MULTI DRUG RESISTANCE

By: ALAGAR . S M.Tech /

COMPUTATIONAL BIOLOGY

Objectives

• What is antimicrobial resistance

• Why antibacterial resistance is a concern

• How antibacterials work

• Mechanisms of resistance to antibacterials

• Indian scenario

• NDM-1

• Factors responsible for Resistance

• Alternate & Trending approaches

Introduction

• Throughout history there has been a continual battle between human beings and multitude of micro-organisms that cause infection and disease

In his 1945 Nobel Prize lecture, Fleming himself warned ofthe danger of resistance –

“It is not difficult to make microbes resistant topenicillin in the laboratory by exposing them toconcentrations not sufficient to kill them, and thesame thing has occasionally happened in the body……and by exposing his microbes to non-lethalquantities of the drug make them resistant.”

History Nobel Lecture, December 11, 1945

Sir Alexander Fleming

The Nobel Prize in Physiology or Medicine 1945

Timeline of Antibiotic Resistance

Why resistance is a concern

• Resistant organisms lead to treatment failure

• Increased mortality

• Resistant bacteria may spread in Community

• Low level resistance can go undetected

• Added burden on healthcare costs

• Threatens to return to pre-antibiotic era

• Selection pressure

Drug resistance occurs in :

BACTERIA—ANTIBIOTIC RESISTANCE Endoparasites Viruses—Resistance to antiviral drugs Fungi Cancer cells

Drug Resistance

What is Multi Drug Resistance

MDROs are microorganisms, predominantly bacteria, that are resistant to one or more classes of antimicrobial agents

Examples of MDROs Methicillin-resistant staphylococcus aureus (MRSA)

Vancomycin-intermediate staphylococcus aureus (VISA)

Vancomycin-resistant staphylococus aureus (VRSA)

Vancomycin-resistant enterococcus (VRE)

Streptococcus pneumoniae resistant to penicillin and other broad-spectrum agents

• The concentration of drug at the site ofinfection must inhibit the organism and alsoremain below the level that is toxic to humancells.

GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS - 11th Ed. (2006)

Antibiotic Resistance

Antibiotic Resistance

Defined as micro-organisms that are not

inhibited by usually achievable systemic

concentration of an antimicrobial agent with

normal dosage schedule and / or fall in the

minimum inhibitory concentration (MIC)

range.Antibiotic Resistance (DR)

= MIC / MCC > Toxic Plasma Concentration

Mechanisms of action of antibiotics

Mechanism Antibiotic Resistance

Intrinsic (Natural) Acquired

Genetic Methods

Chromosomal Methods Mutations

Extra chromosomal Methods Plasmids

Antibiotic Resistance

Some microorganisms may ‘born’ resistant,

some ‘achieve’ resistance by mutation or some

have resistance ‘thrust upon them’ by plasmids

Some are born great, some achieve greatness

or some have greatness thrust upon them

Intrinsic Resistance

1. Lack target :

• No cell wall; innately resistant to penicillin

2. Innate efflux pumps:

• Drug blocked from entering cell or ↑ exportof drug (does not achieve adequate internalconcentration). Eg. E. coli, P. aeruginosa

3. Drug inactivation:

• Cephalosporinase in Klebsiella

It occurs naturally.

Acquired resistance

Mutations

• It refers to the change in DNA structure of the gene.

• Occurs at a frequency of one per ten million cells.

• Eg.Mycobacterium.tuberculosis,Mycobacteriumlepra , MRSA.

• Often mutants have reduced susceptibility

Plasmids

• Extra chromosomal genetic elements can replicateindependently and freely in cytoplasm.

• Plasmids which carry genes resistant ( r-genes) are called R-plasmids.

• These r-genes can be readily transferred from one R-plasmid toanother plasmid or to chromosome.

• Much of the drug resistance encountered in clinical practice isplasmid mediated

Mechanisms of Resistance Gene Transfer

• Transfer of r-genes from one bacterium toanother

Conjugation

Transduction

Transformation

• Transfer of r-genes between plasmids within the bacterium

By transposons

By Integrons

Transfer of r-genes from one bacterium to another

Conjugation : Main mechanism for spread of resistance

The conjugative plasmids make a connecting tubebetween the 2 bacteria through which plasmid itselfcan pass.

Transduction : Less common method

The plasmid DNA enclosed in a bacteriophage is transferred to another bacterium of same species. Seen in Staphylococci , Streptococci

Transformation : least clinical problem.

Free DNA is picked up from the environment (i.e..From a cell belonging to closely related or same strain.

Mechanisms of Resistance Gene Transfer Transposons

Transposons are sequences of DNAthat can move around differentpositions within the genome of singlecell.

The donor plasmid containing theTransposons, co-integrate with acceptorplasmid. They can replicate duringcointegration

Both plasmids then separate and eachcontains the r-gene carrying thetransposon.

Eg ; Staphylococci,Enterococci

Mechanisms of Resistance Gene Transfer

Integrons

Integron is a large mobile DNAcan spread Multidrug resistance

Each Integron is packed withmultiple gene casettes, eachconsisting of a resistance geneattached to a small recognition site.

These genes encode severalbacterial functions includingresistance and virulence.

They cannot promote self transfer

Biochemical mechanisms of antibioticresistance

• Prevention of drug accumulation in the bacterium

• Modification/protection of the target site

• Use of alternative pathways for metabolic / growth requirements

• By producing an enzyme that inactivates the antibiotic

Decreased permeability: Porin Loss

Interior of organism

Cell wall

Porin channel

into organism

Antibiotic

Antibiotics normally enter bacterial cells via porin channels

in the cell wall

Decreased permeability: Porin Loss

Interior of organism

Cell wall

New porin channel

into organism

Antibiotic

New porin channels in the bacterial cell wall do not allow

antibiotics to enter the cells

ATP Binding Cassette

Multidrug and toxic compound exporter

Small multidrug resistance transporters

Resistance-nodulation-division

Major facilitator superfamily

Efflux pumps

• Cytoplasmic membrane transport proteins.

• Major mechanism for resistance in Tetracyclines.

• Some gram -ve bacteria inhibit the plasmidmediated synthesis of porin channels ,whichobstructs the influx of hydrophilic Penicillinseg.ampicillin

Structurally modified antibiotic target site

Interior of organism

Cell wall

Target siteBinding

Antibiotic

Antibiotics normally bind to specific binding proteins on the

bacterial cell surface

Structurally modified antibiotic target site

Interior of organism

Cell wall

Modified target site

Antibiotic

Changed site: blocked binding

Antibiotics are no longer able to bind to modified binding proteins

on the bacterial cell surface

Modification/Protection of the Target site

Resistance resulting from altered target sites :

Target sites Resistant Antibiotics

Ribosomal point mutation Tetracyclines,Macrolides, Clindamycin

Altered DNA gyrase Fluoroquinolones

Modified penicillin binding proteins (Strepto.pneumonia)

Penicillins

Mutation in DNA dependant RNA polymerase (M.tuberculosis)

Rifampicin

Antibiotic inactivation

Interior of organism

Cell wall

Antibiotic

Target siteBindingEnzyme

Inactivating enzymes target antibiotics

Antibiotic inactivation

Interior of organism

Cell wall

Antibiotic

Target siteBindingEnzyme

Enzyme

binding

Enzymes bind to antibiotic molecules

Antibiotic inactivation

Interior of organism

Cell wall

Antibiotic

Target siteEnzyme

Antibiotic

destroyedAntibiotic altered,

binding prevented

Enzymes destroy antibiotics or prevent binding to target sites

By producing enzymes that inactivates antibiotic

a)Inactivation of b-lactam antibiotics•S. aureus, N. gonorrohoea, H.influenza, Produce b-lactamase which cleaves -lactam ring

b)Inactivation of Chloramphenicol• Inactivated by chloramphenicol acetyltransferase .• Gram-ve (enzyme present constitutively hence higherresistance) gram +ve bacteria (enzyme is inducible )

c)Inactivation of Aminoglycosides• Inactivated by acetyl, phospho & adenylyl transferasesPresent in gram +ve and gram –ve .

Use of alternative pathways for metabolic / growth

requirements

• Resistance can also occur by alternate pathway that bypasses the reaction inhibited by the antibiotic.

• Sulfonamide resistance can occur from overproduction of PABA

Drug Mechanism of resistance

Pencillins & Cephalosporiins

B Lactamase cleavage of the Blactam ring

Aminoglycosides Modification by phosphorylating, adenylatingand acetylating enzymes

Chloramphenicol Modification by acetylytion

Erythromycin Change in receptor by methylation of r RNA

Tetracycline Reduced uptake / increased export

SulfonamidesActive export out of the cell & reduced affinity of enzymes

Indian scenario

• Lack of community awareness

• Availability over the counter

• Absence of central monitoring agency

• S. Pneumoniae fully resistant to cotrimoxazole

• Still sensitive to penicillins, macrolides and fluoroquinolones

Enteric pathogens

• Vibrio cholerae :

– resistance to furazolidine, cotrimoxazole, nalidixicacid

– Tetracycline remains effective

• Coliforms

– ESBLs , extensive resistance to Beta lactumantibiotics

• Enteric fever

STD

• Penicillin and fluoroquinolone resistance is widespread to gonorhhoea

• Alternate drugs like Azithromycin and cephalosporins should be used

• Syphilis still susceptible to Penicillins

Gram positive Cocci

• Streptococci other than S. Pneumoniae

– Resistant to tetracycline and macrolides (40%)

– Still sensitive to penicillins

• Staph Aureus

– Methicillin resistance 50%-100%

– Vancomycin resistance also increasing

Mycobacteria

• Multidrug resistance

– Combined resistance to rifampicin and isoniazid

• Extensively drug resistant TB

– Additional acquisition of resistance to a fluroquinolone and one of the three injectablesecond line drugs (capreomycin, kanamycin and amikacin)

• Steady rise in these patients

What is NDM-1?

• NDM-1 stands for New Delhi metallo-beta-lactamase, an enzyme produced by certain strains of bacteria that have recently acquired the genetic ability to make this compound.

• The enzyme is active against other compounds that beta-lactam ring like penicillins, cephalosporins, and the carbapenems.

• bacteria that produce NDM-1 are resistant to all commonly used beta-lactam antibiotics, including carbapenems.

New Delhi metallo-beta-lactamase Why everyone concerned ?

• There are currently no new drugs in the research pipelines that aim to stop NDM-1.To date, some strains of E.coli and Klebseilla pneumoniaeare known carriers of the gene, but the gene can be transmitted from one strain of bacteria to another through horizontal gene transfer.

Naming the strain as New Delhi creates controversy

• The gene was named after New Delhi, the capital city of India, as it was first described by Yong et al. in 2009 in a Swedish national who fell ill with an antibiotic-resistant bacterial infection that he acquired in India . The infection was unsuccessfully treated in a New Delhi hospital and after the patient's repatriation to Sweden, a carbapenem-resistant Klebsiella pneumoniae strain bearing the novel gene was identified. The authors concluded that the new resistance mechanism "clearly arose in India, but there are few data arising from India to suggest how widespread it is."

Treatment

• Many NDM-1 strains are resistant to all antibiotics except for colistin.

• Colistin is an older antibiotic that has not been used much in recent decades, because it is somewhat more toxic than other antibiotics.

• A few NDM-1 strains have been sensitive to tigecycline (Tygacil), but this agent should be used cautiously in serious infections because it does not achieve high levels in the bloodstream.

• A few strains have also been sensitive to aztreonam

The spread of NDM-1 can be contained with

• The spread of NDM-1 within health-care facilities can be curbed through strict infection-control measures, including patient isolation and hand washing.

..

Strategy to Contain Resistance

• Develop new antibiotics

–Bypass the drug resistance

• Judicious use of the existing antibiotics:

–Containment of drug resistance

New Antibiotic Development

• Only 15 antibiotics of 167 under development had a new mechanism of action with the potential to combat of multidrug resistance.

• Lack of incentive for companies to develop antibiotics.

Phage therapy

• Phage Therapy is the therapeutic use of lytic bacteriophages totreat pathogenic bacteria infections

• Bacteriophages are viruses that invade bacterial cells anddisrupt bacterial metabolism and cause the bacterium to lyse.

• Bacteriophage therapy is an important alternative to antibiotics

• The success rate was 80–95% with few gastrointestinal orallergic side effects. British studies also demonstrated significantefficacy of phages against Escherichia coli, Acinetobacter spp.,Pseudomonas spp and Staphylococcus aureus.

Alternate Approaches

Quorum sensing

• Microbes communicate with each other and exchange signaling chemicals (Autoinducers)

• These autoinducers allow bacterial population to coordinate gene expression for virulence, conjugation, apoptosis, mobility and resistance

Why named quorum sensing

• Single autoinducer from single microbe is incapable of inducing any such change

• But when its colony reaches a critical density (quorum), threshold of autoinduction is reached and gene expression starts

• QS signal molecules AHL, AIP, AI-2 & AI-3 have been identified in Gm-ve bacteria

• AI-2 QS –system is shared by GM+ve bacteria also

WHY INHIBIT QUORUM SENSING

Proved to be very potent method for bacterial virulence inhibition.

Several QS inhibitors molecules has been synthesized which include AHL, AIP, and AI-2 analogues

QS inhibitors have been synthesized and have been isolated from several natural extracts such as garlic extract.

QS inhibitors have shown to be potent virulence inhibitor both in in-vitro and in-vivo,using infection animal models.

Factors of Antibiotic Resistance

Environmental Factors

Drug Related Factors

Patient Related Factors

Prescriber Related Factors

Antibiotic Resistance

• Huge populations and overcrowding

• Rapid spread by better transport facility

• Poor sanitation

• Increases community acquired resistance

• Ineffective infection control program

• Widespread use of antibiotics in animal husbandry

and agriculture and as medicated cleansing products

1. Environmental Factors

• Over the counter availability of antimicrobials

• Counterfeit and substandard drug causing sub-optimal blood concentration

• Irrational fixed dose combination of antimicrobials

• Soaring use of antibiotics

2. Drug Related

Policy Decision at Higher level

• Poor adherence of dosage Regimens

• Poverty

• Lack of sanitation concept

• Lack of education

• Self-medication

• Misconception

3. Patient Related

Patient Counseling, Awareness

Program

Prescriber Related

• Inappropriate use of available drugs

• Increased empiric poly-antimicrobial use

• Overuse of antimicrobials

• Inadequate dosing

• Lack of current knowledge and training

Poor Clinical Practice

• Poor clinical practice that fail to incorporate

the pharmacological properties of

antimicrobials amplify the speed of

development of drug resistance.

Faulty Antibiotic Use

• Antimicrobials are over prescribed

• Available without prescription

Over Prescribed Antibiotics

• Clinician should first determine whether antimicrobial therapy is warranted for a given patient

Definitive Treatment

1. Can a narrower spectrum agent be substituted for initial empiric drug?

Definitive Treatment (2)

1. Is one agent or combination of agents necessary?

Examples

• -lactam + Aminoglycosides

• Extended spectum Penicillins + -lactamaseInhibitors

• Anti-tubercular regimen

• Anti-leprotic regimen

• Co-trimoxazole

• Sulphadoxin + pyrimethamine

• Artemisinin based Combination Therapy (ACT) in Malaria

Definitive Treatment

What are the

– optimum dose,

– route of administration and

– duration of therapy?

Definitive treatment

What specific test to identify patients who will not respond to treatment?

Creating Super Bugs

TRENDING APPROACHESNew Antibiotics – New bacteria

Mutant Bacteriophages

Quorum sensing