FLOW OF THE SEMINAR 1. Definition – antibiotic resistance, Multi-resistance, cross-resistance in antibiotics 2. Evolution of resistance 3. Impact of resistance 4. The scenario of resistance: Global, India 5. Factors causing resistance 6. Mechanisms of resistance: Intrinsic and Acquired 7. Acquired mechanism of resistance 8. Quorum sensing 9. Mechanism of resistance in commonly used antibiotics 10. Methods for determining the resistance 11. Strategies to contain resistance 12. Antibiotic stewardship 13. Role of Pharmacologist 14. Initiatives undertaken by India to control resistance

1. ANTIMICROBIAL RESISTANCE
- Dr. Hemant R Kanase1
Antibiotics

2. Definition
Overview of the problem
Mechanisms of
resistance
How to overcome
2

3. 3

4. Antibiotic resistance
• Property of microbe
• Defined as micro-organism not inhibited by usually
achievable systemic concentration of an AMA with normal
dosage schedule and / or fall in the MIC range.
4

5. MIC = 6.25 mcg/mL
MIC
Breakpoint
5

6. Multi
Resistance
• MDR –non-susceptibility
to at least 1 agent in 3
antimicrobial categories
• XDR - non-susceptibility
to at least 1 agent in all
but susceptible to 2 or
fewer antimicrobial
categories
• PDR - Non-susceptibility
to all agents in all
antimicrobial categories
P. aeruginosa
7
Magiorakos A et al. Multidrug-resistant, extensively drug-
resistant and pandrug-resistant bacteria: an international
expert proposal for interim standard definitions for
acquired resistance. Clinical Microbiology and Infection.
2012;18(3):268-281.

7. 8
Cross Resistance
Acquisition of resistance to one AMA conferring
resistance to another AMA, to which the organism has
not been exposed.
• Between chemically related drugs.
Eg. Aminoglycoside-modifying enzymes which may
confer resistance to several members of the
aminoglycoside family.
• Unrelated drugs may show partial cross resistance: a
result of either overlapping drug targets
Eg. Macrolides and Lincosamides
Magiorakos A, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim
standard definitions for acquired resistance. Clinical Microbiology and Infection. 2012;18(3):268-281.

8. Evolution
9
Davies J Davies D. Origins and Evolution of Antibiotic Resistance. Microbiology and Molecular Biology Reviews. 2010;74(3):417-433.

9. Antibiotics Microbes
Past
Present
Future
10

10. Evolution
11Davies J Davies D. Origins and Evolution of Antibiotic Resistance. Microbiology and Molecular Biology Reviews. 2010;74(3):417-433.

11. 12

12. Why do we care?
Longer duration of illness
Longer treatment
Increased mortality
Huge economic impact
Patients to community and health care worker
Negates advances in healthcare
13

13. 14

14. 15

15. Antibiotic resistance: Global
Concern
Three agents of greatest
concern associated with both
hospital- and community
acquired infections.
• E-coli
• Klebsiella pneumoniae
• Staphylococcus aureus
Percentage of Staphylococcus aureus
isolates that are methicillin resistant
(MRSA), by country (2011-14)
16Mackenzie D. Antibiotic resistance hits crisis point. New Scientist. 2016; 232(3104-3106):32

16. Indian scenario
• Methicillin Resistant Staphylococcus Aureus
(MRSA)
• Extended Spectrum Beta Lactamase (ESBL)
- Enterobacteriaceae
- Acinetobacter baumanni
• Carbapenem resistance
- Enterobacteriaceae
- Non fermenting gram negative bacilli (NFGNB)
• Vancomycin Resistant Enterococcus (VRE)
17
Laxminarayan R Chaudhury R. Antibiotic Resistance in India: Drivers and Opportunities for Action. PLOS Medicine. 2016;13(3):e1001974.
Kumar S, et al. Antimicrobial resistance in India: A review. Journal of Natural Science, Biology and Medicine. 2013;4(2):286.

17. Other Infections
• Kala-azar
- 60% resistance in pentavalent antimony
- 25% in pentamidine
• Typhoid fever
- MDR Salmonella Typhi prevalent all over
- FQ resistance in Salmonella typhii, 8% in 2008 to 24% in 2014
• Vibrio cholerae
• resistance to furazolidine, cotrimoxazole, nalidixic acid
• Tetracycline remains effective
• Recent study in Sikkim, India found that MRSA was seen in
39% clinical specimens of S. aureus
18
Laxminarayan R Chaudhury R. Antibiotic Resistance in India: Drivers and Opportunities for Action. PLOS Medicine. 2016;13(3):e1001974.
Kumar S, et al. Antimicrobial resistance in India: A review. Journal of Natural Science, Biology and Medicine. 2013;4(2):286.

18. Glimpse in our hospital
• 10399 neonate – 12% culture positive sepsis
(monobacterial > polymicrobial inf)
• Gm –ve > Gm +ve (Acinetobacter species and Klebsiella
pneumoniae)
• Klebsiella – Sensitive to polymyxin B, colistin (100%
sensitivity) imipenem, amikacin, piperacillin and
cefotaxime.
• Acinetobacter sp. – low sensitivity to all antibiotics except
colistin
• GM +ve : predominant was Staphylcocci spp. (CoNS, more
MRCoNS) (S – Vanco and linezolid) 19

19. What is NDM-1?
• New Delhi Metallo-beta-lactamase-1 (NDM-1) first
described by Yong et al in 2009
• The gene for NDM-1 is a member of a large gene
family that encodes beta-
lactamase enzymes called carbapenemases.
• Bacteria that produce carbapenemases – superbugs
• NDM-1 was first detected in a Klebsiella
pneumoniae isolate from a Swedish patient of Indian
origin in 2008
• Susceptible to few antibiotics (polymyxins,colistin)
20
Kumarsamy K et al. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and
epidemiological study. Lancet Infect Dis. 2010; 10(9): 597–602.

20. This reported 37 cases with NDM-1 isolates in the United Kingdom, 44 in
Chennai, 26 in Haryana, and 73 in various other sites in Pakistan and
India.
An environmental point prevalence study conducted between 26
September and 10 October 2010 found bacteria with the NDM-1 gene in
drinking water and seepage samples in New Delhi. (Johnson and Woodford 2013)21

21. Superbugs* are visible manifestations of our prolonged failure to preserve antibiotics
** Methicillin resistant Staph aureus, MDR-and XDR Mycobacteria, ESBL producing Gram negative bacteria and NDM-1 producing
enterobacteriaceae bacteria are few examples of superbugs because these fail to respond to large number of commonly used antibiotics
Known but neglected.
Need immediate action
Known but
inevitable
22

22. Factors of Antibiotic Resistance
Environmental
Factors
Drug Related
Factors
Patient Related
Factors
Prescriber
Related Factors
Antibiotic
Resistance
23

23. Environmental Causes
24

24. Poor adherence of dosage
Regimens
Poverty
Lack of
sanitation
concept
Lack of
education
Self-
medication
Misconception
Patient Related causes
25

25. Over the
counter
availability of
drugs
Irrational FDC
of
antimicrobials
Counterfeit
substandard
drugs
Soaring
use of
antibiotics
Poor
regulatory
control
Drug related causes
26

26. 27

27. An Audit of antimicrobial use as per policy in various
wards of a tertiary care hospital in Mumbai
Aim: To ensure effective implementation of antibiotic
policy and optimise antimicrobial drug use.
‘Guidelines for Antimicrobial Therapy and Prophylaxis’
(2013)
• Overall antibiotic use was uniform with 70-75%
adherence to the policy with minor finidings.
• Good approach to selection of antibiotics (empirical,
definitive)
28

28. • Over use – few cases of longer duration of rx with
augmentin
• Selection of antibiotic
• Ceftriaxone used in AGE instead of
cipro/doxy/cotrimoxazole (policy)
• Amikacin used as 1st line instead of gentamicin.
• Availability of dug
• Augmentin, imepenem, voriconazole(NA)
• Availability of booklet
• 70% resident + lecturers (50% actually used)
• Indication not included in policy – tubercular
pericarditis
Message: 29

29. Animal Feed
Antibiotic
resistance in
genetically
modified
crops
Antibiotics in
food and
water
Miscellaneous causes

30. Mechanisms of action of
antibiotics
31
Tille, P. M. et. al Bailey & Scott's diagnostic microbiology. 13th ed. St. Louis, Missouri: Elsevier, 2014 p 161

31. Mechanism Antibiotic Resistance
Intrinsic (Natural) Acquired
Genetic
Chromosomal Methods
Mutations
Extra chromosomal
Methods
Plasmids
32

32. Intrinsic mechanism
• Innate ability - to resist activity of a particular
antimicrobial agent through its inherent structural
or functional characteristics
• No clinical significance, only proper drugs to be
selected
33

33. ORGANISMS NATURAL RESISTANCE
AGAINST:
MECHANISM
Anaerobic
bacteria
Aminoglycosides Lack of oxidative metabolism to drive uptake of
aminoglycosides
Aerobic bacteria Metronidazole Inability to anaerobically reduce drug to its active form
Gram + ve
bacteria
Aztreonam Lack of penicillin binding proteins (PBPs) that bind and are
inhibited by this beta lactam antibiotic
Gram –ve
bacteria
Vancomycin Lack of uptake resulting from inability of vancomycin to
penetrate outer membrane
Klebsiella spp. Ampicillin (a beta-lactam) Production of enzymes (beta-lactamases) that destroy
ampicillin before the drug can reach the PBP targets
Pseudomonas
aeruginosa
Sulfonamides,
trimethoprim,
tetracycline, or
chloramphenicol
Lack of uptake resulting from inability of antibiotics to
achieve effective intracellular concentrations
Enterococci Aminoglycosides Lack of sufficient oxidative metabolism to drive uptake of
aminoglycosides
All cephalosporins Lack of PBPs that effectively bind and are inhibited by these
beta lactam antibiotics
34
Dever L. Mechanisms of bacterial resistance to antibiotics. Archives of Internal Medicine. 1991;151(5):886-895.

34. Acquired mechanism of
resistance
Modification
in biochemical
mechanisms
Gene transfer
35van Hoek A et al. Acquired Antibiotic Resistance Genes: An Overview. Frontiers in Microbiology. 2011;2.

35. Biochemical mechanism
36

36. Biochemical mechanism
1. By production of enzymes that inactivates
the antibiotic.
• Beta lactamase enzyme - beta lactam antibiotics
(S. aureus, N. gonorrhea, H. influenza)
• Chloramphenicol acetyl transferase -
chloramphenicol (gram –ve > gram +ve)
• Acetyl transferases, phosphotranferases and
adenyltransferases - aminoglycoside
37

37. 2. Prevention of drug accumulation in the
bacterium
• Efflux pump – cytoplasmic membrane transport
proteins
• E.coli, P.aeruginosa, S. typhi, S. aureus, S.
pneumonia etc.
• Major mechanism of resistance for tetracycline,
increasing for FQ’s
• Inhibition of plasmid mediated synthesis of porin
channels – obstruct influx of hydrophilic
penicillin. Eg. ampicillin
38

38. 3. Modificaton of target sites
• Altered DNA gyrase – FQ
• Altered PBP in Strep. Pneumoniae – Penicillin
4. Use of alternative pathway for growth
requirement
• Bypass the reaction inhibited by the antibiotic
• Eg. Sulfonamide resistance
- Alternate folate metabolism pathway
- over production of PABA
40

39. Quorum sensing
• Mode of communication with each other and exchange
signaling chemicals (Autoinducers)
• Allow bacteria to coordinate gene expression for
virulence, conjugation, apoptosis, mobility and
resistance
• 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
• QS inhibitors - synthesized and isolated from several
natural extracts such as garlic extract. 41

40. Genetic mechanisms
• Chromosomal Mutation
• Spontaneous change in DNA structure of the gene
• Low mutation rate – frequency is 1/mln cells
• Over the course sensitive bacteria die and resistant
survive and multiple
42

41. Types of mutation
Single step
Single gene mutation
confers high degree of
resistance, emerges
rapidly.
Eg. E.coli, Staph to
Rifampicin.
Enterococci to
Streptomycin.
Multi step
A number of gene
modifications are
involved, sensitivity
decreases gradually in
a stepwise manner.
Eg. E. coli to
Fluoroquinolones.
43

42. 2. Extra chromosomal mechanism : Plasmids
• Carry resistant genes to antibiotics (r - genes) aka
R- plasmids
• Transferred from one R- plasmid to another
plasmid or chromosome.
• Horizontal gene transfer
44

43. Conjugation:
most common
• Commonly observed with bacterial
population with high density eg. Gut
Transduction:
Less common
• R- plasmid is transferred to another
bacteria with the help of bacteriophage
• Eg. Staphyloccoci strains, streptococci
strains
Transformation:
least common
• Ability of certain bacteria to pickup free
DNA from environment
Transfer of r – genes between different bacteria
45

44. Transfer of r – genes between plasmids within the
bacterium
• Transposons – mobile genetic elements which can
move anywhere in a genome and transfer r – genes
between plasmids or from plasmid to chromosome.
• Transposons co-integrate with acceptor plasmid and
replicate
• Integrons - Larger mobile genetic elements
• packed with multiple gene cassettes each containing
r- gene
• Cannot promote self transfer like transposons.
46

45. 47
Transposons Integrons

46. Drug Mode of action Gene Gene
function
Role
First-line
Isoniazid Inhibition of mycolic acid
biosynthesis and other metabolic
processes
katG
inhA
ndh
ahpC
Catalase-peroxidase
Enoyl ACP reductase
NADH dehydrogenase II
Alkyl hydroperoxidase
Prodrug activation
Drug target
Activity modulation
Resistance marker
Rifampicin Inhibition of transcription rpoB β-subunit of RNA
polymerase
Drug target
Pyrazinamide Inhibition of trans-translation pncA
rspA
Pyrazinamidase
S1 ribosomal protein
Prodrug activation
Drug target
Ethambutol Inhibition of arabinogalactan
synthesis
embCAB
embR
Arabinosyltransferases
embCAB transcription
regulator
Drug target
Drug target
expression
Streptomycin Inhibition of translation rpsL
rrs
gidB
S12 ribosomal protein
16S rRNA
16S rRNA
methyltransferase
Drug target
Drug target
Target modification
Second-
line
Amikacin/Kanamy
cin
Inhibition of translation rrs
eis
16S rRNA
Acetyltransferase
Drug target
Drug modification
Ethionamide Inhibition of mycolic acid
biosynthesis
ethA
inhA
ethR
ndh
mshA
Flavin monooxygenase
Enoyl ACP reductase
ethA transcription
repressor
NADH dehydrogenase II
Glycosyltransferase
Prodrug activation
Drug target
Prodrug activator
expression
Activity modulation
Prodrug activation
Fluoroquinolones Inhibition of DNA gyrase gyrA
gyrB
DNA gyrase subunit A
DNA gyrase subunit B
Drug target
Drug binding
48Smith T. Molecular Biology of Drug Resistance in Mycobacterium tuberculosis. Curr Top Microbiol Immunol. 2013 ; 374: 53–80

47. Mechanism of resistance
AMA MOA MOR
Sulfonamide Structural analog of PABA
Inhibit folate synthetase – FA not
formed
•Increased production of
PABA
•Low affinity folate
synthetase enzyme
•Alternate folate
metabolism pathway
Co-trimoxazole Inhibits dihydrofolate reductase
(DHFRase)
•Low affinity DHFRase
Fluoroquinolon
es
Inhibits bacterial enzyme DNA
gyrase or topoisomerase IV
•Low affinity DNA gyrase
or topoisomerase IV
•↓ permeability
•↑ efflux
Beta lactams Inhibit transpeptidases (PBPs)–
crosslinking of peptidoglycan
residues does not occur
•β- lactamases
•Altered PBPs
•Active efflux 49

48. AMA MOA MOR
Tetracyclines Bind to 30S ribosomes
– inhibit protein synthesis
•↓ influx
•Active efflux
•Inactivating enzymes
Chloramphenicol Bind to 50S ribosomes
– inhibit protein synthesis
•Acetyl transferase –
inactivates CPC
•↓ influx
•Low affinity ribosomes
Aminoglycosides STM- 30S ribosome
Others – 30S, 50S & 30S- 50S
interface
= inhibit protein synthesis
•Inactivating enzymes
•↓ affinity of ribosomal
proteins •↓ efficiency of
AG transporting mechanism
Macrolides Binds with 50S ribosome
subunits – inhibits protein
synthesis
•↓ permeability
•↑ efflux •Erythromycin
esterase •Alteration in
ribosomal binding by
methylase enzyme
50

49. Methods for Determining
resistance
E-test®
Kirby-Bauer
Disk Diffusion
Agar dilution
Automated
techniques
1.Phenotypic method:-
Estimation of phenotypic
expression of resistance to
antimicrobial drugs.
• Agar screen
• Disc diffusion/ broth
dilution/ E-test
• MIC determination
(breakpoint)
• Automated method (Vitek,
Microscan)
51
Anjum M. Screening methods for the detection of antimicrobial
resistance genes present in bacterial isolates and the microbiota.
Future Microbiology. 2015;10(3):317-320

50. Methods for Determining
resistance
2.Genotypic method:-
Detection of genes or nucleotide sequences
responsible for coding resistance.
• DNA hybridization
• Nucleic acid amplification (PCR) technique
• Microarray techniques
52
Anjum M. Screening methods for the detection of antimicrobial resistance genes present in bacterial isolates and the microbiota. Future
Microbiology. 2015;10(3):317-320

51. How to overcome
Contain exsisting resistance
Avoid future development of resistance
53

52. Judicious use of use antibiotics
54

53. Strategy to Contain Resistance
•Develop new antibiotics
• Bypass the drug
resistance
Problems??
Other options??
55

54. 56Rello J, Bunsow E, Perez A. What if there were no new antibiotics? A look at alternatives. Expert Review of Clinical Pharmacology.
2016;9(12):1547-1555.

55. Antibiotic stewardship
• A program that encourages judicious use of
antibiotics
• Who’s work??
57

56. Who’s Work?
Microbiologist
Physician
Bacterial
sensitivity test
and find out
the possible
causes of
development
Treat Infection58

57. Multidisciplinary team
Microbiologist
Physician
Pharmacologist
New drug development
Surveys and audits
Formulating STG,
Policies for use of AB in
hospital, for govt.
Formulary updation
Essential drug list 59

58. Antibiotic stewardship
at patient level
Control exsisting resistance
Is antibiotic really needed ?
Empiric rx - Clinical
diagnosis, Evidence
Definitive rx – culture
Optimal drug selection –
Dose, ROA, Duration,de-
escalation
Parenteral to oral?
Adjunctive rx – catheter
removal
60. Mendelson M. Role of antibiotic stewardship in extending the age of modern medicine. South African Medical Journal. 2015;105(5):414

59. Antibiotic stewardship at
community level
• Aviod further development of resistance
• Multidisciplinary team
• Generate public awareness - drug resistance, imp. of
vacination, hygiene in preventing infection
• Interventions to improve antibiotic prescribing – CME,
educational sessions, antibiotic prescription charts, hospital
antibiograms
• Conduct surveys and prescription audit with feed back
• Formulate policies – hospital and national use to Curb misuse
of AMA, control the spread of infection and save money
61
Mendelson M. Role of antibiotic stewardship in extending the age of modern medicine. South African Medical Journal. 2015;105(5):414

60. 1. The National Policy for Containment of Antimicrobial
Resistance (2011)
– not yet implemented.
- Strategy to monitor sale of antibiotics (misuse, overuse), AMR
survelliance (lab), promote research, measures to enhance
rational use of antibiotics in hospital and curb its use in
veterinary and industry
2. 12th Five Year Plan- Containment of AMR (2012 - 2017)
– create awareness for rational use of antibiotics
among health care providers and common people
Initiatives taken by India with
respect to AMR
62

61. 3. The Drugs and Cosmetic Rule,
1945 was amended in 2013 to
include a new Schedule H1.
• 46 drugs
• Sold only with prescription of
RMP
• The drug should be labeled with
the symbol Rx in red and warning.
63

62. 4. National Anti-Microbial Resistance
Research and Surveillance Network
(AMRRSN) (ICMR) (2015)
- to compile national data of AMR at
different levels of Health Care
5. National Treatment Guidelines for
Antimicrobial Use in Infectious Diseases
(2016 v1)
6. ICMR - Treatment Guidelines for
Antimicrobial Use in Common Syndromes
7. The Union Health Minister launched
a campaign called “Medicines with the
Red Line” - promote rational use
64

63. Antibiotics are
a precious
resource
We need to
preserve this
resource by
working together
Combating
antimicrobial
resistance: No action
today, no cure
tomorrow
Thank you 65