BIOLOGY

INVESTIGATORY
PROJECT
STUDY OF DRUG RESISTANCE
IN BACTERIA USING
ANTIBIOTICS
 SUNBEAM
SENIOR SECONDARY SCHOOL
VELLORE

BIOLOGY

Name: DWARAKESWARAN U Register No.

Class:XII-D

Certified that this is the bonafide record of assignment

work done by U.DWARAKESWARAN of class 12-D of
SUNBEAM HIGHER SECONDARY SCHOOL, Vellore,
during the year of 2018.

Submitted project on the topic-STUDY OF DRUG

RESISTANCE IN BACTERIA USING ANTIBIOTICS
 CONTENTS

1. Introduction
2. Definition
3. Causes
 Human medicine
 Veterinary medicine
 Natural occurrence
 Water pollution
4. Prevention
 Duration of antibiotics
 and mapping
 Monitoring
 Limiting antibiotic use
 At the hospital level
5. Mechanisms and organisms
 Fundamentals
 Bacteria
 Viruses
 Fungi
 Parasites
6. Further research
 Vaccines
 Development of new drugs
7. Reference
 STUDY OF DRUG RESISTANCE IN BACTERIA USING
ANTIBIOTICS

Introduction

Antimicrobial resistance (AMR or AR) is the ability of a microbe to resist the

effects of medication that once could successfully treat the microbe. The
term antibiotic resistance (AR or ABR) is a subset of AMR, as it applies
only to bacteria becoming resistant to antibiotics. Resistant microbes are
more difficult to treat, requiring alternative medications or higher doses of
antimicrobials. These approaches may be more expensive, more toxic or
both. Microbes resistant to multiple antimicrobials are called multidrug
resistant (MDR). Those considered extensively drug resistant (XDR) or
totally drug resistant (TDR) are sometimes called "superbugs".
Resistance arises through one of three mechanisms: natural resistance in
certain types of bacteria, genetic mutation, or by one species acquiring
resistance from another. All classes of microbes can develop resistance.
Fungi develop antifungal resistance. Viruses develop antiviral resistance.
Protozoa develop antiprotozoal resistance, and bacteria develop antibiotic
resistance. Resistance can appear spontaneously because of random
mutations. However, extended use of antimicrobials appears to encourage
selection for mutations which can render antimicrobials ineffective.
Preventive measures include only using antibiotics when needed, thereby
stopping misuse of antibiotics or antimicrobials.Narrow-spectrum antibiotics
are preferred over broad-spectrum antibiotics when possible, as effectively
and accurately targeting specific organisms is less likely to cause
resistance. For people who take these medications at home, education
about proper use is essential. Health care providers can minimize spread of
resistant infections by use of proper sanitation and hygiene,
including handwashing and disinfecting between patients, and should
encourage the same of the patient, visitors, and family members.
Rising drug resistance is caused mainly by use of antimicrobials in humans
and other animals, and spread of resistant strains between the two.
Growing resistance has also been linked to dumping of inadequately
treated effluents from the pharmaceutical industry, especially in countries
where bulk drugs are manufactured. Antibiotics increase selective
pressure in bacterial populations, causing vulnerable bacteria to die; this
increases the percentage of resistant bacteria which continue growing.
Even at very low levels of antibiotic, resistant bacteria can have a growth
advantage and grow faster than vulnerable bacteria. With resistance to
antibiotics becoming more common there is greater need for alternative
treatments. Calls for new antibiotic therapies have been issued, but new
drug development is becoming rarer.
Antimicrobial resistance is increasing globally because of greater access to
antibiotic drugs in developing countries. Estimates are that 700,000 to
several million deaths result per year. Each year in the United States, at
least 2 million people become infected with bacteria that are resistant to
antibiotics and at least 23,000 people die as a result. There are public calls
for global collective action to address the threat that include proposals
for international treaties on antimicrobial resistance. Worldwide antibiotic
resistance is not completely identified, but poorer countries with weaker
healthcare systems are more affected.
Definition

The WHO defines antimicrobial resistance as a microorganism's resistance

to an antimicrobial drug that was once able to treat an infection by that
microorganism.A person cannot become resistant to antibiotics. Resistance
is a property of the microbe, not a person or other organism infected by a
microbe.
 Causes

Bacteria with resistance to antibiotics predate medical use of antibiotics by

humans. However, widespread antibiotic use has made more bacteria
resistant through the process of evolutionary pressure.
Reasons for the widespread use of antibiotics in human medicine include:

 increasing global availability over time since the 1950s

 uncontrolled sale in many low or middle income countries, where they
can be obtained over the counter without a prescription, potentially
resulting in antibiotics being used when not indicated.This may result in
emergence of resistance in any remaining bacteria.
Other causes include:

 Antibiotic use in livestock feed at low doses for growth promotion is an

accepted practice in many industrialized countries and is known to lead
to increased levels of resistance.
 Releasing large quantities of antibiotics into the environment during
pharmaceutical manufacturing through inadequate wastewater
treatment increases the risk that antibiotic-resistant strains will develop
and spread.
 It is uncertain whether antibacterials in soaps and other products
contribute to antibiotic resistance, but antibacterial soaps are
discouraged for other reasons.

Human medicine:
Increasing bacterial resistance is linked with the volume of antibiotic
prescribed, as well as missing doses when taking antibiotics. Inappropriate
prescribing of antibiotics has been attributed to a number of causes, such
as patients insisting on antibiotics and physicians prescribing them as they
do not have time to explain why they are not necessary. Another cause can
be physicians not knowing when to prescribe antibiotics or being overly
cautious for medical or legal reasons. For example, 70 to 80 percent
of diarrhea is caused by viral pathogens, for which antibiotics are not
effective. But nevertheless, around 40 percent of these cases are
attempted to be treated with antibiotics. In some areas even over 80
percent of such cases are attempted to be treated with antibiotics.
Lower antibiotic concentration contributes to the increase of AMR by
introducing more mutations that support bacterial growth in higher antibiotic
concentration. For example, sub-inhibitory concentration have induced
genetic mutation in bacteria such as Pseudomonas aeruginosa and
Bacteroides fragilis.
Up to half of antibiotics used in humans are unnecessary and
inappropriate. For example, a third of people believe that antibiotics are
effective for the common cold, and the common cold is the most common
reason antibiotics are prescribed even though antibiotics are useless
against viruses. A single regimen of antibiotics even in compliant
individuals leads to a greater risk of resistant organisms to that antibiotic in
the person for a month to possibly a year.
Antibiotic resistance increases with duration of treatment. Therefore, as
long as an effective minimum is kept, shorter courses of antibiotics are
likely to decrease rates of resistance, reduce cost, and have better
outcomes with fewer complications. Short course regimens exist for
community-acquired pneumonia spontaneous bacterial peritonitis,
suspected lung infections in intense care wards, so-called acute abdomen,
middle ear infections, sinusitis and throat infections, and penetrating gut
injuries. In some situations a short course may not cure the infection as
well as a long course. A BMJ editorial recommended that antibiotics can
often be safely stopped 72 hours after symptoms resolve.
Because individuals may feel better before the infection is eradicated,
doctors must provide instructions to them so they know when it is safe to
stop taking a prescription. Some researchers advocate doctors' using a
very short course of antibiotics, reevaluating the patient after a few days,
and stopping treatment if there are no clinical signs of infection.
Certain antibiotic classes result in resistance more than others. Increased
rates of MRSA infections are seen when using glycopeptides,
cephalosporins, and quinolone antibiotics. Cephalosporins, and particularly
quinolones and clindamycin, are more likely to produce colonization with
Clostridium difficile.
Factors within the intensive care unit setting such as mechanical ventilation
and multiple underlying diseases also appear to contribute to bacterial
resistance. Poor hand hygiene by hospital staff has been associated with
the spread of resistant organisms.

Veterinary medicine:
The World Health Organization concluded that inappropriate use of
antibiotics in animal husbandry is an underlying contributor to the
emergence and spread of antibiotic-resistant germs, and that the use of
antibiotics as growth promoters in animal feeds should be
restricted. The World Organisation for Animal Health has added to the
Terrestrial Animal Health Code a series of guidelines with
recommendations to its members for the creation and harmonization of
national antimicrobial resistance surveillance and monitoring
programs, monitoring of the quantities of antibiotics used in animal
husbandry,and recommendations to ensure the proper and prudent use of
antibiotic substances. Another guideline is to implement methodologies that
help to establish associated risk factors and assess the risk of antibiotic
resistance.

Natural occurrence:
Naturally occurring antibiotic resistance is common. Genes for resistance to
antibiotics, like antibiotics themselves, are ancient. The genes that confer
resistance are known as the environmental resistome. These genes may
be transferred from non-disease-causing bacteria to those that do cause
disease, leading to clinically significant antibiotic resistance.
In 1952 it was shown that penicillin-resistant bacteria existed before
penicillin treatment; and also preexistent bacterial resistance
to streptomycin. In 1962, the presence of penicillinase was detected in
dormant endospores of Bacillus licheniformis, revived from dried soil on the
roots of plants, preserved since 1689 in the British Museum.
Six strains of Clostridium, found in the bowels of William Braine and John
Hartnell (members of the Franklin Expedition) showed resistance
to cefoxitin and clindamycin.
Penicillinase may have emerged as a defense mechanism for bacteria in
their habitats, such as the case of penicillinase-rich Staphylococcus
aureus, living with penicillin-producing Trichophyton; however, this may be
circumstantial. Search for a penicillinase ancestor has focused on the class
of proteins that must be a priori capable of specific combination
with penicillin. The resistance to cefoxitin and clindamycin in turn was
attributed to Braine's and Hartnell's contact with microorganisms that
naturally produce them or random mutation in the chromosomes of
Clostridium strains.
There is evidence that heavy metals and other pollutants may select for
antibiotic-resistant bacteria, generating a constant source of them in small
numbers.

Water pollution:
Antibiotic resistance is a growing problem among humans and wildlife in
terrestrial or aquatic environments. In this respect, the spread and
contamination of the environment, especially through water pollution "hot
spots" such as hospital wastewater and untreated urban wastewater, is a
growing and serious public health problem. Antibiotics have been polluting
the environment since their introduction through human waste (medication,
farming), animals, and the pharmaceutical industry. The contribution of the
pharmaceutical industry is so significant that parallels can be drawn
between countries with highest rate of increasing antibiotic resistance and
countries with largest footprint of pharmaceutical industry. China, which
contributes to nearly 90 per cent of the world's active pharmaceutical
ingredient (API) manufacturing, has seen a 22 per cent increase in rate of
antimicrobial resistance in six years, compared to a 6 per cent increase in
the United States.
Along with antibiotic waste, resistant bacteria follow, thus introducing
antibiotic-resistant bacteria into the environment. Already in 2011, mapping
of sewage and water supply samples in New Delhi showed widespread and
uncontrolled infection as indicated by the presence of NDM-1-positive
enteric bacteria (New Delhi metallo-beta-lactamase 1).
As bacteria replicate quickly, the resistant bacteria that enter water bodies
through wastewater replicate their resistance genes as they continue to
divide. In addition, bacteria carrying resistance genes have the ability to
spread those genes to other species via horizontal gene transfer.
Therefore, even if the specific antibiotic is no longer introduced into the
environment, antibiotic-resistance genes will persist through the bacteria
that have since replicated without continuous exposure. Antibiotic
resistance is widespread in marine vertebrates, and they may be important
reservoirs of antibiotic-resistant bacteria in the marine environment.
 Prevention
There have been increasing public calls for global collective action to
address the threat, including a proposal for international treaty on
antimicrobial resistance. Further detail and attention is still needed in order
to recognize and measure trends in resistance on the international level;
the idea of a global tracking system has been suggested but
implementation has yet to occur. A system of this nature would provide
insight to areas of high resistance as well as information necessary for
evaluation of programs and other changes made to fight or reverse
antibiotic resistance.
Five important strategies needed for minimising antibiotic resistance are as
follows:

 Antibiotic stewardship to maintain the value of existing and future

antibiotics
 The timing of prescription to use the effective antibiotics sooner rather
than later
 To develop and approve ten new antibiotics by 2020
 Development of a molecular method for detecting antibiotic resistance
genes
 To avoid the delay in distribution of US$2 billion global antibiotic
resistance innovation fund.
Duration of antibiotics:
Antibiotic treatment duration should be based on the infection and other
health problems a person may have.For many infections once a person has
improved there is little evidence that stopping treatment causes more
resistance.Some therefore feel that stopping early may be reasonable in
some cases.Other infections, however, do require long courses regardless
of whether a person feels better.

Monitoring and mapping:

There are multiple national and international monitoring programs for drug-
resistant threats, including methicillin-resistant Staphylococcus aureus
(MRSA), vancomycin-resistant S. aureus (VRSA), extended spectrum beta-
lactamase (ESBL), vancomycin-resistant Enterococcus (VRE), multidrug-
resistant A. baumannii (MRAB).
ResistanceOpen is an online global map of antimicrobial resistance
developed by HealthMap which displays aggregated data on antimicrobial
resistance from publicly available and user submitted data. The website
can display data for a 25-mile radius from a location. Users may submit
data from antibiograms for individual hospitals or laboratories. European
data is from the EARS-Net (European Antimicrobial Resistance
Surveillance Network), part of the ECDC.
ResistanceMap is a website by the Center for Disease Dynamics,
Economics & Policy and provides data on antimicrobial resistance on a
global level.

Limiting antibiotic use:

Antibiotic stewardship programmes appear useful in reducing rates of
antibiotic resistance.
Excessive antibiotic use has become one of the top contributors to the
development of antibiotic resistance. Since the beginning of the antibiotic
era, antibiotics have been used to treat a wide range of disease. Overuse
of antibiotics has become the primary cause of rising levels of antibiotic
resistance. The main problem is that doctors are willing to prescribe
antibiotics to ill-informed individuals who believe that antibiotics can cure
nearly all illnesses, including viral infections like the common cold. In an
analysis of drug prescriptions, 36% of individuals with a cold or an upper
respiratory infection (both viral in origin) were given prescriptions for
antibiotics. These prescriptions accomplished nothing other than increasing
the risk of further evolution of antibiotic resistant bacteria.
At the hospital level:
Antimicrobial stewardship teams in hospitals are encouraging optimal use
of antimicrobials. The goals of antimicrobial stewardship are to help
practitioners pick the right drug at the right dose and duration of therapy
while preventing misuse and minimizing the development of resistance.
Stewardship may reduce the length of stay by an average of slightly over 1
day while not increasing the risk of death.

Mechanisms and organisms

Fundamentals:
The four main mechanisms by which microorganisms exhibit resistance to
antimicrobials are:

1. Drug inactivation or modification: for example, enzymatic deactivation

of penicillin G in some penicillin-resistant bacteria through the
production of β-lactamases.The emergence of carbapenem-resistant
Gram-negative pathogens poses a serious threat to public health
worldwide. Klebsiella pneumoniae carbapenemases (KPCs) and
carbapenemases of the oxacillinase-48 (OXA-48) type have been
reported worldwide. New Delhi metallo-β-lactamase (NDM)
carbapenemases were originally identified in Sweden in 2008 and
have spread worldwide rapidly. Most commonly, the protective
enzymes produced by the bacterial cell will add an acetyl or
phosphate group to a specific site on the antibiotic, which will reduce
its ability to bind to the bacterial ribosomes and disrupt protein
synthesis.
2. Alteration of target- or binding site: for example, alteration of PBP—
the binding target site of penicillins—in MRSA and other penicillin-
resistant bacteria. Another protective mechanism found among
bacterial species is ribosomal protection proteins. These proteins
protect the bacterial cell from antibiotics that target the cell’s
ribosomes to inhibit protein synthesis. The mechanism involves the
binding of the ribosomal protection proteins to the ribosomes of the
bacterial cell, which in turn changes its conformational shape. This
allows the ribosomes to continue synthesizing proteins essential to
the cell while preventing antibiotics from binding to the ribosome to
inhibit protein synthesis.
3. Alteration of metabolic pathway: for example, some sulfonamide-
resistant bacteria do not require para-aminobenzoic acid (PABA), an
 important precursor for the synthesis of folic acid and nucleic acids in
bacteria inhibited by sulfonamides, instead, like mammalian cells,
they turn to using preformed folic acid.
4. Reduced drug accumulation: by decreasing drug permeability or
increasing active efflux (pumping out) of the drugs across the cell
surface These pumps within the cellular membrane of certain
bacterial species are used to pump antibiotics out of the cell before
they are able to do any damage. They are often activated by a
specific substrate associated with an antibiotic as
in fluoroquinolone resistance.

Bacteria:
Bacteria can often develop antibiotic resistance. Mutations that confer
increased survival are selected for in natural selection, which can happen
quickly in bacteria because lifespans and production of new generations
can be on a timescale of mere hours. A new (de novo) mutation in a parent
cell can quickly become an inherited mutation of widespread prevalence.
Moreover, some adaptive mutations can propagate not only through
inheritance but also through horizontal gene transfer. Very often this is
done via plasmids, however, through means of Transduction
(genetics), Transformation (genetics) and chromosomal Conjugation
(genetics), resistance genes residing on bacterial chromosomes can also
be transferred. If the new DNA is maintained in the receiving bacterium,
this transfer is followed by inheritance of the new resistance from parents to
offspring.
Recent findings show no necessity of large populations of bacteria for the
appearance of antibiotic resistance. Small populations of E. coli in an
antibiotic gradient can become resistant. Any heterogeneous environment
with respect to nutrient and antibiotic gradients may facilitate antibiotic
resistance in small bacterial populations. Researchers hypothesize that the
mechanism of resistance development is based on four SNP mutations in
the genome of E. coli produced by the gradient of antibiotic.
Antibiotic resistance can be introduced artificially into a microorganism
through laboratory protocols, sometimes used as a selectable marker to
examine the mechanisms of gene transfer or to identify individuals that
absorbed a piece of DNA that included the resistance gene and another
gene of interest.
New Delhi metallo-beta-lactamase 1 (NDM-1) is an enzyme that
makes bacteria resistant to a broad range of beta-lactam antibiotics. The
most common bacteria that make this enzyme are gram-negative such
as Escherichia coli and Klebsiella pneumoniae, but the gene for NDM-1
can spread from one strain of bacteria to another by horizontal gene
transfer.

Viruses:
Specific antiviral drugs are used to treat some viral infections. These drugs
prevent viruses from reproducing by inhibiting essential stages of the
virus's replication cycle in infected cells. Antivirals are used to treat HIV,
hepatitis B, hepatitis C, influenza, herpes viruses including varicella zoster
virus, cytomegalovirus and Epstein-Barr virus. With each virus, some
strains have become resistant to the administered drugs.
Resistance to HIV antivirals is problematic, and even multi-drug resistant
strains have evolved. Resistant strains of the HIV virus emerge rapidly if
only one antiviral drug is used. Using three or more drugs together has
helped to control this problem, but new drugs are needed because of the
continuing emergence of drug-resistant HIV strains.

Fungi:
Infections by fungi are a cause of high morbidity and mortality
in immunocompromised persons, such as those with HIV/AIDS,
tuberculosis or receiving chemotherapy. The fungi candida, Cryptococcus
neoformans and Aspergillus fumigatus cause most of these infections and
antifungal resistance occurs in all of them. Multidrug resistance in fungi is
increasing because of the widespread use of antifungal drugs to treat
infections in immunocompromised individuals.
Of particular note, Fluconazole-resistant Candida species have been
highlighted as a growing problem by the CDC. More than 20 species of
Candida can cause Candidiasis infection, the most common of which
is Candida albicans. Candida yeasts normally inhabit the skin and mucous
membranes without causing infection. However, overgrowth of Candida
can lead to Candidiasis. Some Candida strains are becoming resistant to
first-line and second-line antifungal agents such as azoles and
echinocandins.
Parasites:
The protozoan parasites that cause the diseases
malaria,trypanosomiasis,toxoplasmosis,cryptosporidiosis and leishmaniasis
are important human pathogens.
Malarial parasites that are resistant to the drugs that are currently available
to infections are common and this has led to increased efforts to develop
new drugs. Resistance to recently developed drugs such as artemisinin has
also been reported. The problem of drug resistance in malaria has driven
efforts to develop vaccines.
Trypanosomes are parasitic protozoa that cause African
trypanosomiasis and Chagas disease (American trypanosomiasis). There
are no vaccines to prevent these infections so drugs such
as pentamidine and suramin, benznidazole and nifurtimox are used to treat
infections. These drugs are effective but infections caused by resistant
parasites have been reported.
Leishmaniasis is caused by protozoa and is an important public health
problem worldwide, especially in sub-tropical and tropical countries. Drug
resistance has "become a major concern".
Further research
Vaccines:
Microorganisms do not develop resistance to vaccines because a vaccine
enhances the body's immune system, whereas an antibiotic operates
separately from the body's normal defenses. Furthermore, if the use of
vaccines increase, there is evidence that antibiotic resistant strains of
pathogens will decrease; the need for antibiotics will naturally decrease as
vaccines prevent infection before it occurs. However, new strains that
escape immunity induced by vaccines may evolve; for example, an
updated influenza vaccine is needed each year.
While theoretically promising, antistaphylococcal vaccines have shown
limited efficacy, because of immunological variation
between Staphylococcus species, and the limited duration of effectiveness
of the antibodies produced. Development and testing of more effective
vaccines is underway.

Development of new drugs:

Since the discovery of antibiotics, research and development (R&D) efforts
have provided new drugs in time to treat bacteria that became resistant to
older antibiotics, but in the 2000s there has been concern that development
has slowed enough that seriously ill people may run out of treatment
options. Another concern is that doctors may become reluctant to perform
routine surgeries because of the increased risk of harmful infection. Backup
treatments can have serious side-effects; for example, treatment of multi-
drug-resistant tuberculosis can cause deafness or psychological disability.
The potential crisis at hand is the result of a marked decrease in industry
R&D. Poor financial investment in antibiotic research has exacerbated the
situation. The pharmaceutical industry has little incentive to invest in
antibiotics because of the high risk and because the potential financial
returns are less likely to cover the cost of development than for other
pharmaceuticals. In 2011, Pfizer, one of the last major pharmaceutical
companies developing new antibiotics, shut down its primary research
effort, citing poor shareholder returns relative to drugs for chronic
illnesses. However, small and medium-sized pharmaceutical companies
are still active in antibiotic drug research.
In the United States, drug companies and the administration of
President Barack Obama have been proposing changing the standards by
which the FDA approves antibiotics targeted at resistant organisms. On 12
December 2013, the Antibiotic Development to Advance Patient Treatment
(ADAPT) Act of 2013 was introduced in the U.S. Congress. The ADAPT
Act aims to fast-track the drug development in order to combat the growing
public health threat of 'superbugs'. Under this Act, the FDA can approve
antibiotics and antifungals needed for life-threatening infections based on
data from smaller clinical trials. The Centers for Disease Control and
Prevention (CDC) will reinforce the monitoring of the use of antibiotics that
treat serious and life-threatening infections and the emerging resistance,
and make the data publicly available. The FDA antibiotics labeling process,
'Susceptibility Test Interpretive Criteria for Microbial Organisms' or
'breakpoints' is also streamlined to allow the most up-to-date and cutting-
edge data available to healthcare professionals under the new Act.
On 18 September 2014 Obama signed an executive order to implement the
recommendations proposed in a report by the President's Council of
Advisors on Science and Technology (PCAST) which outlines strategies to
stream-line clinical trials and speed up the R&D of new antibiotics. Among
the proposals:

 Create a 'robust, standing national clinical trials network for antibiotic

testing' which will promptly enroll patients once identified to be suffering
from dangerous bacterial infections. The network will allow testing
multiple new agents from different companies simultaneously for their
safety and efficacy.
 Establish a 'Special Medical Use (SMU)' pathway for FDA to approve
new antimicrobial agents for use in limited patient populations, shorten
the approval timeline for new drug so patients with severe infections
could benefit as quickly as possible.
 Provide economic incentives, especially for development of new classes
of antibiotics, to offset the steep R&D costs which drive away the
industry to develop antibiotics.
The executive order also included a $20 million prize to encourage the
development of diagnostic tests to identify highly resistant bacterial
infections.
The U.S. National Institutes of Health plans to fund a new research network
on the issue up to $62 million from 2013 to 2019. Using authority created
by the Pandemic and All Hazards Preparedness Act of 2006,
the Biomedical Advanced Research and Development Authority in the
U.S. Department of Health and Human Services announced that it will
spend between $40 million and $200 million in funding for R&D on new
antibiotic drugs under development by GlaxoSmithKline.
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