DEPARTMENT OF BIOTECHNOLOGY

“IDENTIFICATION OF
NOVEL AMINOGLYCOSIDE
ANTIBIOTIC RESISTANCE
LIKE GENE FROM ISOLATED
PURPLE BACTERIA FROM
MIU CAMPUS LAKE”
FINAL YEAR PROJECT PROPOSAL
DINESH KUMARAN (1107151024)
JUNE, 2017
BSc. (BIOTECHNOLOGY) (HONS)
TABLE OF CONTENTS

NO CHAPTERS PAGES

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 1.0 Introduction 3-5

Literature review
2.0 6-16

2.1 Global statistic of antibiotic resistance

2.2 Statistic of antibiotic resistance in Malaysia
2.3 Aminoglycoside
2.4 Aminoglycoside modifying enzymes
2.5 Identification of antimicrobial resistance gene
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3.0 Problem statement
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4.0 Research objectives
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5.0 Materials & Methodology

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6.0 Expected Outcome

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7.0 Significance

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8.0 Benefit of research
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9.0 Table of research activities
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10.0 References

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1.0 INTRODUCTION
Since the pioneering work of researches like Alexander Fleming, Paul Ehrlich, Gerhard
Domagk, and others on antibiotics about 100 years ago, the beneficial drug has been used in
the treatment of infectious diseases. However, this benefit has been taken for granted in
public health resulting in a dramatic change taking place in terms of the efficacy of the
administered antibiotics. Currently, there are many bacteria developing resistance against
antibiotics. As such, standard treatments become ineffective and infections persist thus
increasing the risk of spreading to others (Chellat, Raguž, & Riedl, 2016). Evolution and
spread of antibiotic resistance in pathogenic bacteria is now the most urgent challenge in
public health. Nevertheless, it still remains controversial whether the extensive diversity of
resistance in environmental reservoirs and pathogenic bacteria is the result of human
activity (Perron et al., 2015).

Research on antibiotic resistance has focused on traditionally pathogenic bacteria isolated

in a clinical setting and the role of antibiotic resistance genes already present in those
species. However, antibiotic resistance phenotype is actually an ancient function of
environmental bacteria, although they were largely absent from human pathogens prior to
the antibiotic age (Pehrsson, Forsberg, Gibson, Ahmadi, & Dantas, 2013). Discovery of
antibiotics were originally from environmental fungi and bacteria which were capable of
killing off other microorganism. As these compounds were efficient at eradicating bacteria, it
was seen that the purpose of antibiotic production was to stave off competing
microorganism. Similarly, when antibiotic resistance genes were discovered in environmental
microorganism, it was believed to have evolved in bacteria producing the antibiotics and
target bacteria in order to protect against the effects of antibiotics. Besides that, evidence
suggests that antibiotic resistance genes function to regulate the responses induced from
sub-inhibitory concentrations of antibiotics and biosynthesis of antibiotics. There are several
examples of antibiotic resistance genes identified in the environmental bacteria strain which
includes sulphonamide resistance genes, trimethoprim resistance genes, quinolone
resistance genes, macrolide resistance genes and vancomycin resistance genes (Berglund,
2015).

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Diverse mechanisms of antibiotic resistance have been discovered in nearly all

environments with new metagenomics study uncovering many examples of resistance genes
previously unreported in public databases. As such, environmental antibiotic resistance is
not only widespread, but also represents the likely origins of the resistance seen in human
pathogens (Pehrsson et al., 2013). A study done by (Hong, Al-Jassim, Ansari, & Mackie, 2013)
discussed about the microbial problems related to the use of reclaimed water as an
alternative water source for agricultural irrigation to alleviate the demand on freshwater
sources. Their concern was regarding the occurrence of antibiotic residues in the reclaimed
water that can select for antibiotic resistance genes among the microbial community. The
antibiotic resistance genes can be associated with mobile genetic elements, which would
allow a promiscuous transfer of resistance traits from one bacterium to another. As a result
of it, pathogens that are present in the reclaimed water and antibiotic resistant bacteria can
potentially exchange mobile genetic elements to create the “perfect microbial storm”.

There are several classes of antibiotics available to clinicians which includes

aminoglycosides, Beta-lactams, glycopeptides, tetracyclines and macrolides. These
compounds target vital microbial biochemistry at vulnerable metabolic and physiologic hubs
like translations, DNA replication, and cell wall biosynthesis. Based on previous studies,
these antibiotics are able to arrest cell growth or kill the cells outright depending on their
mode of action, biochemical and genetic trait (McArthur et al., 2013).

Another research by (Perron et al., 2015) was done using functional metagenomics to
study the antibiotic resistance of bacterial communities isolated from different layers of the
Canadian Artic permafrost. This study focussed to prove that the microbial communities
contain diverse resistance mechanism at least 5000 years ago. Isolation of eight genes
conferring clinical levels of resistance against aminoglycoside, B-lactam and tetracycline
which are naturally produced by the isolated bacterial samples were successful. Besides
that, their study showed that antibiotic resistance genes were functionally diverse prior to

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the earlier use of antibiotics by humans contributing to the evolution of natural reservoirs of
resistance genes.

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Similar research was done by (Rascovan, Telke, Raoult, Rolain, & Desnues, 2016) whereby
they analysed meta-genomic data sets from ancient and remote samples from diverse
environmental sources and observed the presence of all eleven antibiotic resistance gene
groups. As ancient samples are not subjected to modern effects of antibiotic misuse
therefore they represent a clean model to explore the natural diversity of antibiotic
resistance gene in the environment. The results showed that most sequences indicated high
divergence compared with known antibiotic resistant gene thus representing a much larger
information database than the currently known and characterized antibiotic resistant genes.

Horizontal gene transfer is seen as a major mechanism responsible for the spread of
antibiotic resistance. Generally, horizontal gene transfer refers to the transfer of genetic
material within or between species, separate from the vertical descent of genes from parent
to offspring. In bacteria, this gene transfer takes place either through one of the 3 major
mechanism namely, conjugation, transformation and transduction. Study conducted showed
that conjugation as the dominant mode of horizontal gene transfer in the spread of
antibiotic resistance on the global scale. Therefore, more analysis needs to be done as a
critical measurement to develop novel strategies to combat resistance spread through
horizontal gene transfer. (Lopatkin, Sysoeva, & You, 2016)

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2.0 Literature review

2.1 Global statistic of antibiotic resistance

According to (Organisation for Economic Co-operation and Development, 2016)

antimicrobial resistance (AMR) has become a global concern where it is no longer a problem
of medical science being beaten by nature. Resistance to antimicrobial is a natural process
which has been observed since the first antibiotics were discovered. Genes that confer drug
resistance upon some strains of bacteria pre-date antibiotics by millions of years.
Unfortunately, as a result of misuse of antibiotics in the medical, veterinary and agricultural
sectors have contributed to the development of antimicrobial resistance. Furthermore,
global trade and travel have accelerated the spread of antibiotic resistance. Development of
new antibiotics has slowed down due to insufficient incentives thus allowing microorganism
to outpace the development of new drugs. With resistance on the rise, we stand to lose the
immense ground we have gained in the last century which includes fight against life
threatening infectious diseases such as pneumonia, TB, HIV and malaria. Besides that, our
battle against conditions such as cancer, where antibiotics are crucial in helping
chemotherapy patients avoid and fight infection will be at a losing ground. Another
advantage in surgical procedures like organ transplants and caesarean sections, which have
now become routine and relatively low risk, thanks to our ability to effectively treat acute
infections with antibiotics will become ineffective (Organisation for Economic Co-operation
and Development, 2016).

It was stated by (O ’neill, 2016) that a report published in December 2014 showed that
about 700 00 people die each year due to drug resistant strains of common bacterial
infections. This number is highly likely to be an underestimate because of poor reporting and
surveillance. An estimated number of 200 00 people die every year due to multidrug-

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resistant and extreme drug resistant tuberculosis (TB) alone. Furthermore, in India the
antibiotic resistant neonatal infects has caused the deaths of approximately 60 000 new-
borns each year. As such, based on scenarios of rising drug resistance for six pathogens to
2050, we can estimate that unless action is taken, the death toll from antimicrobial
resistance could rise up to 10 million each

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year. This could cost a cumulative amount of 100 trillion USD on the global economic. It is
difficult to predict the path of emerging drug resistance but it is also a trend that has been
on the run in one direction so far thus efforts need to be taken to combat this pandemic.

Figure 1: Death tolls resulted by six different diseases. (Adapted from O ’neill, 2016)

2.2 Statistic of antibiotic resistance in Malaysia

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 In the case of antimicrobial resistance in Malaysia, it was stated by (Institute for Medical
Research Malaysia, 2016) that Malaysian hospitals have been at the forefront of the war
against antimicrobial resistance. A study was conducted by (Al-Marzooq, Mohd Yusof, & Tay,
2015) on the multidrug resistance of Klebsiella pneumoniae isolated from patients attending
to University of Malaya Medical Centre, Kuala Lumpur, Malaysia from 2010-2012. The
samples were tested for antibiotic resistance determinants that includes extended spectrum

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beta-lactamase (ESBLs), aminoglycoside, trimethoprim/sulfamethoxazole resistance genes

and plasmid replicons. Results showed that CTX-M-15 (91.3%) was the predominant ESBL
gene detected with aacC2 gene (67.7%) was the most common gene detected in
aminoglycoside-resistant isolates. Trimethoprim/sulfamethoxazole resistance (90.3%) was
attributed to the presence of sul1 (53.8%) and dfrA (59.1%) genes respectively in the
isolates. As such, the results obtained suggest the diffusion of highly diverse plasmids with
multiple antibiotic resistance determinants among the Malaysian isolates. Therefore, an
effective infection control measures and antibiotic stewardship programs should be adopted
to limit the spread of the multidrug resistant bacteria in healthcare settings.

Besides that, another study was done by (Odeyemi & Ahmad, 2017) to investigate
antibiotics resistance pattern and phenotyping of Aeromonas species isolated from different
aquatic sources in Malaysia. Aeromonas species were isolated from the following sources:
sediment (n = 13), bivalve (n = 10), sea cucumber (n = 16) and sea water (n = 14). In addition
to that, resistance to 12 antibiotics were done namely, tetracycline (30 μg), kanamycin (30
μg), oxytetracycline (30 μg), ampicillin (10 μg), streptomycin (10 μg), gentamicin (10 μg),
sulphamethoxazole (25 μg), nalixidic acid (30 μg), trimethoprim (1.25 μg), novobiocin (5 μg),
penicilin (10 μg) and chloramphenicol (10 μg). The results obtained showed multi drug
resistance pattern among the isolates. All the isolates were completely resistant to
ampicillin, novobiocin, sulphamethoxazole and trimethoprim, respectively but susceptible to
tetracycline (100%), kanamycin (5.7%), gentamicin (5.7%) and oxytetracycline (24.5%). As
such, this study does highlight the presence of antibiotic resistance gene naturally in the
environmental microorganism isolated from aquatic sources in Malaysia.

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 Presence of antibiotic resistance gene in environmental microorganism isolated in Malaysia
can further be understood though a research done by (Ghaderpour et al., 2015). E.coli was
found to be multi-drug resistant and genetically diverse in the Matang mangrove estuaries,
Malaysia. Observation showed that one-third (34%) of the estuarine E. coli were multi-drug
resistant. The highest antibiotic resistance prevalence was observed for aminoglycosides
(83%) and beta-lactams (37%). Phylogenetic groups A and B1, being the most predominant

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E. coli, demonstrated the highest antibiotic resistant level and prevalence of integrons. The
results indicated multi-drug resistance among E. coli circulating in Matang estuaries, which
could be a result of anthropogenic activities and aggravated by bacterial and antibiotic
discharges from village lack of a sewerage system, aquaculture farms and upstream animal
husbandry.

2.3 Aminoglycoside

Aminoglycoside antibiotics are a complex family of compounds which are characterized

through the presence of an aminocyclitol nucleus (streptamine, 2- deoxystreptamine, or
streptidine) linked to amino sugars by glycosidic bonds. Depending on the substitution
pattern, aminoglycosides can be grouped into four different subfamilies namely
monosubstituted, atypical, 4, 5-disubstituted, and 4, 6-disubstituted. This antibiotic has
mainly been used in the treatment of infections caused by Gram-negative aerobic bacilli,
staphylococci, and other Gram-positives. However, when used against Gram-positives,
aminoglycosides are used in combination with other antibiotics such as B-lactams whereby
they exert a synergistic affect probably due to an enhanced uptake. Several life threatening
infections were successfully treated with help of aminoglycosides includes plague, tularemia,
brucellosis, endocarditis caused by enterococci and infections caused by streptococci and
enterococci. The property of aminoglycosides to decrease the fidelity of the eukaryotic
elongation machinery makes them potential candidates to treat nonsense mutation related
genetic disorders such as cystic fibrosis. Furthermore, aminoglycoside-based drugs are also

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inhibitors of reproduction of the HIV virus, showing promise on the treatment of AIDS.
(Ramirez & Tolmasky, 2010).

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Figure 2: Structures of typical and atypical aminoglycosides. (Adapted from Chellat, Raguž, &
Riedl, 2016).

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 Aminoglycoside resistance takes place through several mechanisms which are able to
coexist simultaneously in the same cell. The mechanism which are involved includes the
modification of the target by mutation of the 16S rRNA or ribosomal proteins. Besides that,
another mechanism involved is the methylation of 16S rRNA, a mechanism found in most
aminoglycoside-producing organisms and in clinical strains. Reduced permeability by
modification of outer membrane’s permeability or diminished inner membrane transport is
a different mechanism affecting the cell membrane. Lastly, sequestration of the drug by tight
binding to an acetyltransferase of very low activity and enzymatic inactivation of the
antibiotic molecule, the most prevalent in the clinical setting (Ramirez & Tolmasky, 2010).

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2.4 Aminoglycoside modifying enzymes

Aminoglycoside inactivation via aminoglycoside acetyltransferase (AAC) enzyme was the

second bacterial resistance mechanism identified after penicillinase. Ever since then,
extensive structure-function characterization of these enzymes have been ongoing. AACs
belong to the GCN5 N-acetyl transferase (GNAT) superfamily. This superfamily of enzymes
are able to utilize acetyl-CoA as a cofactor and contains approximately 10 000 members. Out
of these numbers, it was AACs that were first to be identified and were shown to have
conserved sequence motifs with eukaryotic transcription factor GCN5. AACs can be divided
into four groups based on the region specificity of the aminoglycoside modification and
designated AAC (1), (3), (2’) and (6’). (Morar & Wright, 2010).

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Figure 3: Chemical structure of an aminoglycoside antibiotic (ribostamycin) showing the
central aminocyclitol ring and acetyl group modification sites (1, 2′, 3 and 6′). (Adapted from
Chellat, Raguž, & Riedl, 2016).

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AAC (1) enzymes have been found in E. coli, Campylobacter spp. and an Actinomycete.
Isolated AAC (1) from E.coli catalyses acetylation of apramycin, butirosin, lividomycin and
paromomycin at the 1st position as well catalyses di-acetylation of ribostamycin and
neomycin. On the other hand, isolated AAC (1) from an Actinomycete differed in substrate
profile when compared from that isolated from E.coli as apramycin was not acetylated by
this enzyme. Furthermore, paromomycin was preferentially acetylated at position 1 but 1,
2’-di-Nacetylparomomycin and 1, 6’”’-di-N- acetylparomomycin were also found as products
of the enzymatic reaction. Besides that, the studies showed that these modifications were
not followed by a significant reduction of the antibiotic activity. The substrate profile of the
AAC (1) isolated from Campylobacter spp. was similar to that of the E. coli enzyme. Although
all three enzymes have been named AAC (1) but the difference in substrate profile of at least
one of them were seen. (Morar & Wright, 2010)

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Figure 4: Crystal structure of an aminoglycoside acetyltransferase HMB0020 from an
uncultured soil metagenomics sample in complex with trehalose (Adapted from:
10.2210/pdb5f47/pdb)

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Another group AAC (3), has nine recognized subclasses of enzymes which are all in Gram-
negatives. The subclass AAC (3)-V has been eliminated after confirmation that the only
enzyme in this group is identical to AAC (3) - III. As for the subclass AAC (3) – I, it includes five
enzymes that confer resistance to gentamicin, sisomicin and fortimicin. It has been observed
that they are present in large number of Enterobacteriaceae and other Gram-negative
clinical isolates. Subclass AAC (3) - II is characterized by their resistance to gentamicin,
netilmicin, tobramycin, sisomicin, 2’-N-ethylnetilmicin, 6’-N-ethylnetilmicin and dibekacin.
On the other hand, there are three enzymes belonging to the subclass AAC (3) – III which are
all isolated from P. aeruginosa isolates. Besides that the only representative of AAC (3)-IV has
been identified in clinical strains of E. coli, Campylobacter jejuni, and Pseudomonas stutzeri.

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Subclasses AAC (3)-VII, AAC (3)-VIII, AAC (3)-IX, and AAC (3)-X are all represented in strains of
Actinomycetes. (Morar & Wright, 2010)

Figure 5: Crystal structure of aminoglycoside acetyltransferase AAC (3)-Ib (Adapted from

10.2210/pdb4yfj/pdb)

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The enzyme, AAC (2’) have been found in Gram-negatives and Mycobacterium where they
help to mediate modification of several aminoglycosides including gentamicin, tobramycin,
dibekacin, kanamycin and netimicin. A putative AAC (2′) enzyme has been proposed to be
part of multidrug resistance in Stenotrophomonas maltophilia but it has not been named
further. A Blast analysis of the amino acid sequence of this protein against those in GenBank
did not show 100% homology with any of the AAC(2′) know enzymes. (Morar & Wright,
2010)

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Figure 6: Aminoglycoside 2'-N-acetyltransferase from Mycobacterium tuberculosis-Complex
with Coenzyme A and Tobramycin (Adapted from 10.2210/pdb1m4d/pdb)

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Lastly, AAC (6′) enzymes are the most common whereby they are present in Gram
negatives as well as Gram-positives. Besides that, the genes have been found in plasmids
and chromosomes, and are often part of mobile genetic elements. There are two main
subclasses of AAC (6′) enzymes that specify resistance to several aminoglycosides and differ
in their activity against amikacin and gentamicin C1. While AAC (6′)-I shows high activity
against amikacin and gentamicin C1a and C2 but very low towards gentamicin C1.

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Furthermore, AAC (6′)-II enzymes actively mediate acetylation of all three forms of
gentamicin but not amikacin. (Morar & Wright, 2010)

Figure 7: Crystal structure of aminoglycoside N-acetyltransferase AAC (6')-Ib11 (Adapted

from: 10.2210/pdb2pr8/pdb)

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2.4 Identification of antimicrobial resistance gene

According to (Call, Bakko, Krug, & Roberts, 2003), DNA microarray can act as an alternative
method for screening for the presence of a wide diversity of genes. This is done using probe
specific to each gene which are deposited onto a solid substrate in a lattice pattern. DNA
would be then labelled and hybridized to the array and specific target-probe duplexes are
detected with a reporter molecule. The authors studied the feasibility of using DNA
microarrays as a tool to screen bacterial isolates for tetracycline resistance genes.

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Microarray probes for 17 tet genes which were Beta- lactamase gene and a 16S ribosomal
DNA gene (E. coli) were generated from known controls by PCR. They were able to correctly
identify the tet genes carried by 39 test strains but 9 additional strains were not known to
contain any genes represented on the microarray, and these strains were negative for all 17
tet probes as expected. As such, the authors indicated that microarray technology has the
potential for screening a large number of different antibiotic resistance genes by the
relatively low-cost methods used.

GeneHunter is a transposon tool designed for the experimental activation and

identification of silent antibiotic resistance genes. This method allows for the identification
of novel resistance genes that lack previously identified homologues. The author used
Salmonella enterica serovar Typhimurium strain LT2 as a test organism for the in vivo version
of the GeneHunter method, where they were able to activate, clone, and identify two cryptic
antibiotic resistance genes. The genes were aminoglycoside acetyltransferase aac(6′)-Iaa and
the probable Mar-A regulon activator rma. As this method requires electroporation of the
host with an efficiency of at least 1010 transformants per microgram, therefore the in vivo
method is not applicable to most microorganisms. Nevertheless, it is expected that
GeneHunter method will be used to identify potential resistance genes during the
development and testing of novel antibiotics, new variants of existing antibiotics, and drug
inhibitor combinations.(Salipante, Barlow, & Hall, 2003)

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3.0 Problem Statement

Currently, many of the antibiotic resistance genes are being studied in bacteria from the
clinical settings while the naturally occurring antibiotic resistance gene have not been much
looked upon. This action could result in the development of a stronger antibiotic resistance
in the environmental microorganism thus causing a huge pandemic in the future leading to

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the development of superbug. Furthermore, public members are not aware of their actions
of overconsuming antibiotics and discarding them without proper disposal.

4.0 Research Objectives

 To identify the presence of antibiotic resistance gene for aminoglycoside from
isolated purple bacteria from MIU lake
 To isolate and amplify the aminoglycoside antibiotic resistant gene from the isolated
purple bacteria from MIU lake
 To deduce the phylogenetic relationship of different class of aminoglycoside
antibiotic resistance gene.

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5.0 Materials and Methodology

 Purple bacteria from MIU campus lake

 Nutrient Agar
 Aminoglycoside antibiotics
 Antibiotic disks
 NCBI software
 Degenerative forward primer and reverse primer
 PCR (Polymerase Chain Reaction) machine
 Gel electrophoresis

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Step 1:
Identification of
aminoglycosidase
antibiotic resistance gene
Disk diffusion method conducted
Varying concentrations of test antibiotics are
used
Determination of minimal inhibition
concentration

Identify the conserved region of antibiotic

Step 2:
Identification of the
presence of particular
resistance gene
resistance gene through Multiple sequence
allignment (MSA)
Design primers to be used in Polymerase chain
reaction (PCR)
Gel electrophoresis of PCR product

Identified antibiotic resistance gene is sent for

Step 3 : gene sequencing
Confirmation of the class Blasting and deduce the phylogenetic
relationship of isolated aminglycoside
of aminoglyoside acetyltransferase like gene with others
antibiotic resistance gene

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6.0 Expected Outcomes

At the end of the research, identification of the presence of antibiotic resistance gene for
aminoglycoside from isolated purple bacteria from MIU lake can be done. Besides that,
isolation and amplification of the aminoglycoside antibiotic resistant gene from the isolated
purple bacteria from MIU lake can be successfully conducted. Lastly, the phylogenetic
relationship of different class of aminoglycoside antibiotic resistance gene can be deduced.

7.0 Significance

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It will help to provide an understanding of the current antibiotic resistance gene evolving
naturally in the environment. Besides that, many different aspects of laboratory skills can be
enhanced by conducting this research. Finally, it will encourage a better understanding on
the functions of gene especially antibiotic resistance gene present in the microorganism.

8.0 Benefits of research

This research will help in educating the public on the importance of taking moderate amount
of antibiotics. Besides that, the importance of understanding about antibiotic resistance
prevalence will encourage many to discard the antibiotics properly and not polluting the
environment. In addition to that, this research will allow current researchers to conduct
further test to identify the significance of the antibiotic resistance gene found naturally in
order to combat the antibiotic resistance pandemic in the clinical sector.

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9.0 Table of Research Activity

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10.0 References

Al-Marzooq, F., Mohd Yusof, M. Y., & Tay, S. T. (2015). Molecular Analysis of Antibiotic

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 Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant
Klebsiella pneumoniae. PloS One, 10(7), e0133654; Retrieved from: https://doi.org /
10.1371/journal.pone.0133654
Berglund, B. (2015). Environmental dissemination of antibiotic resistance genes and
correlation to anthropogenic contamination with antibiotics. Infection Ecology &
Epidemiology, 5, 28564; Retrieved from: https://doi.org/10.3402/iee.v5.28564
Call, D. R., Bakko, M. K., Krug, M. J., & Roberts, M. C. (2003). Identifying antimicrobial
resistance genes with DNA microarrays. Antimicrobial Agents and Chemotherapy,
47(10), 3290–5; Retrieved from: https://doi.org/10.1128/aac.47.10.3290-3295.2003
Chellat, M. F., Raguž, L., & Riedl, R. (2016). Targeting Antibiotic Resistance. Angewandte
Chemie International Edition, 55(23), 6600–6626; Retrieved from: https://doi.org /10.
1002/anie.201506818
Ghaderpour, A., Ho, W. S., Chew, L.-L., Bong, C. W., Chong, V. C., Thong, K.-L., & Chai, L. C.
(2015). Diverse and abundant multi-drug resistant E. coli in Matang mangrove
estuaries , Malaysia. Frontiers in Microbiology, 6, 977; Retrieved from: https://doi.org/
10.3389 /fmicb.2015.00977
Hong, P.-Y., Al-Jassim, N., Ansari, M. I., & Mackie, R. I. (2013). Environmental and Public
Health Implications of Water Reuse: Antibiotics, Antibiotic Resistant Bacteria, and
Antibiotic Resistance Genes. Antibiotics (Basel, Switzerland), 2(3), 367–99; Retrieved
from: https://doi.org/10.3390/antibiotics2030367
Institute for Medical Research Malaysia. (2016). National antibiotic resistance surveillance
report 2016. Retrieved from: http://www.imr.gov.my /images/uploads/NSAR /NSAR
_2016/NSAR_report_2016.pdf
Lopatkin, A. J., Sysoeva, T. A., & You, L. (2016). Dissecting the effects of antibiotics on
horizontal gene transfer: Analysis suggests a critical role of selection dynamics.
BioEssays, 38(12), 1283–1292; Retrieved from:
https://doi.org/10.1002/bies.201600133
McArthur, A. G., Waglechner, N., Nizam, F., Yan, A., Azad, M. A., Baylay, A. J., Wright, G. D.
(2013). The comprehensive antibiotic resistance database. Antimicrobial Agents and
Chemotherapy, 57(7), 3348–57; Retrieved from: https://doi.org/10.1128/AAC.00419-13
Morar, M., & Wright, G. D. (2010). The Genomic Enzymology of Antibiotic Resistance. Annual
Review of Genetics, 44(1), 25–51; Retrieved from: https://doi.org/10.1146 /annurev-
genet-102209-163517
O ’neill, J. (2016). TACKLING DRUG-RESISTANT INFECTIONS GLOBALLY: FINAL REPORT AND
RECOMMENDATIONS THE REVIEW ON ANTIMICROBIAL RESISTANCE. Retrieved from
https://amr-review.org/sites/default/files/160518_Final paper_with cover.pdf
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Odeyemi, O. A., & Ahmad, A. (2017). Antibiotic resistance profiling and phenotyping of
Aeromonas species isolated from aquatic sources. Saudi Journal of Biological Sciences,
24(1), 65–70; Retrieved from: https://doi.org/10.1016/j.sjbs.2015.09.016
Organisation for Economic Co-operation and Development. (n.d.). Antimicrobial resistance;
Retrieved from www.oecd.org/health/antimicrobial-resistance.htm
Pehrsson, E. C., Forsberg, K. J., Gibson, M. K., Ahmadi, S., & Dantas, G. (2013). Novel
resistance functions uncovered using functional metagenomic investigations of
resistance reservoirs. Frontiers in Microbiology, 4, 145; Retrieved from: https://doi.org /
10.3389/fmicb.2013.00145
Perron, G. G., Whyte, L., Turnbaugh, P. J., Goordial, J., Hanage, W. P., Dantas, G., & Desai, M.
M. (2015). Functional characterization of bacteria isolated from ancient arctic soil
exposes diverse resistance mechanisms to modern antibiotics. PloS One, 10(3),
e0069533; Retrieved from: https://doi.org/10.1371/journal.pone.0069533
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