1.

1 INTRODUCTION
The global use and misuse of antibiotics has led to the evolution and spread of
bacterial resistance to all routinely used antibiotics. In order to effectively tackle the
spread of antimicrobial resistance (AMR) through antimicrobial stewardship or
development of novel antimicrobial compounds, understanding how bacteria adapt
and evolve to survive antibiotic treatments is crucial (Bottery et al., 2021).

According to WHO, Antimicrobial Resistance (AMR) occurs when bacteria, viruses,

fungi and parasites change over time and no longer respond to medicines making
infections harder to treat and increasing the risk of disease spread, severe illness and
death (WHO, 2021). As a result of drug resistance, antibiotics and other antimicrobial
medicines become ineffective and infections become increasingly difficult or
impossible to treat. Antimicrobial resistance (AMR) has become a global health and
development threat and it requires urgent multisectoral actions in order to achieve
the Sustainable Development Goals (SDGs) (WHO, 2021).

Any substance that inhibits the growth and replication of a bacterium or kills it
outright can be called an antibiotic. Antibiotics are a type of antimicrobial designed
to target bacterial infections within (or on) the body. An antimicrobial agent is
defined as a natural or synthetic substance that kills or inhibits the growth of
microorganisms such as bacteria, fungi and algae. Resistance is the ability of a
bacteria against the antagonizing effect of an antibacterial agent upon reproduction
prevention or bactericidal. The development of resistance to antibiotics in bacteria
often develops as a result of unnecessary and inappropriate use of antibiotics.
Through the intense use of antibiotics, resistant microorganisms have emerged over
the years, and problems were started to be experienced for the treatment of these
infections emerged with these resistant microorganisms. Today, on the one hand
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trying to develop new drugs, on the other hand, there are difficulties in treatment as
a result of development of resistance to these drugs rapidly. The main four types of
resistance to antibiotics develops; Natural (Intrinsic) resistance, Acquired resistance,
Cross-resistance, multi-drug resistance and pan-resistance (Salih & Ali 2013).

1.2 ANTIMICROBIAL RESISTANCE EVOLUTION

Antimicrobial resistance (AMR) occurs when microbes evolve mechanisms that

protect them from the effects of antimicrobials. Antibiotic resistance is a subset of
AMR that applies specifically to bacteria that become resistant to antibiotics.
Infections due to AMR cause millions of deaths each year. Infections caused by
resistant microbes are more difficult to treat, requiring higher doses of antimicrobial
drugs, or alternative medications which may prove more toxic. These approaches
may also be more expensive. Microbes resistant to multiple antimicrobials are called
multidrug resistant (MDR) (Magiorakos et al., 2012).

All classes of microbes can evolve resistance. Fungi evolve antifungal resistance.
Viruses evolve antiviral resistance. Protozoa evolve antiprotozoal resistance, and
bacteria evolve antibiotic resistance. Those bacteria that are considered extensively
drug resistant (XDR) or totally drug-resistant (TDR) are sometimes called
"superbugs". Resistance in bacteria can arise naturally by genetic mutation or by one
species acquiring resistance from another. Resistance can appear spontaneously
because of random mutations. However, extended use of antimicrobials appears to
encourage selection for mutations which can render antimicrobials ineffective. The
prevention of antibiotic misuse, which can lead to antibiotic resistance, includes
taking antibiotics only when prescribed. Narrow-spectrum antibiotics are preferred
over broad-spectrum antibiotics when possible, as effectively and accurately

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targeting specific organisms is less likely to cause resistance, as well as side effects.
(Gerber et al., 2017). 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.

Fig 1: Natural development of resistance; source: Gerber et al., 2017.

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 releasing 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
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grow faster than vulnerable bacteria. (Gullberg et al., 2011). As resistance to
antibiotics becomes more common there is greater need for alternative treatments.
Calls for new antibiotic therapies have been issued, but new drug development is
becoming rarer (Cassir , et al., 2014)

Antimicrobial resistance is increasing globally due to increased prescription and

dispensing of antibiotic drugs in developing countries. Estimates are that 700,000 to
several million deaths result per year and continue to pose a major public health
threat worldwide (Dadgostar, 2019). Each year in the United States, at least
2.8 million people become infected with bacteria that are resistant to antibiotics and
at least 35,000 people die and US$55 billion in increased health care costs and lost
productivity. According to World Health Organization (WHO) estimates, 350 million
deaths could be caused by AMR by 2050. (Chanel & Doherty, 2020). By then, the
yearly death toll will be 10 million, according to a United Nations report.

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. During the COVID-19 pandemic, action against
antimicrobial resistance slowed due to scientists focusing more on SARS-CoV-2
research (Hoffman et al., 2017).

2.1 Antimicrobial Development

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The 1950s to 1970s represented the golden age of antibiotic discovery, where
countless new classes of antibiotics were discovered to treat previously incurable
diseases such as tuberculosis and syphilis (Aminov, 2010). However, since that time
the discovery of new classes of antibiotics has been almost nonexistent, and
represents a situation that is especially problematic considering the resiliency of
bacteria shown over time and the continued misuse and overuse of antibiotics in
treatment (WHO, 2014)

The phenomenon of antimicrobial resistance caused by overuse of antibiotics was

predicted as early as 1945 by Alexander Fleming who said "The time may come when
penicillin can be bought by anyone in the shops. Then there is the danger that the
ignorant man may easily under-dose himself and by exposing his microbes to
nonlethal quantities of the drug makes them resistant”. Without the creation of new
and stronger antibiotics an era where common infections and minor injuries can kill,
and where complex procedures such as surgery and chemotherapy become too risky,
is a very real possibility. Antimicrobial resistance threatens the world as we know it,
and can lead to epidemics of enormous proportions if preventive actions are not
taken. In this day and age, current antimicrobial resistance leads to longer hospital
stays, higher medical costs and increased mortality (WHO 2014).

2.2 Mechanism of Antimicrobial Resistance

Antimicrobial resistance mechanisms fall into four main categories: limiting uptake
of a drug; modifying a drug target; inactivating a drug and active drug efflux. Intrinsic
resistance may make use of limiting uptake, drug inactivation, and drug efflux;
acquired resistance mechanisms used may be drug target modification, drug
inactivation, and drug efflux. Because of differences in structure, etc., there is
variation in the types of mechanisms used by gram negative bacteria versus gram
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positive bacteria. Gram negative bacteria make use of all four main mechanisms,
whereas gram positive bacteria less commonly use limiting the uptake of a drug.

Fig 2: The general antimicrobial resistance mechanisms, Source: Erik-Wistrand et al.,

2018)

2.1.1 Enzyme –Based Antimicrobial –Inactivation system

The enzymatic mechanisms of antibiotic resistance include hydrolysis, group
transfer, and redox processes. In terms of diversity, evolution and spread, antibiotic
resistance enzymes contribute remarkably to the bacterial ability to overcome
antibiotic pressure (Varela, et al., 2021). Bacteria that produce antibiotics apparently
require mechanisms to overcome the lethal effects of the compounds and these are
in the form of concurrent production of degradative enzymes, mutations in targets of
antibiotics or active extrusion of antibiotics of antibiotics from the cell so that the
antibiotic-producing cell is protected ((Varela, et al., 2021). The β-lactams constitute
the largest group of clinically used antibiotics, comprising of penicillins,
cephalosporins of different generations, monobactams, and carbapenems, all of
which are characterized by the presence of 3-carbon, 1-nitrogen containing β-lactam
ring. The β-lactam antibiotics inhibit the bacterial proteins known as penicillin-

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binding proteins (PBPs), which perform the critical role of peptide cross-linking
during peptidoglycan cell wall biosynthesis. The structural mimicry of the D-Ala-D-
Ala terminal fragment of cross-linking peptide by β-lactams facilitates competitive
inhibition of PBPs, which stops the cell wall synthesis leading to bacterial cell lysis
and death (Bush, & Jacoby, 2010). However, bacteria gain resistance to lactam
antibiotics by modifying their PBPs, which are no longer susceptible to binding by the
antibiotic. Alternatively, bacteria produce powerful lactamases that degrade
antibiotics before they can bind with the PBPs.
2.1.2 Alteration of Antimicrobial Targets

As bacterial enzymes alter drug structures, the drug targets may likewise be altered,
preventing drug binding and, thus, conferring resistance. Antimicrobial targets play
vital roles in microbial growth or survival and, thus, serve as potential useful targets
for mitigating infection. These targets must differ or be completely absent from
humans or the animal species being treated with an antimicrobial to allow for a
selective mode of action (Varela, et al., 2021). A good example of such a target is
peptidoglycan. Peptidoglycan is essential to the growth and survival of many
bacterial species and has a chemical structure that is not present in the mammalian
hosts they infect. This allows for the targeting of enzymes responsible for the
synthesis and assembly of peptidoglycan. The alteration in target sites has been
attributed to ribosomes, nucleic acid enzymes, and lipopolysaccharides. Pertinently,
peptidoglycan inhibition by glycopeptides involves the binding of the peptidyl-D-
alanyl-D-alanine terminus of peptidoglycan precursors. This binding prevents
integration via the transglycosylase activity of these precursors into the cell wall. The
alteration in the binding site of the drug to the peptidoglycan can lead to
antimicrobial resistance (Varela, et al., 2021).

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2.1.3 Active Efflux Pumps of Antimicrobial Agents

In cases where intact antimicrobial agents enter bacterial cells and drug targets are
freely accessible, active drug efflux systems can come into play. Bacteria that are
pathogenic frequently make use of integral membrane proteins that function as
transporters of antimicrobial agents. Such bacterial transport proteins serve to
actively export structurally distinctive antimicrobial agents from the cytoplasm,
where drug targets reside, to the extracellular milieu, where their molecular targets
are lacking (Kumar, et al., 2020) Efflux pumps are present in all bacteria and are
integral parts of bacterial physiology, being involved in diverse functions such as the
expulsion of toxic products of metabolism, and maintenance of homeostasis.
However, antibiotics as incidental substrates of efflux pumps have resulted in them
being viewed largely as bacterial mechanisms of antimicrobial resistance (Kumar,
&Varela, 2012).
2.1.4 Reduction of Antimicrobial Permeability into Bacterial Cells

An important mechanism of bacterial resistance to antimicrobial agents involves

preventing drug permeability and access to the internal milieu of the pathogenic
cells. Strains of Gram-negative pathogenic bacterial species, such as Escherichia coli,
Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella spp., and Salmonella enterica, are
particularly troublesome. The molecular systems involved in reduced permeability of
antimicrobial agents include resistance mechanisms at the bacterial cell wall. The
extensive structural nature of the lipopolysaccharide layer constitutes a formidable
barrier to the passage of small molecules, especially those that are growth inhibitory
in their properties. Another important molecular mechanism for conferring
resistance via permeability reduction involves porins, which are integral outer
membrane proteins with water-filled pore-like channels that permit the passage of
8
molecules with definitive sizes and charges. The reduction in the permission to the
amount of drugs that enter the organism can lead to drug resistance (Varela, et al.,
2021).

Chapter three

9
3.1 Causes of Antimicrobial Resistance

Antimicrobial resistance is mainly caused by the overuse of antimicrobials. This leads

to microbes either evolving a defense against drugs used to treat them, or certain
strains of microbes that have a natural resistance to antimicrobials becoming much
more prevalent than the ones that are easily defeated with medication.  While
antimicrobial resistance does occur naturally over time, the use of antimicrobial
agents in a variety of settings both within the healthcare industry and outside has led
to antimicrobial resistance becoming increasingly more prevalent (Holmes AH et al.,
2016).
3.1.1 Natural Occurrence
Antimicrobial resistance can evolve naturally due to continued exposure to
antimicrobials. Natural selection means that organisms that are able to adapt to their
environment, survive, and continue to produce offspring. As a result, the types of
microorganisms that are able to survive over time with continued attack by certain
antimicrobial agents will naturally become more prevalent in the environment, and
those without this resistance will become obsolete. Over time most of the strains of
bacteria and infections present will be the type resistant to the antimicrobial agent
being used to treat them, making this agent now ineffective to defeat most
microbes. With the increased use of antimicrobial agents, there is a speeding up of
this natural process (Ferri M et al., 2017). The natural processes include:

 Selective Pressure

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In the presence of an antimicrobial, microbes are either killed or, if they carry
resistance genes, survive. These survivors will replicate, and their progeny will quickly
become the dominant type throughout the microbial population.

Fig 3: Multiplication of pathogen after survive of antimicrobial

 Mutation

Most microbes reproduce by dividing every few hours, allowing them to evolve
rapidly and adapt quickly to new environmental conditions. During replication,
mutations arise and some of these mutations may help an individual microbe survive
exposure to an antimicrobial.

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Fig 4: Diagram showing when bacteria multiplying while some will mutate.

 Gene Transfer

Microbes also may get genes from each other, including genes that make
the microbe drug resistant. Bacteria multiply by the billions. Bacteria that have drug-
resistant DNA may transfer a copy of these genes to other bacteria. Non-resistant
bacteria receive the new DNA and become resistant to drugs. In the presence of
drugs, only drug-resistant bacteria survive. The drug-resistant bacteria multiply and
thrive.

Fig 5: Gene transfer

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3.1.2 Self-medication

Self-medication by consumers is defined as "the taking of medicines on one's own

initiative or on another person's suggestion, who is not a certified medical
professional", and it has been identified as one of the primary reasons for the
evolution of antimicrobial resistance. (Rather IA, et al., 2017). In an effort to manage
their own illness, patients take the advice of false media sources, friends, and family
causing them to take antimicrobials unnecessarily or in excess. Many people resort to
this out of necessity, when they have a limited amount of money to see a doctor, or
in many developing countries a poorly developed economy and lack of doctors are
the cause of self-medication. In these developing countries, governments resort to
allowing the sale of antimicrobials as over the counter medications so people could
have access to them without having to find or pay to see a medical professional
(Ayukekbong JA et al., 2017). This increased access makes it extremely easy to obtain
antimicrobials without the advice of a physician, and as a result many antimicrobials
are taken incorrectly leading to resistant microbial strains. One major example of a
place that faces these challenges is India; where in the state of Punjab 73% of the
population resorted to treating their minor health issues and chronic illnesses
through self-medication (Rather IA, et al., 2017)

The major issue with self-medication is the lack of knowledge of the public on the
dangerous effects of antimicrobial resistance, and how they can contribute to it
through mistreating or misdiagnosing themselves.  In order to determine the public's
knowledge and preconceived notions on antibiotic resistance, a major type of
antimicrobial resistance, a screening of 3537 articles published in Europe, Asia, and
North America was done.  Of the 55,225 total people surveyed, 70% had heard of
antibiotic resistance previously, but 88% of those people thought it referred to some
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type of physical change in the body. With so many people around the world with the
ability to self-medicate using antibiotics, and a vast majority unaware of what
antimicrobial resistance is, it makes the increase of antimicrobial resistance much
more likely (Ayukekbong J A et al., 2017).

3.1.3 Clinical misuse

Clinical misuse by healthcare professionals is another cause leading to increased

antimicrobial resistance. Studies done by the CDC show that the indication for
treatment of antibiotics, choice of the agent used, and the duration of therapy was
incorrect in up to 50% of the cases studied.  In another study done in an intensive
care unit in a major hospital in France, it was shown that 30% to 60% of prescribed
antibiotics were unnecessary (Ventola CL, 2015). These inappropriate uses of
antimicrobial agents promote the evolution of antimicrobial resistance by supporting
the bacteria in developing genetic alterations that lead to resistance. In a study done
by the American Journal of Infection Control aimed to evaluate physicians’ attitudes
and knowledge on antimicrobial resistance in ambulatory settings, only 63% of those
surveyed reported antibiotic resistance as a problem in their local practices, while
23% reported the aggressive prescription of antibiotics as necessary to avoid failing
to provide adequate care (Harris A et al., 2019). This demonstrates how a majority of
doctors underestimate the impact that their own prescribing habits have on
antimicrobial resistance as a whole. It also confirms that some physicians may be
overly cautious when it comes to prescribing antibiotics for both medical and legal
reasons, even when indication for use for these medications is not always confirmed.
This can lead to unnecessary antimicrobial use.

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Studies have shown that common misconceptions about the effectiveness and
necessity of antibiotics to treat common mild illnesses contribute to their overuse
(Blaser MJ et al., 2020)

3.1.4 Environmental pollution

Untreated effluents from pharmaceutical manufacturing industries, hospitals and

clinics, and inappropriate disposal of unused or expired medication can expose
microbes in the environment to antibiotics and trigger the evolution of resistance
(Ahmad A et al., 2017).
3.2 Preventive strategies to reduce antimicrobial resistance

 Improve awareness and understanding of AMR through effective

communication, education, and training.

AMR should be included as a core component of professional education, not only for
medical practitioners but also for pharmacists and agricultural training. In addition to
training professionals, public education efforts should attempt to increase awareness
(Ahmed M et al., 2015). Studies have shown that pharmaceutical drug promotions
can have an impact on the request of prescription drugs and that physician
prescription can be affected by interactions with pharmaceutical companies. A
review study of the interactions of the pharmaceutical industry with physicians from
developing countries showed that physicians view interaction favorably as a means
of acquiring new and reliable information about the drugs they prescribed but few
acknowledged that their prescribing habits can be influenced (Ahmed M et al., 2015).

 Strengthen knowledge and evidence through surveillance and research

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Active surveillance methods regarding incidence, prevalence, and geographic
patterns should be implemented at the local, national, and regional levels to guide
best treatment practices and detect emergence of new resistance. Basic science
research into the mechanisms of resistance may offer new tools to diagnose
resistance or treatment modalities. Understanding the relationship of human
pathogens to animal and environmental microbes may inform smarter policies (Pérez
G et al., 2016).

Overall, reviews in prevention strategies acknowledge the lack of data regarding the
various approaches in preventing MDRO infections. Methodological study designs
are needed when randomized controlled trial designs are not feasible. Additionally,
more studies are needed in district and rural hospitals in developing countries, and
this will require substantial improvements in bacteriological services and surveillance
in such settings. Surveillance over time is similarly important, as resistance frequently
evolves (Sutter DE et al., 2016).

 Reduce the incidence of infection through effective sanitation, hygiene,

and infection prevention measures

Recognizing that the most serious resistant infections are related to healthcare
facilities, implementing and monitoring hygiene and infection control methods
continue to be mainstay of reducing spread of resistant pathogens. Critical to
controlling the spread of MDROs within hospital environments is the reduction of
hospital-acquired infections (HAIs). Resource-limited countries have higher rates of
central line-associated bloodstream infections (CLABSIs), catheter-associated
urinary tract infections, and ventilator-associated pneumonias (VAPs) than high-
income countries (Rosenthal VD et al., 2012). Some factors contributing to these
16
higher rates include crowded wards, insufficient hand hygiene infrastructure, open
stopcocks, and use of multiuse vials. The International Nosocomial Infection Control
Consortium (INICC) adapted recommendations published by the Society for
Healthcare Epidemiology of America (SHEA) and the Infectious Diseases Society of
America (IDSA) into a protocol that can be implemented in the developing
world.These include practice bundles of infection control practices, education,
surveillance programs, as well as performance feedback of the implemented
infection control practices (Rosenthal V D et al., 2012)..

Vaccination should be encouraged to reduce infections that drive antimicrobial use.

Reduction or elimination of major diseases can shift epidemiology such that choices
of empiric treatment can change. Sanitation and water safety is a key to reducing
environmental reservoirs. Untreated waste water, particularly in urban areas,
contributes to environmental presence of drug-resistant bacteria, including the
super-resistance to last-resort antibiotics. This can potentially lead to asymptomatic
colonization with these organisms in a population (Woolhouse M. et al., 2015)

 Optimize the use of antimicrobial medicines in human and animal health

Excessive antibiotic use is the primary driver of resistance. Empiric therapy when
diagnostics are not available, over-prescription, under-dosing, self-treatment, and
unregulated agricultural use have all been implicated. Antimicrobial stewardship
should include evolving practice guidelines to better match local epidemiology. The
use of antibiotics that are not prescribed by a provider is common practice in
developing countries. Patients may purchase antibiotics based on their own
knowledge or as recommended by a pharmacist. This is especially problematic when
a patient has limited finances to purchase a full course, leading to incomplete
17
treatment or under-dosing. A systematic review and meta-analysis of the factors that
contribute to self-medication found that level of education, income, and past
successful use were contributors. (Chioro A et al., 2015)

 Sustainable investment in new medicines, diagnostic tools, vaccines, and

other interventions

Stimulating investment in new medicines, diagnostic tools, and vaccines may require
funding driven by a public health approach rather than continuing to rely on sales
revenues. Economic impact assessments are direly needed. Priority should be given
to those agents relevant to high-burden countries and funding schemes to make
these agents available. Diagnostic tools have potential to play perhaps the most
crucial role in prevention. If excessive or inappropriate antibiotic use is driven by
empiric treatment of suspected bacterial infections, rapidly identifying nonbacterial
illnesses and accurate and early identification of bacterial pathogens are the keys to
curbing it. As infection surveillance programs in resource-limited settings consider
how to thoughtfully allocate laboratory resources, it is worthwhile to examine the
cost-efficiency of traditional culture-based systems versus molecular testing that
could allow for rapid diagnosis (Diaz, A., Antonara, S. & Barton, T 2018).

3.3 Drug Discovery and Development as a method of combating antimicrobial

resistance
Drug discovery is a multifaceted process, which involves identification of a drug
chemical therapeutically useful in treating and management of a disease condition.
Typically, researchers find out new drugs through new visions into a disease process
that permit investigator to design a medicine to stopover or contrary the effects of
the disease. The process of drug discovery includes the identification of drug
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candidates, synthesis, characterization, screening, and assays for therapeutic
efficacy. When a molecule avails its satisfactory results in these investigations, it will
commence the process of drug development subsequent to clinical trials. (Deore, A B
et al., 2019).
Stages of drug discovery and development include: Target identification, Target
validation, lead identification, lead optimization, Product characterization,
Formulation and development, Preclinical research, Investigational New Drug,
Clinical trials, New Drug Application and Approval (Deore, A B et al., 2019).

Fig 6: Drug discovery and Development process

3.4 Molecular Strategies for overcoming Antibiotics Resistance in Bacteria

3.4.1 Bioinformatics Strategies

Bioinformatics is a discipline that employs a lot of computation techniques including

sequence and structural alignment, analyses of large collections of biological data,
such as genetic sequences, cell populations, or protein samples, to make new
predictions or discover new biology. Nowadays bioinformatics techniques such as
molecular docking have been used to evaluate ligand–protein interaction and also to
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estimate binding energy during the docking process. Bioinformatics revolutionizes
molecular studies to determine the protein structure, gene structure or sequence,
molecular markers and relate them to other previously known structures (H. Hemlata
and A. Tiwari (2016). Bioinformatics studies have provided an important system of
modelling a biological living cell (with proven DNA sequence) and docking proteins
that enabled scientists to discover effective drug strategies to combat the
diversifying problem of antibiotic resistance that is becoming one of the major public
health challenges. It can be used to explore the connections between classical
mathematical modelling (at different scales) and predictions of omic scope along
with specific aspects of the immune system. Some commonly used software in
bioinformatics include; Swiss-model (online software for homology modelling),
Autodock Vina (for ligand-protein docking) Avogadro (for ligand energy
minimisation) and Chimera (to prepare and view 3D docked complex), among others.
In the recent time, bioinformatics strategy at preventing antibiotic resistance has
evolved, this includes; (Umar Ndagi et al., 2020)

3.4.2 Whole Genome Sequencing Bioinformatics Strategies of Curbing

Antimicrobial Resistance

In recent times, whole-genome sequencing (WGS) of pathogens has become a more

accessible and affordable tool for genotyping and plays an important role in human
and agricultural research. Analysis of the entire bacteria genome using WGS could
provide insight into related lineages of bacterial and can revolutionize outbreak
analysis in hospitals. The recent development in bioinformatics has changed the
understanding of clinicians and perhaps financial burden been perceived on the
patients. Sequencing technologies and analysis tools have rapidly increased the

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output and analysis speed as well as reduced the overall costs of WGS. Genome
sequencing provides a good regime for improvement in scientific research,
particularly in biomolecular modelling and drug design with an emphasis on
antibiotics resistance. In addition to identifying pathogens more rapidly and precisely
than traditional methods, high-throughput technologies and bioinformatics can
provide new insights into disease transmission, virulence, and antimicrobial
resistance (Umar Ndagi et al., 2020).

Deoxyribonucleic acid (DNA) sequencing provides a good platform for protein

modelling and drug design. Also, remarkable progress made in genome sequencing,
protein expression, high-throughput crystallography, and nuclear magnetic
resonance (NMR) has radically transformed the opportunities to use protein three-
dimensional structures to accelerate drug discovery useful in combating
antimicrobial resistance (J. L. Jones et al., 2019).

3.4.3 In Silico Analysis of Serovar, Serogroup, and Antigenic Profile

Development of whole-genome sequencing technology takes center stage in

genotyping, as serovar prediction by traditional serotyping got replaced by molecular
serotyping. An existing in silico serovar prediction approaches utilize surface antigen.
A recent study by Zhang et al. (2019) designed and evaluated an in silicon serovar
prediction approach by screening 1089 genomes representing 106 serovars against a
set of 131 serovar-specific gene markers. This approach was reportedly a useful
diagnostic tool for culture-independent and met genomic methods as well as
providing the best alternative of confirming other genome-based analyses. This set
of bioinformatics procedure is useful in identifying a specific type of gene markers

21
and may also be useful in developing more cost-effective molecular assays designed
to detect specific gene markers of the all major serovars (T. F. Jesus et al., 2019)

3.4.4 In Silicon Plasmid Identification

Plasmids are extra chromosomal genetic elements which are also known as mobile
genetic elements (MGEs), they play an important role in horizontal gene transfer and
characterized by their ability to self-replicate and transfer between bacterial. They
exist in different sizes and they are carriers of genes with selective advantage to the
host under specific conditions. Plasmids are essential MGEs for the acquisition and
spread of antibiotic resistance and are important in transmitting virulence traits (T. F.
Jesus et al., 2019).

Different types of tools are readily available for use in plasmid extraction and
assembling from high throughput sequencing (HTS) data. These tools could be used
to access plasmid data by searching for specific markers or exploring the distinctive
nature of plasmid sequence. The tools are assumptions based on relative failure
because plasmid can exist in single-copy in the cell or linear DNA molecules (Umar
Ndagi et al., 2020). This technology has been devised in order to combat
antimicrobial resistance.

3.4.5 Metagenomics for Antimicrobial Surveillance

Antimicrobial resistance (AMR) surveillance has focused mainly on a few pathogens

based on passive reporting of certain phenotypes from laboratory results. This limits
the spectrum to a few selected pathogens as in the Danish Monitoring System
(DANMAP) leading to a narrow pathogen spectrum that does not capture all relevant

22
AMR genes. The majority of AMR genes might be found in the commensal bacterial
or of healthy humans and animals or the environment (R. S. Hendriksen et al., 2019).

Metagenomics techniques use short-read next-generation sequencing data, with the

capacity to quantify thousands of transmissible resistance genes in a single sample
without any predetermined genes. Therefore, it can provide more information about
bacterial species presence, pathogens, and virulence genes, the generated data can
then be re-analyzed, if novel genes of interest are identified. Recently,
metagenomics is superior to conventional methods of AMR surveillance in pig herds,
successfully been used for comparing AMR in livestock (L. Van Gompel et al., 2019)

The prospect of metagenomics as a tool for AMR surveillance in the future is quite
bright because of its enormous advantage, one of which involves direct application
on samples from healthy and clinically ill individuals and animals as well as the
potential reservoir. This might result in the ultimate one goal surveillance of AMR
allowing determination of all resistance genes and their context in all reservoirs (R. S.
Hendriksen et al., 2019).

3.4.6 Use of Comprehensive Antibiotic Resistance Database

The Comprehensive Antibiotic Resistance Database (CARD; is a rigorously curated

resource providing reference DNA and protein sequences, detection models, and
bioinformatics tools on the molecular basis of bacterial antimicrobial resistance
(AMR). CARD is used to focus on providing high-quality reference data and molecular
sequences within a controlled vocabulary (Umar Ndagi et al., 2020). The Antibiotic
Resistance Ontology (ARO), designed by the CARD biocuration team to integrate
with so ware development efforts for resistome analysis and prediction includes
CARD's Resistance Gene Identifier (RGI) so ware (P. A. Brian et al., 2019).
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 In 2017, the use of CARD expanded through extensive curation of reference
sequences, revision of the ontological structure, curation of over 500 new AMR
detection models, development of a new classification paradigm and expansion of
analytical tools. Most importantly are the available new module (Resistomes &
Variants) that provides analysis and statistical summary of in silico predicted
resistance variants from 82 pathogens and over 100 000 genomes. By adding these
resistance variants to CARD, predicted resistance could be summarized using the
information included in CARD, identify trends in AMR mobility and determine
previously undescribed and novel resistance variants (P. A. Brian et al., 2019).

This approach has proven to have several limitations; however, bioinformatics

approaches are required for analysis of genome sequence that is tolerant of missing
data such as unsampled sequence while simultaneously replacing simple sequence
similarity with total evidence when predicting antibiogram. One possible way is the
development of Probabilistic Graphic Models (PGMs), which are a mathematical
framework for incorporating uncertainty and probability when making predictions
from limited or noisy data and that has proven very successful in genomics research,
particularly in the analysis of regulatory networks, genetic association studies, and
genetic architecture of complex diseases (A. G. McArthur and G. D. Wright 2015).

24
 Chapter four

Conclusion

Any substance that inhibits the growth and replication of a bacterium or kills it
outright can be called an antibiotic. Antibiotics are types of antimicrobial designed to
target bacterial infections within (or on) the body. An antimicrobial agent is defined
as a natural or synthetic substance that kills or inhibits the growth of microorganisms
such as bacteria, fungi and algae. Resistance is the ability of a bacteria against the
antagonizing effect of an antibacterial agent upon reproduction prevention or
bactericidal. Antimicrobial resistance (AMR) occurs when microbes evolve
mechanisms that protect them from the effects of antimicrobials. Antibiotic
resistance is a subset of AMR that applies specifically to bacteria that become
resistant to antibiotics. Infections due to AMR cause millions of deaths each year.
Microorganisms use the following mechanisms to resist antimicrobial: Enzyme –
Based Antimicrobial –Inactivation system, alteration of antimicrobial targets active,
efflux pumps of antimicrobial agents and reduction of antimicrobial permeability into
bacterial Cells. Causes of Antimicrobial Resistance include natural occurrence, self-
medication, clinical misuse and environmental pollution. Improve awareness and
understanding of AMR through effective communication, education, and training,
strengthen knowledge and evidence through surveillance and research, reduced
incidence of infection through effective sanitation, hygiene, and infection prevention
measures, optimize the use of antimicrobial medicines in human and animal health,
sustainable investment in new medicines, diagnostic tools, vaccines, and other
interventions and molecular technologies can be the possible means of combating
antimicrobial resistance.
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