated from Diarrhoeic Calves in Gujarat, India. Zoonoses and public health, 55(2), 89-98. 8. Baurhoo, B., Phillip, L., & Ruiz-Feria, C. A. (2007). Effects of purified lignin and mannan oligosaccharides on intestinal integrity and microbial populations in the ceca and litter of broiler chickens. Poultry science, 86(6), 1070-1078.
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9. Bérdy, J. (2012). Thoughts and facts about antibiotics: where we are now and where we are heading. The Journal of antibiotics, 65(8), 385-395.
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10. Buret, A. G. (2010). Immuno-modulation and anti-inflammatory benefits of antibiotics: the example of tilmicosin. Canadian Journal of Veterinary Research, 74(1), 1-10.
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11. CIBA Special Publication No. 83, Responsible Use of Antimicrobials in Indian Aquaculture, Opportunities and Challenges, 7th December 2016, ICAR - CIBA, Chennai
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