Faculty of Pharmacy

Research Project Title

Antibiotic Resistance Genes
Research Project Code “PHM2201”R4
Research Group N°149

Submitted by:
Student Name: ‫ﻋﺒﺪاﻟﺮﺣﻤﻦ ﻋﻼء ﻋﺒﺪه أﺣﻤﺪ اﻟﺸﻮرﺑﺠﻲ‬ Student ID: 101601327

PHM 2201-Microbiology

Supervised by:
Dr. Aya Ahmed Ali

2020
 List of Contents
List of Abbreviations ................................................................................................i
List of Figures .......................................................................................................... ii
Abstract .....................................................................................................................1
Introduction ..............................................................................................................2
Definition...................................................................................................................2
Mechanisms of Antibiotic Resistance.....................................................................2
Antibiotic Drug Molecule Modification .................................................................................................... 3
Changing Permeability to Antibiotic Drugs ............................................................................................... 5
Development of Efflux Pumps .................................................................................................................. 5
Changing of Antibiotic Target Sites ........................................................................................................... 6
Alteration of Metabolic Pathways ............................................................................................................ 6
Horizontal Gene Transfer .......................................................................................................................... 6

Transmission.............................................................................................................7
Prevention .................................................................................................................7
Examples of Antibiotic Resistance .........................................................................8
Methicillin-Resistant Staphylococcus aureus (MRSA) .............................................................................. 8
Vancomycin Resistant Staphylococcus aureus (VRSA) ............................................................................. 8
Vancomycin-Resistant Enterococcus ........................................................................................................ 8
Carbapenem-Resistant Enterobacteriaceae (CRE) ................................................................................... 8
Rifampicin-Resistant Mycobacterium tuberculosis .................................................................................. 9

Conclusion.................................................................................................................9
References ...............................................................................................................10
 List of Abbreviations

AME Aminoglycoside Modifying Enzyme

CRE Carbapenem-Resistant Enterobacteriaceae
DNA Deoxyribonucleic Acid
EDTA Ethylenediaminetetraacetic acid
HGT Horizontal Gene Transfer
MGE Mobile Genetic Elements
MPC Mutation Prevention Concentration
MRSA Methicillin-Resistant Staphylococcus aureus
M. tuberculosis Mycobacterium tuberculosis
PBP Penicillin Binding Protein
RNA Ribonucleic Acid
S. aureus Staphylococcus aureus
TMP-SMX Trimethoprim/sulfamethoxazole
VRE Vancomycin-Resistant Enterococcus
VRSA Vancomycin Resistant Staphylococcus aureus

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 List of Figures
Figure 1. Summary of antibiotic resistance mechanisms................................................................ 3
Figure 2. Structures of AMEs ......................................................................................................... 4
Figure 3. Bush-Jacoby and Ambler classification system. ............................................................. 4
Figure 4. Difference in cell walls of different types of bacteria. .................................................... 5
Figure 5. Different types of efflux pumps in bacteria..................................................................... 5
Figure 6. Illustration of mutated target. .......................................................................................... 6
Figure 7. Bacterial transduction schematic illustration................................................................... 7

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 Abstract
Resistance of bacteria to antibiotics is an issue crucial to public health and safety. This paper
defines antibiotic resistance, explains its mechanisms, outlines its transmission and prevention and
provides five detailed examples of microbial resistance. Resistance to antibiotics can be intrinsic
meaning that a microorganism naturally has an advantage like the additional cell wall layer in gram
negative bacteria or acquired meaning that selective pressure due to exposure to antibiotics causes
genetic changes. Mechanisms of resistance include eliciting change in drug molecule, changing
bacterial target sites, modifying permeability to drug, or changing metabolic pathways.
Transmission can occur between animals, humans and inanimate objects. Prevention requires
careful use of antibiotics and personal and general hygiene and safety practices. Due to the
pathogenic and contagious nature of many microorganisms the development of efficient antibiotic
drugs is an endeavor that requires significant resources.

Keywords
Antibiotic resistance
Bacterial mutation
CRE
Horizontal gene transfer
MRSA
VRSA

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Introduction
Bacterial resistance to drugs is becoming an increasingly difficult problem to solve, and there is a
great need for understanding the mechanisms and solutions to microbial resistance to drugs in
order to improve healthcare outcomes. Many new dangerous “superbugs” are becoming
increasingly common in hospitals where antibiotic drugs of last resort are used, and this can pose
a serious problem to public health and safety.
Definition
From a microbiological point of view, resistance is defined as a state in which an isolate has a
resistance mechanism rendering it less susceptible than other members of the same species lacking
any resistance mechanism. This definition is valid irrespective of the level of resistance (i.e. low
or high level of resistance) and does not necessarily correlate with clinical resistance. From a
clinical point of view, resistance is defined as a state in which a patient, when infected with a
specific pathogen, is treated with an adequate antimicrobial dosage and administration schedule,
but clinical criteria of cure (at a clinical and/or a microbiological level) are not reached. Cross-
resistance is the resistance of a microorganism to a drug with a similar mechanism of action or
structure to the original drug which it is resistant to1.
Mechanisms of Antibiotic Resistance
Resistance to a class of antibiotics can be attained via numerous biochemical pathways, and a
single bacterium may be able to use a variety of mechanisms of resistance. For example,
fluoroquinolone resistance can happen via three routes, within the same bacteria at the same time
(creating a synergistic effect and increasing the levels of resistance)2.
Alternatively, bacteria can develop a preference for a particular mechanism of resistance over
others. For example, the prevalent resistance mechanism of gram-negative bacteria to to β-lactams
in is the synthesis of β-lactamases, whereas gram-positive organisms develop modifications in
penicillin-binding proteins (PBPs). The difference in the methods of both types of bacteria is due
to the fact that gram-negative bacteria have an outer membrane to control the entry of drug
molecules (B-lactams require porins to reach PBPs on the inner membrane, and B-lactamase
synthesis can target drugs trapped in periplasmic space) while gram-positive bacteria do not have
an outer membrane2.
Antibiotic resistance can be genetic. Chromosomal resistance occurs due to a mutation that
develops spontaneously in a locus that governs antibiotic susceptibility. The presence of antibiotics
acts as a selective pressure for this mutation by suppressing organisms that are susceptible and
favoring mutant growth. These types of mutations occur at a low frequency although are a common
cause of rifampin resistance. The phenotypic consequence of this mutation is a structural change
in drug receptor, for example gene mutation can cause loss of P12 protein on ribosomal 30S
subunit (streptomycin resistance) or loss of PBPs (B-lactam resistance). Extrachromosomal
resistance occurs via plasmids, which are extranuclear genetic materials, can carry genes of
antibiotic resistance. These genes usually control synthesis of enzymes able to destroy
cephalosporins and penicillins, such as B-lactamases, as well as other functions such as active
transport of tetracyclines across bacterial membranes and aminoglycoside phosphorylation2.

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Antibiotic resistance can also be nongenetic. Some bacteria may not be in state of active replication
so are not affected by antibiotics. This is called phenotypic resistance. Some mycobacteria can
persist in tissues for years after infection as multiplication is prevented by host immune system
but, when an opportunity arises to multiply, they regain their susceptibility to antibiotics. Some
types of resistance can be conferred to several generation of a bacterial population but be lost again
due to the loss of a specific structure that is targeted by antibiotics. Some microorganisms
susceptible to penicillin may become cell-wall deficient L forms for a few generations so become
resistant to cell wall inhibitors like cephalosporins and penicillins. Microorganisms can be present
in sites within the host where antibiotics are not active or cannot reach. Gentamicin and other
aminoglycosides cannot treat enteric fevers caused by salmonella as the bacteria enter the host cell.
Legionella pneumophila which cause legionnaires disease are antibiotic resistance due to their
intracellular placement. Figure 1 shows a general summary of antibiotic resistance mechanisms2.

Figure 1. Summary of antibiotic resistance mechanisms.

Antibiotic Drug Molecule Modification

The modification of antibiotic drug molecules can occur via enzyme secretion to modify antibiotic
drugs or to destroy antibiotic drug3.
Enzymes can alter dugs via adenylation (lincosamides and aminoglycosides), acetylation
(streptogramins, chloramphenicol and aminoglycosides) or phosphorylation (aminoglycosides and

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chloramphenicol). For example, aminoglycoside modifying enzymes or AMEs such as
acetyltransferase, phosphotransferase and adenyl transferase can covalently modify amino groups
and hydroxyl groups of aminoglycoside antibiotics (figure 2)3.

Figure 2. Structures of AMEs

Penicillin G-resistant staphylococci produce B-lactamase which breaks down B-lactam antibiotics,
by destroying the amide bond of a B-lactam ring. When new B-lactam antibiotics were released to
the market with less susceptibility to enzymes, newer bacterial enzymes appeared, starting a cycle
of antibiotic-driven adaptive evolution. Thousands of B-lactamases exist and are categorized into
four major classes, based on substrate specificity in the Bush-Jacoby system, and based on amino
acid sequence in the Ambler system (figure 3)3.
Class A enzymes have a serine residue for the catalytic site and are inhibited by clavulanic acid,
and they are active against monobactams with the exception of cephamycins. Class B enzymes
require a metal ion like zinc as a cofactor and are inhibited by ion-chelating agents like EDTA,
they are active against carbapenems. Class C enzymes are active against all penicillins and
cephalosporins and are not inhibited by clavulanic acid3.

Figure 3. Bush-Jacoby and Ambler classification system.

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Changing Permeability to Antibiotic Drugs
Many antibiotics have bacterial intracellular targets or inner membrane targets (gram negative
bacteria). Hydrophilic drugs like tetracyclines, B-lactams and some fluoroquinolones enter the cell
via porins which are aqueous diffusion channels. Examples of major mutated porins are PhoE,
OmpC and OmpF in E. coli and OprD in Pseudomonas aeruginosa. Porin alteration can occur via
change in amount of porin expression, change in type of porin expression and impairment of porin
function4.
Resistant bacteria can undergo outer membrane changes that alter permeability to resist
tetracycline and polymyxin entry and accumulation, or to impair active transport to prevent
amikacin entry. This is partly overcome by use of cell-wall active agents like penicillin. Figure 4
shows the difference between gram-positive and gram-negative bacteria4.

Figure 4. Difference in cell walls of different types of bacteria.

Development of Efflux Pumps

Efflux pumps transport antibiotics outside the bacterial cell and can belong to 5 different families:
the small multidrug resistance family, the multidrug and toxic compound extrusion family, the
major facilitator superfamily, the ATP-binding cassette family and the resistance-nodulation-cell-
division family (figure 5). Many gram-positive and gram-negative bacteria have acquired this
method to resist mostly tetracyclines but also fluoroquinolones and macrolides4.

Figure 5. Different types of efflux pumps in bacteria.

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Changing of Antibiotic Target Sites
Protection of target site involves the synthesis of proteins which can compete for drug, binding
sites. Modification of antibiotic target site can occur via point mutations in genes that encode
binding site, enzymatic alteration of target site and or replacement of original binding site (figure
6)1.
Erythromycin resistant bacteria have an altered receptor in the 50S ribosomal subunit, this is due
to 23S ribosomal RNA methylation. Altered PBPs can cause resistance to penicillins and
cephalosporins1.

Figure 6. Illustration of mutated target.

Alteration of Metabolic Pathways

Development of alternative metabolic pathways to bypass metabolic reaction inhibited by
antibiotic drug can be realized by overproducing the drug target (such as trimethoprim-
sulfamethoxazole or TMP-SMX resistance), modification of bacterial enzymes so as to carry out
metabolic functions but not be affected by drug (sulfonamide-resistant bacteria do not need
extracellular PABA but can use preformed folic acid and trimethoprim-resistant bacteria have
modified dihydrofolic acid reductase)2.
Horizontal Gene Transfer
Is an important aspect of bacterial evolution and a major cause of antibiotic resistance. It is much
quicker than mutation and consists of transformation (incorporation of naked DNA), transduction
(phage mediated transfer) (figure 7) and conjugation which occurs frequently in the
gastrointestinal tract and requires cell-to-cell contact. Mobile genetic elements (MGEs) act as
vehicles to transport genetic information, and direct chromosome-to-chromosome sharing can also
occur. Common MGEs are transposons, integrons and plasmids. Plasmids are extrachromosomal
DNA that can replicate independently and contain genes that are helpful to microorganisms under
certain conditions. Transposons are DNA sequences that can change their position within a single
genome or move genetic material between plasmid and chromosome. Integrons are elements that
can carry and transfer genes amongst different species of bacteria. They consist of a gene encoding
an integrase enzyme, a recombination site and a promoter that leads transcription of one or more
“gene cassettes” that have been caught by the integron. Gene cassettes usually encode
an individual protein. Integrase catalyzes the excision of a cassette from an integron. The cassette
is then acquired by another integron via its integrase enzyme. If a gene
cassette is obtained by an integron within a conjugative plasmid, it can be transferred to other cells
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through conjugation. Integrons can carry genes that encode resistance against several antibiotics,
thus overuse of a non-crucial drug can lead to resistance against multiple drugs3.

Figure 7. Bacterial transduction schematic illustration.

Transmission
Animals can receive antibiotics thus develop and carry antibiotic resistant bacteria. Vegetables can
become contaminated with antibiotic-resistant bacteria from fertilizer made of animal. Antibiotic-
resistant bacteria can then spread to humans via food or direct interaction with animals. Bacteria
build resistance to antibiotics as a natural, adaptive mechanism, and resistant strains can then
spread one person to another. Hospitals are hotbeds for transmission of resistant bacterial strains,
especially via contaminated surfaces such as door handles and medical equipment such as
stethoscopes and catheters. Humans can also acquire antibiotic resistance from foreign travel to an
area with high resistance prevalence3
Prevention
Maintenance of high drug levels in patients to inhibit the original bacterial population as well as
first step mutants. Administration of two antibiotics, which are not cross-resistant, simultaneously
thus delaying the appearance of mutations to resist either drug. Avoiding exposure of valuable
drugs to microorganisms such as limiting their use to hospitals. Use of bacteriostatic rather than
bactericidal drugs so as to apply the lowest antibacterial load possible for the immune system to
be able to elimination the infection. Some antibiotics are concentration dependent (such as

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bactericidals targeting protein synthesis) or time dependent such as bacteriostatics which target
cell wall structure and they have to applied for an appropriate period of time at levels best above
MPC. The mutant prevention concentration antibiotic drug level above which bacterial mutations
that occur in a single step cannot develop further, and development of resistance requires two or
more concurrent mutations of which there is a rarer chance. The drug concentration between the
mutant prevention concentration and the minimum inhibitory concentration is called the mutant
selection window3.
Examples of Antibiotic Resistance
Methicillin-Resistant Staphylococcus aureus (MRSA)
Initially isolated in hospitals, strains of MRSA have become spread out within the community,
thwarting the managing of this occasionally-fatal pathogen. Methicillin resistance in S. aureus is
facilitated by the mecA gene, which encodes for a new PBP called PBP-2a. When MRSA is
exposed to methicillin it inactivates the four PBPs usually present. PBP-2a, which has a low
binding affinity for methicillin, assumes the roles of the four PBPs, allowing cell growth.
Vancomycin is the drug of choice for MRSA infections. Combinations of vancomycin with ss-
lactam drugs has the potential to be synergistic against MRSA strains, even those with weak
susceptibility to vancomycin4.
Vancomycin Resistant Staphylococcus aureus (VRSA)
Vancomycin is a glycopeptide antibiotic used to treat gram-positive infections. MRSA strains with
decreased susceptibility to vancomycin (vancomycin intermediate-resistant S. aureus [VISA]) and
newly with completed vancomycin resistance (VRSA) have been found even though vancomycin
is used as a drug of last resort. VRSA carry the transposon Tn1546, obtained from vancomycin-
resistant Enterococcus faecalis, which is capable of altering cell wall structure and metabolism5.
Vancomycin-Resistant Enterococcus

Enterococci are not usually extremely virulent pathogens. But, antibiotic resistance makes difficult
the treatment of enterococcal infections. Acquired resistance to large concentrations of
glycopeptide antibiotics, especially vancomycin, has aggravated this issue. The mechanism
underlying this resistance is due to the encoding of an alternative biosynthetic pathway in order to
produce cell wall precursors that vancomycin binds to very poorly. Normal peptidoglycan
prcursors have D-alnyl-D-alanine dipeptide termini but, resistant enterococci have D-alanyl-D-
lactate, to which vancomycin binds to with a much lower affinity. This biosynthetic pathway is
triggered when VanS protein which is a histidine kinase sensor and VanR protein which is a
response regulator sense the presence of an antibiotic, in which case VanS autophophorylates the
phosphorylates VanR. Doxycycline, chloramphenicol, and rifampin in different combinations can
be used to treat VRE such as Enterococcus faecalis and Enterococcus faecium3.

Carbapenem-Resistant Enterobacteriaceae (CRE)

Enterobacteriaceae that are resistant to at least a single carbapenem drug or produce a
carbapenemase are called CRE, like the following species: Proteus, Morganella and Providencia

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Enterobacteriaceae can be resistant to carbapenems by enzyme production, efflux pumps and
porin mutations. Three major enzyme groups cause most cases of resistance: KPC (Klebsiella
pneumoniae carbapenemase) (Ambler class A), MBLs (Metallo-ß-Lactamases) (Ambler class B)
and OXA-48-like (Ambler class D). Enterobacteriaceae can also make ß-lactamases like AmpC-
type ß-lactamases, which do not degrade carbapenems but bond with the drug molecule, stopping
it from gaining access to its target. Antibiotics like aminoglycosides, polymyxin(s), tigecycline
can be used to treat CRE4.
Rifampicin-Resistant Mycobacterium tuberculosis
The mode of action of rifampicin in M. tuberculosis is by binding to the β-subunit of the RNA
polymerase, inhibiting the elongation of messenger RNA. The majority of rifampicin-resistant
clinical isolates of M. tuberculosis harbor mutations in the rpoB gene that codes for the β-subunit
of the RNA polymerase. As a result of this, conformational changes occur that decrease the affinity
for the drug and results in the development of resistance7.
Conclusion
Antibiotic resistance can be acquired through exposure to environmental factors or intrinsically, it
can also be genetic or nongenetic and chromosomal or non-chromosomal. Mechanisms of
resistance include destroying or modifying drug molecules, producing different pathways to allow
cell growth that are non-susceptible to drugs, altering cell membrane permeability and
development of efflux pumps. People should be careful in personal or hospital settings to maintain
adequate hygiene and cleanliness to prevent transmission and should be careful in interactions with
animals and selection of food.

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References
1. Munita J, Arias C. Mechanisms of Antibiotic Resistance. Microbiology Spectrum. 2016;4(2).
2. C Reygaert W. An overview of the antimicrobial resistance mechanisms of bacteria. AIMS
Microbiology. 2018;4(3):482-501.
3. Peterson E, Kaur P. Antibiotic Resistance Mechanisms in Bacteria: Relationships Between
Resistance Determinants of Antibiotic Producers, Environmental Bacteria, and Clinical
Pathogens. Frontiers in Microbiology. 2018
4. David M, Boyle-Vavra S, Zychowski D, Daum R. Methicillin-Susceptible Staphylococcus
aureus as a Predominantly Healthcare-Associated Pathogen: A Possible Reversal of Roles?.
PLoS ONE. 2011;6(4):e18217.
5. Gardete S, Tomasz A. Mechanisms of vancomycin resistance in Staphylococcus aureus. Journal
of Clinical Investigation. 2014;124(7):2836-2840.
6. Palomino J, Martin A. Drug Resistance Mechanisms in Mycobacterium tuberculosis.
Antibiotics. 2014;3(3):317-340.

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