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Schematic representation of how antibiotic resistance evolves via natural selection. The top
section represents a population of bacteria before exposure to an antibiotic. The middle section shows the
population directly after exposure, the phase in which selection took place. The last section shows the
distribution of resistance in a new generation of bacteria. The legend indicates the resistance levels of
individuals.
Antibiotic resistance can be a result of horizontal gene transfer, and also of unlinked point mutations in
the pathogen genome and a rate of about 1 in 108 per chromosomal replication. The antibiotic action
against the pathogen can be seen as an environmental pressure; those bacteria which have a mutation
allowing them to survive will live on to reproduce. They will then pass this trait to their offspring, which
will result in a fully resistant colony.
Several studies have demonstrated that patterns of antibiotic usage greatly affect the number of resistant
organisms which develop. Overuse of broad-spectrum antibiotics, such as second- and third-generation
cephalosporins, greatly hastens the development of methicillin resistance. Other factors contributing
towards resistance include incorrect diagnosis, unnecessary prescriptions, improper use of antibiotics by
patients, the impregnation of household items and children's toys with low levels of antibiotics, and the
administration of antibiotics by mouth in livestock for growth promotion. Also unsound practices in the
pharmaceutical manufacturing industry can contribute towards the likeliness of creation antibiotic
resistant strains. Researchers have recently demonstrated the bacterial protein LexA may play a key role
in the acquisition of bacterial mutations.
Drug resistance occurs in several classes of pathogens:
1. bacteria—antibiotic resistance
2. endoparasites
3. viruses—resistance to antiviral drugs
4. fungi
5. cancer cells
Mechanisms
The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are:
1. Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some
penicillin-resistant bacteria through the
production of β-lactamases. Antibiotic
modification is the best known: the
resistant bacteria retain the same
sensitive target as antibiotic sensitive
strains, but the antibiotic is prevented
from reaching it. This happens, for
example, with lactamases the
lactamase enzymatically cleaves the four
membered lactam ring, rendering the
antibiotic inactive. Over 200 types of
lactamase have been described (table).
Most lactamases act to some degree
against both penicillins and
cephalosporins; others are more specific
namely, cephalosporinases (for
example, AmpC enzyme found in
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Molecular Mechanism of Drug Resistence
2. Alteration of target site: e.g. alteration of PBP—the binding target site of penicillins—in MRSA
and other penicillin-resistant bacteria. Alterations in the primary site of action may mean that the
antibiotic penetrates the cell and reaches the target site but is unable to inhibit the activity of the
target because of structural changes in the molecule. Enterococci are regarded as being inherently
resistant to cephalosporins because the enzymes responsible for cell wall synthesis (production of
the polymer peptidoglycan) known as penicillin binding proteins have a low affinity for them
and therefore are not inhibited. Most strains of Streptococcus pneumoniae are highly susceptible to
both penicillins and cephalosporins but can acquire DNA from other bacteria, which changes the
enzyme so that they develop a low affinity for penicillins and hence become resistant to inhibition
by penicillins.3 The altered enzyme still synthesises peptidoglycan but it now has a different
structure.4 Mutants of Streptococcus pyogenes that are resistant to penicillin and express altered
penicillin binding proteins can be selected in the laboratory, but they have not been seen in
patients, possibly because the cell wall can no longer bind the anti-phagocytic M protein.
3. Alteration of metabolic pathway: e.g. some sulfonamide-resistant bacteria do not require para-
aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic
acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing
preformed folic acid.
4. Quick Efflux: Active efflux is a mechanism responsible for extrusion of toxic substances and
antibiotics outside the cell, this is considered to be a vital part of xenobiotic metabolism. This
mechanism is important in medicine as it can contribute to bacterial antibiotic resistance.Efflux
systems function via an energy-dependent mechanism (Active transport) to pump out unwanted
toxic substances through specific efflux pumps. Some efflux systems are drug-specific while
others may accommodate multiple drugs, and thus contribute to bacterial multidrug resistance
(MDR).
There are three known mechanisms of fluoroquinolone resistance. Some types of efflux pumps can act to
decrease intracellular quinolone concentration. In gram-negative bacteria, plasmid-mediated resistance
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Molecular Mechanism of Drug Resistence
genes produce proteins that can bind to DNA gyrase, protecting it from the action of quinolones. Finally,
mutations at key sites in DNA gyrase or Topoisomerase IV can decrease their binding affinity to
quinolones, decreasing the drug's effectiveness.
Bacterial efflux pumps are proteinaceous transporters localized in the cytoplasmic membrane of
all kinds of cells. They are active transporters meaning that they require a source of chemical energy to
perform their function. Some are primary active transporters utilizing Adenosine triphosphate hydrolysis
as a source of energy, while others are secondary active transporters (uniporters, symporters or
antiporters) in which transport is coupled to an electrochemical potential difference created by pumping
out hydrogen or sodium ions outside the cell.Bacterial efflux transporters are classified into five major
superfamilies, based on the amino acid sequence and the energy source used to export their substrates:
1. The major facilitator superfamily (MFS);
2. The ATP-binding cassette superfamily (ABC);
3. The small multidrug resistance family (SMR);
4. The resistance-nodulation-cell division superfamily (RND); and
5. The Multi antimicrobial extrusion protein family (MATE).
Of these only the ABC superfamily are primary transporters, the rest being secondary transporters
utilizing proton or sodium gradient as a source of energy. While MFS dominates in Gram positive
bacteria , the RND family is unique to Gram-negatives.
In the case of imipenem resistant Pseudomonas aeruginosa, lack of the specific D2 porin confers
resistance, as imipenem cannot penetrate the cell. This mechanism is also seen with low level resistance to
fluoroquinolones and aminoglycosides. Increased efflux via an energy-requiring transport pump is a well
recognised mechanism for resistance to tetracyclines and is encoded by a wide range of related genes, such
as tet(A), that have become distributed in the enterobacteriaceae.
Function
Although antibiotics are the most clinically important substrates of efflux systems, it is probable that most
efflux pumps have other natural physiological functions. Examples include:
• The E.coli AcrAB efflux system which has a physiologic role of pumping out bile acids and fatty
acids to lower their toxicity.
• The MFS family Ptr pump in Streptomyces pristinaespiralis appears to be an autoimmunity pump
for this organism when it turns on production of pristinamycins I and II.
• The AcrAB–TolC system in E.coli is suspected to have a role in the transport of the calcium-
channel components in the E. coli membrane.
• The MtrCDE system plays a protective role by providing resistance to faecal lipids in rectal
isolates of Neisseria gonorrhoeae.
• The AcrAB efflux system of Erwinia amylovora is important for this organism's virulence, plant
(host) colonization and resistance to plant toxins.
The ability of efflux systems to recognize a large number of compounds other than their natural substrates
is probably because substrate recognition is based on physicochemical properties, such as hydrophobicity,
aromaticity and ionizable character rather than on defined chemical properties, as in classical enzyme-
substrate or ligand-receptor recognition. Because most antibiotics are amphiphilic molecules - possessing
both hydrophilic and hydrophobic characters, they are easily recognized by many efflux pumps.
Impact on antimicrobial resistance
The impact of efflux mechanisms on antimicrobial resistance is large, this is usually attributed to the
following:
• The genetic elements encoding efflux pumps may be encoded on chromosomes and/or plasmids,
thus contributing to both intrinsic (natural) and acquired resistance respectively. As an intrinsic
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Molecular Mechanism of Drug Resistence
mechanism of resistance, efflux pump genes can survive a hostile environment ( for example in
the presence of antibiotics) which allows for the selection of mutants that over-express these
genes. Being located on transpoable genetic elements as plasmids or transposons is also
advantageous for the microorganisms as it allows for the easy spread of efflux genes between
distant species.
• Antibiotics can act as inducers and regulators of the expression of some efflux pumps.
• Expression of several efflux pumps in a given bacterial species may lead to a broad spectrum of
resistance when considering the shared substrates of some multi-drug efflux pumps, where one
efflux pump may confer resistance to a wide range of antimicrobials.
agricultural hosts throughout the world. There is support for the notion that determinants of antibiotic
resistance were not derived from the currently observed bacterial host in which the resistance plasmid is
seen. DNA sequencing studies of lactamases and aminoglycoside inactivating enzymes show that despite
similarities within the protein studies of the two families, there are substantial sequence differences. 12 13
As the evolutionary time frame has to be less than 50 years it is not possible to derive a model in which
evolution could have occurred by mutation alone from common ancestral genes. They must have been
derived from a large and diverse gene pool presumably already occurring in environmental bacteria. Many
bacteria and fungi that produce antibiotics possess resistance determinants that are similar to those found
in clinical bacteria.10 Gene exchange might occur in soil or, more likely, in the gut of humans or animals.
It has been discovered that commercial antibiotic preparations contain DNA from the producing organism,
and antibiotic resistance gene sequences can be identified by the polymerase chain reaction.14
Genes either exist in nature already or can emerge by mutation rapidly. Rapid mutation has been seen
with (a) the TEM lactamase, resulting in an extension of the substrate profile to include third generation
cephalosporins (first reported in Athens in 1963, one year after the introduction of ampicillin) and (b) the
IMI-1 lactamase (reported from a Californian hospital before imipenem was approved for use in the
United States).15 The selection pressure is heavy, and injudicious use of antibiotics, largely in medical
practice, is probably responsible although agricultural and veterinary use contributes to resistance in
human pathogens. The addition of antibiotics to animal feed or water, either for growth promotion or,
more significantly, for mass treatment or prophylaxis (or both treatment and prophylaxis) in factory
farmed animals, is having an unquantified effect on resistance levels.16 Bacteria clearly have a wondrous
array of biochemical and genetic systems for ensuring the evolution and dissemination of antibiotic
resistance.
The bla gene encoding the TEM-1 ß-lactamase is the most encountered AmpR marker used in molecular
biology (pBR and pUC plasmids). TEM-1 is a widespread plasmidic ß-lactamase that attacks narrow-
spectrum cephalosporins, cefamandole, and cefoperazone and all the anti-gram-negative-bacterium
penicillins except temocillin. Aminothiazol chephalosporins, cephamycins, monobactams and
carbapenems are resistant to its action. It belongs to the Bush-Jacoby-Medeiros group 2b and the
molecular class A. The TEM-1 enzyme was first reported from an E. coli isolate in 1965 and is now the
commonest ß-lactamase found in enterobacteriaceae. Resistance in more than 50% of AmpR E. coli
clinical isolates is due to TEM-1. Most extended-spectrum ß-lactamases (ESBLs) derive from TEM-1,
TEM-2 and SHV-1 by mutations generating 1- to 4-amino-acid sequence substitutions.
2. Aminoglycoside resistance
Aminoglycosides (Streptomycin, kanamycin, tobramycin, amikacin,...) are compounds that are
characterized by the presense of an aminocyclitol ring linked to aminosugars in their structure. Their
bactericidal activity is attributed to the irreversible binding to the ribosomes although their interaction
with other cellular structures and metabolic processes has also been considered. They have a broad
antimicrobial spectrum. They are active against aerobic and facultative aerobic Gram-negative bacilli and
some Gram-positive bacteria of which staphylococci. Aminoglycosides are not active against anaerobes
and rikettsia. Spectinomycin which is an aminocyclitol devoided of aminosugars is by extension included
in the familiy of aminoglycosides. It also differs from them by its bacteriostatic ativity and by its way of
action. Spectinomycin acts on protein synthesis during the mRNA-ribosome interaction and it does not
lead to mistranslation like aminoglycosides do. Three mechanisms of resistance have been recognized,
namely ribosome alteration, decreased permeability, and inactivation of the drugs by aminoglycoside
modifying enzymes. The latter mechanism is of most clinical importance since the genes encoding
aminoglycoside modifying enzymes can be disseminated by plasmids or transposons.
Ribosome alteration
High level resistance to streptomycin and spectinomycin can result from single step mutations in
chromosomal genes encoding ribosomal proteins: rpsL (or strA), rpsD (or ramA or sud2), rpsE (eps or spc
or spcA). Mutations in strC (or strB) generate a low-level streptomycin resistance.
Decreased permeability
Absence of or alteration in the aminoglycoside transport system, inadequate membrane potential,
modification in the LPS (lipopolysacchaccarides) phenotype can result in a cross resistance to all
aminoglycosides.
Inactivation of aminoglycosides
These enzymes are classified into three major classes according to the type modification: AAC
(acetyltransferases), ANT (nucleotidyltransferases or adenyltransferases), APH (phosphotransferases).
This classification was extensively reviewed by Shaw et al. (1993).
Commonly used aminoglycoside resistance markers in molecular biology
ant(3'')-Ia (synonyms: aadA, aad(3'')(9))confers resistance to streptomycin and spectinomycin. The gene
has been found in association with several transposons (Tn7, Tn21, ...) and is ubiquitous among gram-
negative bacteria.aph(3')-II (synonyms: aphA-2, nptII) confers resistance to Km (Kanamycin), Neo
(Neomycin), Prm (Paromomycin), Rsm (Ribostamycin), But (Butirosin), GmB (GentamycinB). This gene
is rarely found in clinical isolates. aph(3')-II is associated with transposon Tn5 and observed in gram-
negative bacteria and Pseudomonas sp. However, its relative abundance in environmental KanR isolates
seems to be low (Recorbet et al., 1992; Leff et al., 1993; Smalla et al., 1993).aph(3')-III (synonyms:
nptIII) confers resistance to Km (Kanamycin), Neo (Neomycin), Prm (Paromomycin), Rsm
(Ribostamycin), Lvdm (Lividomycin), But (Butirosin), GmB (GentamycinB). Amk (Amikacin) and Isp
(Isepamicin) are also modified in vitro, but according to the susceptibility standards established by
NCCLS resistance is only expressed at a low level by many strains. aph(3')-III is commonly distributed
among gram-positive bacteria but has also been observed in Campylobacter spp.
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Molecular Mechanism of Drug Resistence
nptIII is not frequent in molecular biology but can be found on some Agrobacterium vectors for plant
transformation (Bevan, 1984).
3. Tetracycline resistance
Tetracyclines (tetracycline, doxycycline, minocycline, oxtetracycline) are antibiotics which inhibit the
bacterial growth by stopping protein synthesis. They have been widely used for the past forty years as
therapeutic agent in human and veterinary medicine but also as growth promotor in animal husbandry.
The emergence of bacterial resistances to these antibiotics has nowadays limited their use. Three different
specific mechanisms of tetracycline resistance have been identified so far: tetracycline efflux, ribosome
protection and tetracycline modification.
Tetracycline efflux is achieved by an export protein from the major facilitator superfamily (MFS). The
export protein was shown to function as an electroneutral antiport system which catalyzes the exchange of
tetracycline-divalent-metal-cation complex for a proton. In Gram-negative bacteria the export protein
contains 12 TMS (transmembrane fragments) whereas in Gram-positive bacteria it displays 14 TMS.
Ribosome protection is mediated by a soluble protein which shares homolgy with the GTPases
participating in protein synthesis, namely EF-Tu and EF-G. The third mechanism involves a cytoplasmic
protein that chemically modifies tetracycline. This reaction takes only place in the presence of oxygen and
NADPH and does not function in the natural host (Bacteroides). The two first mechanisms are the most
widespread and most of their genes are normally acquired via transferable plasmids and/or transposons.
These two mechanisms were observed both in aerobic and anaerobic Gram-negative or Gram-positive
bacteria demonstrating their wide distribution among the bacterial kingdom. To date, about sixty-one
tetracycline resistance genes have been sequenced and thirty-two classes of genes identified in non-
producers and producers (Streptomyces). Each new class is identified by its inability to hybridize with any
of the known tet genes under stringent conditions. A new nomenclature for the resistance determinants
has been proposed for the future with the S. B. Levy group to coordinate the naming of the
Commonly used tetracycline resistance markers in molecular biology
Several tetracycline resistance determinants are currently used in molecular biology. The most
encountered are the tetA genes of classes A (RP1, RP4 or Tn1721 derivatives), B (Tn10 derivatives) and
C (pSC101 or pBR322 derivatives) encoding a tetracycline efflux system. These genes are regulated by a
repressor protein (TetR). This feature has also been exploited to construct tightly regulated, high level
mammalian expression systems by using the regulatory elements of the Tn10 tetracycline operon (Tet-
OffTM and Tet-OnTM Expression Systems & Cell Lines,Clontech).The tetM gene from Tn916 which can be
expressed both in Gram-positive and Gram-negative bacteria is also frequently used. Several
Bacteroides/Escherichia shuttle vectors contain the tetQ gene. tetM and tetQ encode a soluble protein
protecting the ribosome from the inhibiting effects of tetracycline. The distribution of these genes is given
in the pages relating to the determinant classification.
Some Resistant pathogens
Staphylococcus aureus:
Staphylococcus aureus (colloquially known as "Staph aureus" or a Staph infection) is one of the major
resistant pathogens. Found on the mucous membranes and the skin of around a third of the population, it
is extremely adaptable to antibiotic pressure. It was the first bacterium in which penicillin resistance was
found—in 1947, just four years after the drug started being mass-produced. Methicillin was then the
antibiotic of choice, but has since been replaced by oxacillin due to significant kidney toxicity. MRSA
(methicillin-resistant Staphylococcus aureus) was first detected in Britain in 1961 and is now "quite
common" in hospitals. MRSA was responsible for 37% of fatal cases of blood poisoning in the UK in
1999, up from 4% in 1991. Half of all S. aureus infections in the US are resistant to penicillin, methicillin,
tetracycline and erythromycin.
Methicillin Resistant Staphylococcus Aureus (MRSA) is acknowledged to be a human commensal and
pathogen. MRSA has been found in cats, dogs and horses, where it can cause the same problems as it does
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Molecular Mechanism of Drug Resistence
in humans. Owners can transfer the organism to their pets and vice-versa, and MRSA in animals is
generally believed to be derived from humans.
This left vancomycin as the only effective agent available at the time. However, strains with intermediate
(4-8 ug/ml) levels of resistance, termed GISA (glycopeptide intermediate Staphylococcus aureus) or
VISA (vancomycin intermediate Staphylococcus aureus), began appearing in the late 1990s. The first
identified case was in Japan in 1996, and strains have since been found in hospitals in England, France
and the US. The first documented strain with complete (>16 ug/ml) resistance to vancomycin, termed
VRSA (Vancomycin-resistant Staphylococcus aureus) appeared in the United States in 2002.
A new class of antibiotics, oxazolidinones, became available in the 1990s, and the first commercially
available oxazolidinone, linezolid, is comparable to vancomycin in effectiveness against MRSA.
Linezolid-resistance in Staphylococcus aureus was reported in 2003.
CA-MRSA (Community-acquired MRSA) has now emerged as an epidemic that is responsible for rapidly
progressive, fatal diseases including necrotizing pneumonia, severe sepsis and necrotizing fasciitis.
Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequently identified antimicrobial drug-
resistant pathogen in US hospitals. The epidemiology of infections caused by MRSA is rapidly changing.
In the past 10 years, infections caused by this organism have emerged in the community. The 2 MRSA
clones in the United States most closely associated with community outbreaks, USA400 (MW2 strain,
ST1 lineage) and USA300, often contain Panton-Valentine leukocidin (PVL) genes and, more frequently,
have been associated with skin and soft tissue infections. Outbreaks of community-associated (CA)-
MRSA infections have been reported in correctional facilities, among athletic teams, among military
recruits, in newborn nurseries, and among active homosexual men. CA-MRSA infections now appear to
be endemic in many urban regions and cause most CA-S. aureus infections.
Streptococcus and Enterococcus
Streptococcus pyogenes (Group A Streptococcus: GAS) infections can usually be treated with many
different antibiotics. Early treatment may reduce the risk of death from invasive group A streptococcal
disease. However, even the best medical care does not prevent death in every case. For those with very
severe illness, supportive care in an intensive care unit may be needed. For persons with necrotizing
fasciitis, surgery often is needed to remove damaged tissue. Strains of S. pyogenes resistant to macrolide
antibiotics have emerged, however all strains remain uniformly sensitive to penicillin.
Resistance of Streptococcus pneumoniae to penicillin and other beta-lactams is increasing worldwide. The
major mechanism of resistance involves the introduction of mutations in genes encoding penicillin-
binding proteins. Selective pressure is thought to play an important role, and use of beta-lactam antibiotics
has been implicated as a risk factor for infection and colonization. Streptococcus pneumoniae is
responsible for pneumonia, bacteremia, otitis media, meningitis, sinusitis, peritonitis and arthritis.
Penicillin-resistant pneumonia caused by Streptococcus pneumoniae (commonly known as
pneumococcus), was first detected in 1967, as was penicillin-resistant gonorrhea. Resistance to penicillin
substitutes is also known as beyond S. aureus. By 1993 Escherichia coli was resistant to five
fluoroquinolone variants. Mycobacterium tuberculosis is commonly resistant to isoniazid and rifampin
and sometimes universally resistant to the common treatments. Other pathogens showing some resistance
include Salmonella, Campylobacter, and Streptococci.
Enterococcus faecium is another superbug found in hospitals. Penicillin-Resistant Enterococcus was seen
in 1983, vancomycin-resistant enterococcus (VRE) in 1987, and Linezolid-Resistant Enterococcus (LRE)
in the late 1990s.
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a highly prelevant opportunistic pathogen. One of the most worrisome
characteristics of P. aeruginosa consists in its low antibiotic susceptibility. This low susceptibility is
attributable to a concerted action of multidrug efflux pumps with chromosomally-encoded antibiotic
resistance genes (e.g. mexAB-oprM, mexXY etc) and the low permeability of the bacterial cellular
envelopes. Besides intrinsic resistance, P. aeruginosa easily develop acquired resistance either by
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Molecular Mechanism of Drug Resistence
• Resistance occurs by means of four main mechanisms more than one may be present in a
single bacterium
• Resistance mechanisms have probably evolved from genes present in organisms producing
antibiotics
• Resistance genes occur not only in bacteria that carry disease but also in commensal
bacteria, to which we are continuously exposed and which are found in food, the
environment, and animals
• The plethora of genetic mechanisms for evolution and reassortment of antibiotic resistance
genes ensures that useful genes will be disseminated rapidly
• Action must be taken to slow the rate of evolution and spread of antibiotic resistance
genes, in which the biggest single factor is the amount of antibiotics used in human
medicine and agriculture