Resistance to anti-microbial agents

Antimicrobial
Resistance
By: Dr. Manjeeta Gupta
06-08-2015
MIMER Medical College Talegaon
Department of Pharmacology
1

•History
•Introduction
•What is antimicrobial resistance
•Why antibacterial resistance is a concern
•Mechanisms of resistance
•NDM-1
•Factors causing antimicrobial resistance
•Present status of development of antimicrobials
•Strategies to contain resistance
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Objective
s

In his 1945 Nobel Prize lecture, Fleming himself warned of
danger of resistance –
“It is not difficult to make microbes resistant to penicillin in
laboratory by exposing them to concentrations not sufficient to kill
them, and same thing has occasionally happened in body…
…and by exposing microbes to non-lethal quantities of drug
make them resistant.”
History
(Nobel 1945)
Sir Alexander Fleming
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• Magic Bullets (Miracle cures)
•Present scenario Resistance to every major
class of antimicrobial agents
•Golden age  End of Antimicrobial Era
Introduction
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“Concentration of drug at site
of infection must inhibit
organism & also remain below
level that is toxic to human
cells.“
Antimicrobial
resistance

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Resistance in malaria
The emergence of P. falciparum multidrug resistance, including resistance to ACTs, in the Greater Mekong subregion is an urgent public health
concern that is threatening the ongoing global effort to reduce the burden of malaria. Routine monitoring of therapeutic efficacy is essential to
guide and adjust treatment policies. It can also help to detect early changes in P. falciparum sensitivity to antimalarial drugs.
Resistance in HIV
HIV drug resistance emerges when HIV replicates in the body of a person infected with the virus who is taking antiretroviral drugs. Even when
antiretroviral therapy (ART) programmes are very well-managed, some degree of HIV drug resistance will emerge.
Available data suggest that continued expansion of access to ART is associated with a rise in HIV drug resistance. In 2013, 12.9 million people
living with HIV were receiving antiretroviral therapy globally, of which 11.7 million were in low- and middle-income countries.
HIV drug resistance may rise to such a level that the first-line and second-line ART regimens currently used to treat HIV become ineffective,
jeopardizing people’s lives and threatening national and global investments in ART programmes.
As of 2010, levels of HIV drug resistance among adults who had not begun treatment in countries scaling up ART were found to be about 5%
globally. However, since 2010, there are reports suggesting that pre-treatment resistance is increasing, peaking at 22% in some areas.
Continuous surveillance of HIV drug resistance is of paramount importance to inform global and national decisions on the selection of first and
second-line ART and to maximize overall population level treatment effectiveness.
Resistance in influenza
Over the past 10 years, antiviral drugs have become important tools for treatment of epidemic and pandemic influenza. Several countries have
developed national guidance on their use and have stockpiled the drugs for pandemic preparedness. The constantly evolving nature of influenza
means that resistance to antiviral drugs is continuously emerging.
By 2012, virtually all influenza A viruses circulating in humans were resistant to drugs frequently used for the prevention of influenza (amantadine and rimantadine). However, the
frequency of resistance to the neuraminidase inhibitor oseltamivir remains low (1-2%). Antiviral susceptibility is constantly monitored through the WHO Global Surveillance and Response
System.
Present situation (Updated April 2015)
Resistance in bacteria
WHO’s 2014 report on global surveillance of antimicrobial resistance revealed that antibiotic resistance is no longer a prediction for the future; it
is happening right now, across the world, and is putting at risk the ability to treat common infections in the community and hospitals. Without
urgent, coordinated action, the world is heading towards a post-antibiotic era, in which common infections and minor injuries, which have been
treatable for decades, can once again kill.
•Treatment failure to the drug of last resort for gonorrhoea – third-generation cephalosporins – has been confirmed in several countries.
Untreatable gonococcal infections result in increased rates of illness and complications, such as infertility, adverse pregnancy outcomes and
neonatal blindness, and has the potential to reverse the gains made in the control of this sexually transmitted infection.
•Resistance to one of the most widely used antibacterial drugs for the oral treatment of urinary tract infections caused by E. coli –
fluoroquinolones – is very widespread.
•Resistance to first-line drugs to treat infections caused by Staphlylococcus aureus – a common cause of severe infections acquired both in
health-care facilities and in the community – is also widespread.
•Resistance to the treatment of last resort for life-threatening infections caused by common intestinal bacteria – carbapenem antibiotics – has
spread to all regions of the world. Key tools to tackle antibiotic resistance – such as basic systems to track and monitor the problem – reveal
considerable gaps. In many countries, they do not even seem to exist.
Resistance in tuberculosis
In 2013, there were an estimated 480 000 new cases of MDR-TB in the world. Globally, 3.5% of new TB cases and 20.5% of previously treated
TB cases are estimated to have MDR-TB, with substantial differences in the frequency of MDR-TB among countries. Extensively drug-resistant
TB (XDR-TB, defined as MDR-TB plus resistance to any fluoroquinolone and any second-line injectable drug) has been identified in 100
countries, in all regions of the world.

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ICMR programme on Antibiotic Stewardship, Prevention of Infection & Control (ASPIC)
Indian J Med Res. 2014 Feb; 139(2): 226–230.
Combating Antibiotic-Resistant Bacteria—issued by President Barack Obama on September 18, 2014 —
National Action Plan
FDA is partnering with Center for Disease Control and Prevention (CDC) on "Get Smart: Know When Antibiotics
Work."
In November 2012 an important paper from India was published that was ‘Chennai Declaration’.
J Antimicrob Chemother doi:10.1093/jac/dkt062.

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Selection pressure

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Mechanism of
Resistance
”Some microorganisms are ‘born’ resistant,
Some ‘achieve’ resistance by mutation and
Some have resistance ‘thrust upon them’ by
plasmids.”

Intrinsic Resistance
1. Lack target :
M. tuberculosis resistant to common antibiotics
2. Innate inability to cross outer membrane or bind to target:
E. coli, P. aeruginosa
3. Drug inactivation:
Cephalosporinase in Klebsiella
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Acquired
resistance
1. Mutation (Vertical transfer)
• Occurs in 1/10 million cells
• Single or multiple step
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2. Plasmid mediated
(Horizontal transfer)
• Most common
• Extra chromosomal genetic elements
can replicate independently & freely in
cytoplasm
• R-plasmids
Carry resistant genes ( r-genes)
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Mechanisms of Resistance Gene Transfer
 Conjugation
 Transduction
 Transformation
 Transposons
 Integrons
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 Conjugation : (Main mechanism for spread of resistance)
Conjugative plasmids makes a connecting tube between 2 bacteria through which
plasmid itself can pass
 Transduction : (Less common)
Plasmid DNA is enclosed in bacteriophage  transferred to another bacterium of
same species.
Seen in Staphylococci , Streptococci
 Transformation : (Least common)
Free DNA is picked up from environment
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 Insertion sequences of mobile DNA
 Cannot self replicate
 Resistance gene can ‘hitch hike’
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Transposons

 Large DNA packed with multiple
gene cassettes
 Located within transposon,
plasmid or mobile
 DO NOT SELF REPLICATE
 Encode as Integrase
provide specific site for gene
cassettes integration
 Multidrug resistance & virulence06-08-2015
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Integrons

• When subset of total microbial population is
resistant, despite total population being
considered susceptible on testing
• Vancomycin in S. aureus, E. faecium
Anti-tubercular drugs in TB
Fluconazole in Cryptococcus neoformans,
Candida albicans
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Hetero-
resistance

• Viral replication: more error prone
•Viral evolution under drug &
immune pressure: relatively easy
•Quasi species (drug resistant
subpopulations)
•Failure of antiretroviral therapy
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Viral quasi species

• Ability to protect genetic information & allow changes by causing defects in repair
mechanisms:
•Insertion of correct base pair by DNA polymerase III
•Proof reading by polymerase
•Post replicative repair
 Adaptation & Emergence of multidrug-resistant strains of M. tuberculosis
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Mutator (Mut)
phenotypes
Hypermutable phenotypes

• Prevention of drug accumulation in bacteria
• Modification of target site
• Use of alternative pathways for metabolic /
growth requirements
• By producing an enzyme that inactivates
antibiotic
• Quorum sensing
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Biochemical
mechanisms

Interior of organism
Cell wall
Porin channel
Antimicrobial
Antimicrobials normally enter bacterial cells via porin
channels in cell wall
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Decreased permeability: Porin Loss

Cell wall
New porin channel
Antimicrobial
New porin channels in the bacterial cell wall do not allow
Antimicrobials to enter cells
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• Cytoplasmic membrane
transport proteins
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Efflux
pumps

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Cell wall
Target siteBinding
Antimicrobial
Antimicrobials normally bind to specific binding proteins on
bacterial cell surface
Structurally modified antimicrobial target
site

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Cell wall
Modified target site
Antimicrobial
Changed site: blocked binding
Antimicrobials are no longer able to bind to modified binding
proteins on bacterial cell surface

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Cell wall
Antimicrobial
Target siteBinding
Enzyme
Inactivating enzymes target Antimicrobial
Antimicrobial inactivation

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Cell wall
Antimicrobial
Target siteBindingEnzyme
Enzyme
binding
Enzymes bind to Antimicrobial molecules

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Cell wall
Antimicrobial
Target siteEnzyme
Antimicrobial
destroyed
Antimicrobial altered,
binding prevented
Enzymes destroy antimicrobials or prevent binding to target sites

•Microbes communicate with each other & exchange signals (Autoinducers)
•Single autoinducer Incapable of induction
•When its colony reaches a critical density (quorum),
threshold of auto-induction is reached gene expression
•Allows bacteria to coordinate gene expression for virulence, conjugation,
apoptosis, mobility & resistance
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Quorum sensing

• Some of the best-known examples of quorum sensing come from studies of bacteria. Bacteria use quorum sensing to
coordinate certain behaviors such as biofilm formation,virulence, and antibiotic resistance, based on the local density of
the bacterial population. Quorum sensing can occur within a single bacterial species as well as between diverse species,
and can regulate a host of different processes, in essence, serving as a simple indicator of population density or the
diffusion rate of the cell's immediate environment. A variety of different molecules can be used as signals. Common
classes of signaling molecules are oligopeptides in Gram-positive bacteria, N-Acyl Homoserine Lactones (AHL) inGram-
negative bacteria, and a family of autoinducers known as autoinducer-2 (AI-2) in both Gram-negative and Gram-positive
bacteria.[1]
• Bacteria that use quorum sensing constitutively produce and secrete certain signaling
molecules (called autoinducers or pheromones). These bacteria also have a receptor that can specifically detect the
signaling molecule (inducer). When the inducer binds the receptor, it activates transcription of certain genes, including
those for inducer synthesis. There is a low likelihood of a bacterium detecting its own secreted inducer. Thus, in order for
gene transcription to be activated, the cell must encounter signaling molecules secreted by other cells in its
environment. When only a few other bacteria of the same kind are in the vicinity, diffusion reduces the concentration of
the inducer in the surrounding medium to almost zero, so the bacteria produce little inducer. However, as the population
grows, the concentration of the inducer passes a threshold, causing more inducer to be synthesized. This forms a positive
feedback loop, and the receptor becomes fully activated. Activation of the receptor induces the up-regulation of other
specific genes, causing all of the cells to begin transcription at approximately the same time. This coordinated behavior
of bacterial cells can be useful in a variety of situations. For instance, the bioluminescent luciferase produced by Vibrio
fischeri would not be visible if it were produced by a single cell. By using quorum sensing to limit the production of
luciferase to situations when cell populations are large, V. fischeri cells are able to avoid wasting energy on the
production of useless product.
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Antimicrobials Mechanisms Effect
Beta lactams
(Penicillins,
Cephalosporins,
Carbapenems)
Altered high mol. wt. PBP (MecA gene)
Altered no. & size of porins
Active efflux pumps
β lactamase enzyme
(Gram +veInducible, Gram –veConstitutive)
↓ affinity
↓ permeability
Efflux of drug
Cleavage of β lactam ring
Inactivation
Quinolones Altered DNA gyrase (gyrA gene, gyrB gene)
Altered Topoisomerase IV (parC gene, parE gene)
Efflux Pumps
↓ Affinity
Efflux of drug
Aminoglycosides
(Gentamicin,
Streptomycin)
Drug modifying enzymes (acetylase, phosphorylase &
adenylase)
Altered ribosomal structure
Drug inactivation
↓ permeability
(Gram +ve & Gram –ve)
Antibacterial agents
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Chloramphenicol Acetyltransferase enzyme
Altered ribosomal target
Inactivation
(Gram -ve constitutive,
Gram +ve inducible)
↓ permeability & binding
Macrolides
(Azithromycin,
Erythromycin)
Esterases (Enterobactericeae)
Altered ribosomal target (ermA, B, C gene)
Efflux pumps (mefA, msrA, mefE gene)
Hydrolysis
↓ binding
↓ drug concentration
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Tetracyclines Efflux pumps (tetK gene)
Altered ribosomal target (tetM gene)
Drug modifying enzymes (tetX)
↓ binding
Inactivation

Sulphonamides Point mutations in DHPS gene
Altered metabolic pathway
↑ efflux
Overproduction of PABA
↓ affinity & ↓ bacterial
permeability
↓ inhibition of DHPS
↓ drug effect
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Lincosamide
(Clindamycin)
Streptogramins
(Quinupristin,
Dalfopristin)
Altered ribosomal target (erm gene)
Altered ribosomal target (ermA, B, C gene)
Lactonases (vgbB gene)
Acetyltransferases (vatB, C, D, satA gene)
Efflux pumps (msrA gene)
↓ binding
↓ binding
Inactivation
↓ Drug concentration
Glycopeptides
(Vancomycin)
Altered target (D-alanyl-D-alanine to D-alanyl-D-
lactate/serine)
↓ binding

Pyrazinamide Point mutation (pncA gene) ↓ affinity of Pyrazinamidase
Isoniazid
(1 in 106 bacilli)
Mutation (katG/kasA gene)
Overexpression of inhA gene & ahpC promoter gene
Induction of efflux pumps
Inactivation
↓ binding of INH-NAD
inhibitor ↓ killing
Ethambutol Mutation (embB gene)
↑ activity of efflux pumps
Inactivation
Streptomycin Mutation (rpsL & rrs, gidB gene)
↑ activity of efflux pumps
Inactivation
Anti tubercular
agents
Rifampicin
(1 in 107 to 108
bacilli)
DNA polymerase target altered (rpoB gene)
Induction of dnaE2 gene
↓ binding
Error prone DNA repair
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Atovaquone Single point mutation (cyt b gene)
(mitochondrial chromosome)
Inhibits binding of drug to cyt bc1
complex
Pyrimethamine
Proguanil
Multiple mutation in plasmodium DHFR gene ↓ binding affinity
Chloroquine
(common)
Mutation (K76T) in pfCRT polymorphic gene
Efflux of drug, ↓ MOA
Mefloquine
Quinine
Gene amplification & point mutation of pfmdr1
Mutation of PfNHE
Antimalarial agents
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Benznidazole Mutation in β tubulin gene
↓ expression of NADH dependent mitochondrial
nitroreductase
↓ affinity for β tubulin
No activation of drug
Ivermectin Mutation in ATP dependent P-glycoprotein,
Glutamate/GABA- gated Cl - channels
Loss of MOA
Antihelminthic
agents
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Metronidazole ↓ transcription of the Ferredoxin gene
↑ expression of SOD
↑ expression of nim (resistance) genes
Loss of function mutations in NADPH nitroreductase
(rdxA gene)
Low levels of PFOR
Impaired O2 scavenging
capability
No formation of reactive
nitroso group

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Miltefosine Point mutation in P-type ATPase
(aminophospholipid translocase subfamily)
↓ drug uptake
Pentamidine
Melarsoprol
Point mutation of P2 transporter
Mutation of HAPT1 transporter
Loss of transporter, no
uptake of drug

Antifungal
agents
Flucytosine Loss of Permease
↓ activity of UPRTase/cytosine deaminase
↓ transport
↓ conversion to 5- FUMP
Azoles Mutation (ERG11 gene)
(codes for 14 α demethylase) & ERG3 (C5,6 sterol
reductase)
Efflux pumps (ABC)
↑ production of 14 α sterol demethylase
↓ binding, cross resistance
↓ effect
Antibiotic Mechanisms Effect
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Amphotericin B Replaces Ergosterol with other precursor sterols ↓ binding
Echinocandins Mutation of FKS1 gene (essential component of
1,3-β-D-glucan synthase complex)
↓ MOA

Lamivudine Mutation of HBV DNA polymerase No MOA
Zidovudine NNRTIs
Stavudine
NRTIs
Altered target site
Multiple mutation of RT gene (TAMs)
Extrusion of nucleoside analogue
Promote excision of
incorporated nucleotide by,
cross resistance
Protease inhibitors Multiple mutation of HIV protease gene No MOA & cross resistance
Maraviroc Shift in tropism from CCR5  CXCR4
Specific mutation in V3 loop of gp120
↑ IC50 & ↓ maximum %
inhibition of virus replication
Enfuvirtide
Integrase inhibitors
Specific mutation at binding domain of gp41
Mutation of integrase gene ↑ IC50 & Cross resistance
Anti retroviral
agents
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Entry
inhibitors

Acyclovir
Ganciclovir
Penciclovir
Famciclovir
Impaired production of thymidine kinase
Altered thymidine kinase substrate specificity
Altered viral DNA polymerase
No MOA
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Antiviral
agents
Cidofovir
Foscarnet
Point mutation in viral DNA polymerase No MOA, cross resistance
Amantadine
Rimantidine
Mutation in RNA sequence encoding M2
protein
No MOA
Oseltamivir
Zanamivir
Neuraminidase mutation No MOA
Adefovir Point mutation in HBV polymerase No MOA

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NDM-1 New Delhi metallo-beta-lactamase

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• Transmission from one strain of
bacteria to another plasmid
mediated
• Resistant to all except:
1. Colistin
2. Tigecycline
3. Aztreonam

Factors of Antibiotic Resistance
EnvironmentalDrug Related
Patient Related Prescriber Related
Antibiotic
Resistance
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New Antibiotic Development
•Only 15 antibiotics of 167
under development had
new MOA with potential to
combat of multidrug
resistance
•Lack of incentive

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References
1. Goodman and Gilman’s The Pharmacological Basis of Therapeutics 12th Edition
2. Rang and Dale’s Pharmacology 7th Edition
3. K. D. Tripathi Essentials of Medical Pharmacology 7th Edition
4. K. K. Sharma Principles of Pharmacology 2nd Edition
5. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology 6th Edition
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Thank You

Resistance to anti-microbial agents

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