Multi Drug Resistance

1. MULTI DRUG RESISTANCE
By: ALAGAR . S
M.Tech /
COMPUTATIONAL
BIOLOGY

2. 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

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

4. In his 1945 Nobel Prize lecture, Fleming himself warned of
the danger of resistance –
“It is not difficult to make microbes resistant to
penicillin in the laboratory by exposing them to
concentrations not sufficient to kill them, and the
same thing has occasionally happened in the body…
…and by exposing his microbes to non-lethal
quantities of the drug make them resistant.”
History
Nobel Lecture, December 11, 1945
Sir Alexander Fleming
The Nobel Prize in Physiology or Medicine 1945

5. Timeline of Antibiotic Resistance

6. 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

7. Drug resistance occurs in :
 BACTERIA—ANTIBIOTIC RESISTANCE
 Endoparasites
 Viruses—Resistance to antiviral drugs
 Fungi
 Cancer cells
Drug Resistance

8. 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

9. • The concentration of drug at the site of
infection must inhibit the organism and also
remain below the level that is toxic to human
cells.
GOODMAN & GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS - 11th Ed. (2006)
Antibiotic Resistance

10. 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

11. Mechanisms of action of antibiotics

12. Mechanism Antibiotic Resistance
Intrinsic (Natural) Acquired
Genetic Methods
Chromosomal Methods
Mutations
Extra chromosomal Methods
Plasmids

13. 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

14. Intrinsic Resistance
1. Lack target :
• No cell wall; innately resistant to penicillin
2. Innate efflux pumps:
• Drug blocked from entering cell or ↑ export
of drug (does not achieve adequate internal
concentration). Eg. E. coli, P. aeruginosa
3. Drug inactivation:
• Cephalosporinase in Klebsiella
It occurs naturally.

15. 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,Mycobacterium
lepra , MRSA.
• Often mutants have reduced susceptibility

16. Plasmids
• Extra chromosomal genetic elements can replicate
independently 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 to
another plasmid or to chromosome.
• Much of the drug resistance encountered in clinical practice is
plasmid mediated

17. Mechanisms of Resistance Gene Transfer
• Transfer of r-genes from one bacterium to
another
 Conjugation
 Transduction
 Transformation
• Transfer of r-genes between plasmids within
the bacterium
 By transposons
 By Integrons

18. Transfer of r-genes from one bacterium to another
 Conjugation : Main mechanism for spread of resistance
The conjugative plasmids make a connecting tube
between the 2 bacteria through which plasmid itself
can 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.

19. Mechanisms of Resistance Gene Transfer
Transposons
Transposons are sequences of DNA
that can move around different
positions within the genome of single
cell.
 The donor plasmid containing the
Transposons, co-integrate with acceptor
plasmid. They can replicate during
cointegration
Both plasmids then separate and each
contains the r-gene carrying the
transposon.
Eg ; Staphylococci,Enterococci

21. Mechanisms of Resistance Gene Transfer
Integrons
Integron is a large mobile DNA
can spread Multidrug resistance
Each Integron is packed with
multiple gene casettes, each
consisting of a resistance gene
attached to a small recognition site.
These genes encode several
bacterial functions including
resistance and virulence.
They cannot promote self transfer

22. Biochemical mechanisms of antibiotic
resistance
• 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

23. 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

24. 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

25. 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 plasmid
mediated synthesis of porin channels ,which
obstructs the influx of hydrophilic Penicillins
eg.ampicillin

26. 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

27. 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

28. 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

29. Antibiotic inactivation
Interior of organism
Cell wall
Antibiotic
Target siteBinding
Enzyme
Inactivating enzymes target antibiotics

30. Antibiotic inactivation
Interior of organism
Cell wall
Antibiotic
Target siteBindingEnzyme
Enzyme
binding
Enzymes bind to antibiotic molecules

31. Antibiotic inactivation
Interior of organism
Cell wall
Antibiotic
Target siteEnzyme
Antibiotic
destroyed
Antibiotic altered,
binding prevented
Enzymes destroy antibiotics or prevent binding to target sites

32. 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 higher
resistance) gram +ve bacteria (enzyme is inducible )
c)Inactivation of Aminoglycosides
• Inactivated by acetyl, phospho & adenylyl transferases
Present in gram +ve and gram –ve .

33. 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

34. Drug Mechanism of resistance
Pencillins &
Cephalosporiins
B Lactamase cleavage of the Blactam ring
Aminoglycosides Modification by phosphorylating, adenylating
and acetylating enzymes
Chloramphenicol Modification by acetylytion
Erythromycin Change in receptor by methylation of r RNA
Tetracycline Reduced uptake / increased export
Sulfonamides
Active export out of the cell & reduced affinity
of enzymes

35. 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

36. Enteric pathogens
• Vibrio cholerae :
– resistance to furazolidine, cotrimoxazole, nalidixic
acid
– Tetracycline remains effective
• Coliforms
– ESBLs , extensive resistance to Beta lactum
antibiotics
• Enteric fever

37. STD
• Penicillin and fluoroquinolone resistance is
widespread to gonorhhoea
• Alternate drugs like Azithromycin and
cephalosporins should be used
• Syphilis still susceptible to Penicillins

38. 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

39. 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 injectable
second line drugs (capreomycin, kanamycin and
amikacin)
• Steady rise in these patients

40. 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.

41. 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 pneumoniae
are known carriers of the
gene, but the gene can be
transmitted from one
strain of bacteria to
another through horizontal
gene transfer.

42. 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."

43. 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

44. 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.
..

45. Strategy to Contain Resistance
• Develop new antibiotics
–Bypass the drug resistance
• Judicious use of the existing
antibiotics:
–Containment of drug resistance

46. 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.

47. Phage therapy
• Phage Therapy is the therapeutic use of lytic bacteriophages to
treat pathogenic bacteria infections
• Bacteriophages are viruses that invade bacterial cells and
disrupt 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 or
allergic side effects. British studies also demonstrated significant
efficacy of phages against Escherichia coli, Acinetobacter spp.,
Pseudomonas spp and Staphylococcus aureus.
Alternate Approaches

48. 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

49. 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

50. 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.

51. Factors of Antibiotic Resistance
Environmental
Factors
Drug Related
Factors
Patient Related
Factors
Prescriber
Related Factors
Antibiotic
Resistance

52. • 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

53. • 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

54. • Poor adherence of dosage Regimens
• Poverty
• Lack of sanitation concept
• Lack of education
• Self-medication
• Misconception
3. Patient Related
Patient
Counseling,
Awareness
Program

55. Prescriber Related
• Inappropriate use of available drugs
• Increased empiric poly-antimicrobial use
• Overuse of antimicrobials
• Inadequate dosing
• Lack of current knowledge and training

56. Poor Clinical Practice
• Poor clinical practice that fail to incorporate
the pharmacological properties of
antimicrobials amplify the speed of
development of drug resistance.

57. Faulty Antibiotic Use
• Antimicrobials are over prescribed
• Available without prescription

58. Over Prescribed Antibiotics
• Clinician should first determine whether
antimicrobial therapy is warranted for a given
patient

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

60. Definitive Treatment (2)
1. Is one agent or combination of agents
necessary?

61. Examples
• -lactam + Aminoglycosides
• Extended spectum Penicillins + -lactamase
Inhibitors
• Anti-tubercular regimen
• Anti-leprotic regimen
• Co-trimoxazole
• Sulphadoxin + pyrimethamine
• Artemisinin based Combination Therapy (ACT) in
Malaria

62. Definitive Treatment
What are the
– optimum dose,
– route of administration and
– duration of therapy?

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

64. Creating Super Bugs

65. TRENDING APPROACHES
New Antibiotics – New bacteria

66. Mutant Bacteriophages

67. Quorum sensing