Drug Resistance Mechanism

1. Santosh Yadav
Santosh Yadav
M.Sc. Clinical Microbiology
Dept. of Microbiology
Institute of Medicine
Tribhuvan Univarsity Teaching Hospital, Nepal

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Brief History of Antibiotics

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Mahon CR, Lehman DC, Manuselis G. A Textbook of Diagnostic Microbiology 5th Ed

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Definition :-
 Drug resistance (Antimicrobial resistance) is the
reduction in effectiveness of a drug in curing a disease.
 When the drug is not intended to kill or inhibit a
pathogen, then the term is equivalent to dosage failure
or drug tolerance.
 Antibiotic resistance is the ability of bacteria to resist
the effect of an antibiotics, i.e. the bacteria is not killed
or their growth is not stopped.
 Resistance bacteria survive exposure to the antibiotics
and continue to multiply in the body, potentially
causing more harm and spreading to other animals or
people.

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Evolution of resistance
 Origin of resistance genes could be due a natural process
 The resistance genes are maintained in nature because of the
presence of antibiotics producing bacteria in soil
 These antibiotics act on other bacterial species other than the
producer bacteria
 There has to be a mechanism of protection in the host bacteria
against the antibiotics that it produces
 Which could be the source of genes encoding resistance
BennettPM,HoweTGB.BacterialandBacteriophage..In:Topley&wWilson’smicrobiologyandmicrobialinfections.

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Settings that promote drug-resistance
Day-care centers
Long term care
facilities
Homeless shelters
Jails

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Locations contributing to Drug
Resistance
 Intensive care
units
 Oncology units
 Dialysis units
 Rehab units
 Transplant units
 Burn units

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Development of resistance to newly introduced
antimicrobials
Agent Year of FDA approval First reported
resistance
Penicillin 1943 1940
Streptomycin 1947 1947
Tetracycline 1952 1956
Methicillin 1960 1961
Nalidixic acid 1964 1966
Gentamycin 1967 1969
Vancomycin 1972 1987
Cefotaxime 1981 1981(AmpC)
1983(ESBL)
Linezolid 2000 1999
BushK.ASMnews..2004;70:282-287

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Multidrug-resistant organisms (MDROs)
 Multiple drug resistance or Multidrug- resistance is a
condition enabling a disease causing organism to resist
distinct drugs or chemicals of a wide variety of structure
and function targeted to eradicate the organism
Multidrug-resistance (MDR)
• Multidrug-resistant organisms are bacteria that have become
resistant to certain antibiotics, and these antibiotics can no
longer be used to control or kill the bacteria

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MDR is defined as acquired non-susceptibility to at least one agent in three or
more antimicrobial categories.
European Society of Clinical Microbiology and Infectious Diseases, CMI.,2012; 18: 268–281

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Multi-resistance
 Multi-resistance
 Multi-resistance to different antibiotics generally
results from a combination of different independent
mechanisms of resistance
 P. aeruginosa is a type of multi-resistant bacteria
 It is resistant to β-lactams, (natural resistance)
including third-generation cephalosporins,
quinolones, chloramphenicol, and Tetracycline
 Methicillin-resistant strains have become resistant
to most antibiotics and with a frequency of high
resistance (acquired)

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 Cross-resistance
 Cross-resistance occurs generally in antibiotics of
the same family, to which bacteria may not have
been exposed
 Cross-resistance between penicillin's, more widely
between all the β- lactams
 Colistin and polymyxin B
 Ciprofloxacin and ofloxacin

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Biologic versus clinical resistance
 Development of bacterial resistance to antimicrobial
agents to which they were originally susceptible requires
alterations in the cell’s physiology or structure. Biologic
resistance refers to changes that result in the organism
being less susceptible to a particular antimicrobial agent
than has been previously observed.
 When antimicrobial susceptibility has been lost to such an
extent that the drug is no longer effective for clinical use,
the organism has achieved clinical resistance.

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Antibiotics promote resistance
 If a patient taking a course of
antibiotic treatment does not
complete it
•Or forgets to take the doses
regularly, then resistant strains get
a chance to build up

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Antibiotic Pressure and Resistance in Bacteria
 The antibiotics also kill innocent
non-pathogens
 This reduces the competition for
the resistant pathogens
 The use of antibiotics also promotes
antibiotic resistance in non-
pathogens too
 These non-pathogens may later pass
their resistance genes on to
pathogens

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Transposons & Integrons
 Resistance genes are often associated with transposons,
genes that easily move from one bacterium to another
• Many bacteria also possess integrons, pieces of DNA that
accumulate new genes
• Gradually a strain of a bacterium can build up a whole
range of resistance genes
• This is multiple resistance
• These may then be passed on in a group to other strains or
other species

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Plasmids

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Selective pressure :-Microbes that carry
resistance genes survive to replicate themselves. The
progeny of these resistant microbes will eventually
become the dominant type.
Mutation: when microbes replicate themselves,
genetic mutations can occur. Sometimes, these
mutations can lead to the creation of a microbe with
genes that aid it in surviving exposure to antimicrobial
agents.
Gene transfer: microbes can also acquire genes from
other microbes. Genes that have drug-resistant
qualities can be transferred between microbes easily.

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Mechanism of antibiotic resistance
Intrinsic (natural resistance) resistance:-
 Innate ability of a bacterial species to resist the activity of a
particular antimicrobial agent through inherent structural or
functional characteristics, allowing tolerance to a particular
drug or antimicrobial class.
 Intrinsic mechanisms of resistance are an innate characteristic
of the microorganism and are transmitted to progeny
vertically(i.e. during cell division)
 Eg. Anaerobes are intrinsically resistant to aminoglycosides.

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Acquired resistance:-
 Acquired mechanisms of resistance are caused by changes
in the usual genetic makeup of a microorganism and by the
results of altered cellular physiology and structure.
 These bacteria develop resistance due to mutation
following exposure to antimicrobial agents or receive
resistant property passively from resistant organisms.
 Eg. Methicillin resistance in S. aureus

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Mechanism of intrinsic resistance
1.Impermeability ,
2. Biofilm ,
3. Efflux .and
4.Enzymatic inactivation and destruction

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1.Impermeability
 For antibiotics to affect internal cellular processes, they must
penetrate the cell wall of bacteria to reach their target.
Influx(entry) of antibiotics through the cell wall depends on
the chemical nature of the antibiotic and the structural
characteristics of the cell wall.
 Eg. gram-negative bacteria are intrinsic resistance to
vancomycin bacause their outer membrane protein(porin) is
impermeable to large, rigid, and hydrophobic glycopeptide
molecule of vancomycin.
 Pseudomonas aeruginosa is intrinsically resistant to a wide
variety of antimicrobial agents, including β-lactams, β-lactam
inhibitors, sulfonamides, trimethoprim, tetracycline, and
chloramphenicol due to impermeable OMP.

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2. Biofilm
 Biofilms are sessile bacterial communities that are
irreversibly attached to a solid surface and are embedded
in an exopolysaccharide matrix and are prevalent in the
clinical setting, found on numerous environmental
surfaces and indwelling medical devices.
 Due to decreased penetration of antibiotics into the
physical or chemical barriers of biofilm.
 Also induction by the antimicrobial agent itself, resulting
in differential resistance gene expression throughout the
biofilm community

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3.Efflux pump
 Functions as transporter proteins for the extrusion of
toxic substances and antibiotics from the interior of
the cell to the external environment.
 Efflux pumps are naturally occurring and are present in
susceptible and resistant microorganisms.
 Efflux mechanisms can confer resistance to a particular
antimicrobial agent, a class of agents, or a number of
unrelated antimicrobials, resulting in MDR.
 Due to mutation there is increased expression of the
porin pump(RND) in P. aeruginosa, this results in greater
exit portal for numerous antibiotics including quinolones,
tetracyclines, macrolides, chloramphenicol, β-lactams,
and meropenem, but not imipenem.

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4.Enzymatic inactivation
 Bacteria can produce enzymes that destroy the
antimicrobial agents before they are able to reach the
targets. Enzymatic inactivation of antimicrobial agents is
one of most commonly acquired and intrinsic resistance
mechanism for β-lactam antibiotics.
 β-Lactamases hydrolyze β-lactam antibiotics using two
distinct mechanisms, a metallo-based mechanism of
action and a serine-based mechanism of action. They are
typically grouped into four classes, A to D, on the basis of
amino acid sequence similarity. Class A, C, and D
enzymes use serine for β-lactam hydrolysis, whereas class
B metalloenzymes require divalent zinc ions for substrate
hydrolysis

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 Citrobacter freundii, Enterobacter aerogenes, and P.
aeruginosa are clinically important nosocomial pathogens
encoding chromosomal versions of class C β-lactamases.
 Although these enzymes are commonly found on the
chromosome, they can escape to plasmids and become
transmissible.

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Mechanism of acquired resistance
1. Efflux,
2. Target site modification,
3. Acquisition of new taget,
4. Enzymatic inactivation and destrucion, and
5. Adaptation of alternative metabolic pathway

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1.Efflux
 Although efflux plays a major role in intrinsic resistance, changes
in these cell wall proteins can also result in novel acquired traits.
 In addition, some efflux pumps have translocated to plasmids,
which can be acquired by horizontal gene exchange.
 Eg..An efflux pump encoded by the mef gene in Streptococcus is
an example of an acquired macrolide resistance.
 Many plasmid-encoded tetracycline resistance efflux genes Tet
(A), Tet (B), Tet (C), Tet (D), Tet (E), Tet (G), Tet (H), Tet (J), Tet
(K), Tet (L), Tet (Y), Tet (Z), Tet (30), and Tet (39) have been
detected in many gram negative bacteria

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2.Target site modification
 Modification of a target can reduce the binding affinity
of the antibiotic to the target.
 Modification of target sites occurs primarily by
chromosomal mutation, and enzymatic alteration
of target sites.

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Contd….
A. Chromosomal mutation:-
 Quinolones target DNA gyrase and topoisomerase IV, inhibit
DNA synthesis.
 Mutation in GyrA and ParC gene result in DNA topoisomerases
that have low affinity to quinolones.
 β-Lactam antibiotics kill S. pneumoniae by targeting
endogenous high-molecular-weight PBPs :-PBP1A, -1B, -2A, -2B,
and -2X. Mutations in these PBPs lead to alteration of PBPs,
which result in reducing affinity to β-lactam antibiotics.

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B. Enzymatic alteration of target site :-
 Enzymatic alterations of antibiotic targets result in
reduced affinity of antibiotics for their microbial
targets.
 Macrolides such as erythromycin bind the 50S subunit
of the ribosome at the peptidyltransferase near
adenine residue of 23S rRNA. Monomethylation or
dimethylation of the amino group in the adenine
residue of 23S rRNA results in reduced affinity of the
macrolide for its target site and in elevated MICs
 Proteins encoded by the vanA and vanB genes confer
resistance to vancomycin, which is associated with
alteration of the vancomycin-binding site in the cell
wall and is clinically important in enterococcal species.

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3.Acquisition of new target site
 Microorganisms also adapt to become resistant by
acquiring cellular targets with reduced affinity for the
antibiotic.
 The mecA is a gene responsible for methicillin
resistance and encodes a new PBP, PBP2A (also
PBP2A′), a bifunctional transglycosylasetranspeptidase
with reduced affinity for β-lactam antibiotics,
including penicillins, cephalosporins and
carbapenems.
 Resistance in gram-negative and gram-positive
bacteria to sulfonamide is usually caused by the
acquisition of a new enzyme that is unaffected by
sulfonamides (i.e. altered dihydropteorate synthase).

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4. Enzymatic inactivation and destruction of drugs
 The acquisition of enzymes that inactivate antibiotics
directly is one of the first mechanisms of resistance
identified in bacteria and is a successful strategy used
by many microorganisms to survive the action of many
antibiotic classes.
 Class A β-lactamases are primarily penicillinases
produced by gramnegative and gram-positive bacteria
capable of hydrolyzing penicillin class antibiotic
substrates.
 Also, ESBLs can hydrolyze specific sets of penicillins,
cephalosporins, and monobactams, although not all
ESBLs are capable of hydrolyzing all cephalosporins
equally well.

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Bacterial enzymes for resistance
 Penicillinase
 Cloxacillin
 β-lactamases
 1st and 2nd generations of cephalosporins
 ESBLs
 3rd and 4th generations cephalosporins
 Carbapenemases
 Carbapenems

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 There are three classes of aminoglycoside-modifying
enzymes—
N-acetyltransferase (ACC),
 O-adenyltransferase (ANT), and
O-phosphotransferase (APH).
 Inactivation of the aminoglycoside by the
aminoglycoside-modifying enzymes is a result of the
transfer of a functional group to the aminoglycoside;
 AAC transfers the acetyl group, ANT transfers the
nucleotide triphosphate, and APH transfers the
phosphoryl group to aminoglycosides and inactivate
them.

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5. Adaptation of alternative metabolic pathway
 Some sulfonamide-resistant bacteria do not require
extracellular PABA but, like mammalian cells, they
can utilize preformed folic acid.
 A mutational loss in bacteria make them dependent
on an external supply of thymine, which contributes to
trimethoprim resistance. A mutational change in H.
influenzae results in overproduction of dihydrofolate
reductases, leading to trimethoprim resistance.

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Detection of drug resistance
1.Phenotypic method:-
Estimation of phenotypic expression of resistance to one or
several antimicrobial drugs.
 Agar screen
 Disc diffusion
 MIC determination
 Automated method
2.Genotypic method:-
Detection of genes or nucleotide sequences responsible for
coding antimicrobial resistance.
 DNA hybridization,
 nucleic acid amplification (PCR) technique
 or by microarray techniques.