2.  Relative or complete lack of effect of
antimicrobial against a previously susceptible
microbe
 Increase in MIC

3. Figure 20.20

4. Horizontal Gene Transfer
A = Transformation; B = Conjugation; C = Transduction

5. • Enzymatic destruction of drug
• Prevention of penetration of drug
• Alteration of drug's target site
• Rapid ejection of the drug

6.  Clinical resistance vs actual resistance
 Resistance can arise by mutation or by gene transfer (e.g.
acquisition of a plasmid)
 Resistance provides a selective advantage
 Resistance can result from single or multiple steps
 Cross resistance vs multiple resistance
› Cross resistance -- Single mechanism-- closely related
antibiotics
› Multiple resistance -- Multiple mechanisms -- unrelated
antibiotics

8. Terminologies
Resistant organism
MICs of organism are higher than achieved drug
concentrations in tissues
Intermediately resistant
the antibiotic may still be effective but higher
doses should be used
Highly resistant
the antibiotic tissue concentrations are likely not
to exceed MICs of the microorganisms

9. Types of resistance
Intrinsic or natural resistance
G-neg bacteria are resistant to vancomycin (large
molecule)
Tetracyclines are hydrophobic, G-neg bacilli are
resistant
Acquired resistance
Mutations (PBP)
Disseminated by plasmids and transposons
Spontaneous mutations

10. Mechanisms of antibiotic resistance
1. Production of enzymes
destroying and modifying AB
ß-lactamases AG modifying
enzymes
2. Decrease of cell
membrane permeability
3. Active efflux of AB from
cell
4. Modification of AB target
sites

11. Genetics and spread of drug resistance
Viridans Streptococci
S.pneumoniae
S.Epidermidis
S.aureus
E.faecium
S.aureus

12. Transposon .
genes moving from one point to another (jumping genes)
Bacteriophage
virus, infecting bacteria (virus of bacteria)
Integron
slice(s) of DNA, cassette of gene that may be entered into
other cell
Plasmid
circular double stranded DNA molecule, located separately
of the chromosomal RNA

13. (1) Mechanisms of resistance
Production of enzymes inactivating (destroying)
antibiotics
ß-lactamases
Main mechanism of resistance in ß-lactam
antibiotics
Penicillin-resistant S.aureus
Ampicillin-resistant E.coli
Production of enzymes modifying antibiotics
Aminoglycosides, chloramphenicol

14. Resistance mechanisms: inactivating enzymes (2)
Degrading enzymes will bind to the Blocking enzymes attach side chains
antibiotic and essentially degrade it to the antibiotic that inhibit its function.
or make the antibiotic inactive E.g. ß-lactamases

15. PBP & ß-lactamase
Serine proteases (PBP) a metalloenzymes (Zn-binding thiole group as
coenzyme)
200 different enzymes e.g. penicillinases, cephalosporinases, ESBL,
AmpC
ESBL - extended spectrum ß-lactamases (broad spectrum of activity);
encoded in plasmids, can be transferred from organism to organism

16. Production of ß-lactamases: mechanism of
action
Examples
TEM-1 is a
widespread ß-
lactamase of
Enterobacteriaciae
that attacks
Penicillin G and
narrow spectrum
cephalosporins
>50% AmpR
E.coli isolates are
caused by TEM-1

17.  Altered
permeability
› Altered influx
 Gram negative
bacteria

18. Efflux Mechanisms of resistance
Antibiotics are removed via active efflux pump
Universal efflux pump
specific efflux pump
quinolones, tetracyclines, chloramphenicol

19. Resistance mechanisms: efflux pump
The efflux pump is a membrane bound protein that
"pumps" the antibiotic out of the bacterial cell

20. Microbe Library
American Society for Microbiology
www.microbelibrary.org

21.  Altered
permeability
› Altered efflux
 tetracycline
Microbe Library
American Society for
Microbiology
www.microbelibrary.org

22.  Inactivation
› ß-lactamase
› Chloramphenicol acetyl
transferase
Microbe Library
American Society for
Microbiology
www.microbelibrary.org

23. Mechanisms of resistance
Modification of target sites
altered PBP (PRSP)
new PBP (MRSE, MRSA)
Modification in ribosomes (macrolideresistant
S.pneumoniae)

24.  Altered target site
› Penicillin binding
proteins (penicillins)
› RNA polymerase
(rifampin)
Microbe Library
American Society for
› 30S ribosome Microbiology
(streptomycin) www.microbelibrary.org

25. Modification of AB target sites:
disruption in protein synthesis

26. Important terms among drug
resistant microorganisms
VRE . vancomycin-resistant enterococci
70% of E. faecium strains in USA
GISA . glycopeptide intermediately susceptible S.aureus
VISA . vancomycin intermediately susceptible S.aureus
VRSA & VRSE . vancomycin-resistant S.aureus and S.epidermidis
 (MIC> 32 mcg/ml; 1st clinical case described in 2002 in USA)
ESBL producing K.pneumoniae . Extended spectrum ß-lactamase
producing K. pneumoniae
PRSP penicillin-resistant S. pneumoniae

28. ß-lactam antibiotics:
penicillins
cephalosporines
carbapenems

29. Alexander Fleming
P. chrysogenum
(original strain of Fleming)
destroy Staphylococcus aureus
1928

30. ß-lactam structure is presented in red and blue
Side chain is presented in black

31. Penicillins
Carbapenems
Cephalosporins

32. Mechanism of action of ß-lactam antibiotics
1ß-lactam ab
binds to PBP
2. Inhibition of
peptidoglycan
synthesis
3. Cell death

33. Structure of peptidoglycan
ß-lactams inhibit synthesis of crosslinks

34. Penicillins

35. Cephalosporins
Initially isolated form
the mould Cephalosporium
Compared with penicillins:
More resistant to ß-
lactamase hydrolysis
Wider antibacterial spectrum
Improved PK-properties

36. Resistance to ß-lactam
antibiotics

38. Resistance to ß-lactam antibiotics
Production of ß-lactamases
Penicillin-resistant S.aureus (>95%) - Synthetic
Penicillins
ESBL K.pneumoniae - IV generation cephalosporins,
carbapenems
Ampicillin-resistant E.coli – cephalosporins
Changes in the structure of PBP
(altered PBP) Penicillin-resistant S.pneumoniae -
larger doses of penicillin
New PBP - MRSA, MRSE . vancomycin

39. Disruption of bacterial cell wall
Glycopeptides
vancomycin
teicoplanin

40. Vancomycin: mechanism of action
Mechanism - vancomycin inhibits cross linkage between
peptidoglycan layers
Vancomycin can bind only to D-Ala-D-Ala and not to D-Ala-D-lac

41.  Originally obtained form
Streptomyces orientalis
 Active only against G+
bacteria (large molecule
unable to penetrate outer
membrane of G+ bacteria)
 Used for treatment of
oxacillin resistant G+
infections

42.  Intrinsic resistance (pentapetide end with D-Ala-D-Lac)
 Leuconostoc, Lactobacillus, Pediococcus
Or with D-Ala-D-Ser
 Enetrococcus gallinarum, Enetercoccus caselliflavus
 Acquired resistance
 A thickening of the PG layer, and
 Modification of the PG termini from D-Ala--D-Ala to D-Ala--D-lactate
 Gene (vanA, B, C, D, G, E) is carried on plasmids & may be
transferred from organism to organism
 Importance
 VRE - vancomycin resistant E. faecium, E.faecalis
 VISA - vancomycin intermediately resistant S.aureus
 GISA - glycopeptide intermediately resistant S.aureus
 VRSA - vancomycin resistant S.aureus (MIC> 32 µg/ml; 1st
clinical case reported 2002 in US)

43. Mechanism of Resistance to Vancomycin

44.  Bacitracin (cyclic peptides) is isolated form Bacillus
licheniformis
 Topically applied agent against G+ bacteria
 Interferes with the dephoshorylation and recycling of the
lipid carrier responsible for moving peptidoglycan
precursors
 Polymyxin (cyclic polypeptides) derived from Bacillus
polymyxa
 Interact with the lipopolysaccharides and phospholipids in
the outer membrane and thus increase cell permeability
 Mostly active against G- bacilli (G+ bacilli do not have
outer membrane)

45. Activity of antibiotics to bacterial
cell wall
polypeptides ß-lactams
glycopeptides
G-negative
G-positive

46. Inhibition of protein synthesis
Aminoglycosides
Tetracyclines
Oxazolidones
Chloramphenicol
Macrolides
Clindamycin
Streptogramins

47. Protein synthesis

48. Substance binding to 30S subunit

49. Antibiotics that act at the level of protein
synthesis initiation

50. Antibiotics that act at the level of the
elongation phase of protein synthesis

51.  Consists of aminosugars that are
linked through glycosidic rings
 Origin
 Streptomyces - streptomycin,
 neomycin, kanamycin, tobramycin
 Micromonospora - gentamicin,
Sisomicin
 Synthetic derivates
 Amikacin = kanamycin
 Netilmycin = sisomycin
Mainly active against G-negative
bacteria
Gentamycin

52. Aminoglycoside: mode of action
AG pass through cell wall,
cytoplasmic membrane to
cytoplasma (mainly of Gbacteria,
no penetration through cytoplasmic
membrane of strepto- and
entrococci)
Bind irreversible to the 30S
subunit of bacterial ribosomes and
block the attachment of the 50S
subunit to the initiation complex
As a result production of
aberrant proteins and misreading
of RNA occurs

53. Aminoglycoside: mode of action
1. Passage through cytoplasmic membrane of G- bacteria (no penetration
through cytoplasmic membrane of strepto- and enterococci)
2. Binding to 30S subunit
3. Misreading the codon along mRNA
4. Inhibition of protein synthesis

54. Aminoglycoside resistance
Enzymatic modification (common) of the drug
 High level resistance
>50 enzymes identified
Genes encoding resistance located in plasmids
Gene transfer occurs across species
 Reduced uptake or decreased permeability of bacterial
cell wall
 Resistance in anaerobes (transport through
cytoplasmic membrane depends on anaerobic respiration)
Altered ribosome binding sites (rare)
Microbes bind to multiple sites
Low level resistance

55. Tetracyclines
Origin
Tetracyclin, oxytetracyclin isolated from Streptomyces
 Minocyclin, doxycyclin are synthetic
 Broad spectrum bacteriostatic antibiotics
Antibacterial spectrum similar to macrolides (incl. Clamydia,
Mycoplasma, Rickettsia)
Resistance (widespread)
Energy dependent efflux pump (most common)
Alteration of ribosomal target (ribosome protection)
Enzymatic change

56. Tetracyclines
The tetracyclines block
bacterial translation by binding
reversibly to the 30S subunit and
distorting it in such a way that the
anticodons of the charged tRNAs
cannot align properly with the
codons of the mRNA

57. Oxazolidones: linezolid
Newest class of antibiotics; completely synthetic
Narrow spectrum of activity (G+ bacteria, includingVRE,
MRSA)
G-neg bacteria resistant due to efflux pump
Mode of action: unique mechanism among antibiotics;
interferes with the initiation complex at the 50S ribosome
subunit (V domain of 23S rRNA)
Resistance confers to mutation at 23S rRNA
Resistance is rare; cross-resistance unlikely because 23S
rRNA is encoded by several genes

58. Oxazolidones: mode of action
Inhibit the formation of an initiation complex by binding to the 50S
ribosomal subunit (domain V of the 23S rRNA), disrupting the preliminary
phases of protein synthesis

59. Chloramphenicol
Binds irreversible to peptidyl transferase component of 50S
ribosome and blocks peptide elongation, thus interferes with
protein synthesis
Bacteriostatic antibiotic with broad spectrum of antibacterial
activity
Interferes with the protein synthesis of bone marrow cells
causing aplastic anaemia
Limited clinical use in Western world due to side Effect
Resistance is associated with producing
acetyltransferase which catalyses acetylation of 3-hydroxy
group of chloramphenicol

60. Macrolides (1)
Erythromycin was derived from Streptomyces erythreus
The basic structure is a lactone ring
14-membered lactone ring . erthromycin, clarithromycin, roxithromycin,
telithromyin (ketolide)
15-membered lactone ring . Azithromycin
16-membered lactone ring . spiramycin, josamycin
Acitivity .
broad spectrum G+ bacteria and some G- bacteria including
Chlamydia, Mycoplasma, Legionella, Rickettsia, Neisseria
Azithromycin, Clarithromycin active against some mycobacteria

61. Macrolides: mode of action
Blocking Translation during Bacterial Protein
Synthesis erythromycin
The macrolides bind reversibly to the 50S subunit.
They can inhibit elongation of the protein by the peptidyltransferase, the
enzyme that forms peptide bonds between the amino acids.

62. Mode of Action of Macrolides in Blocking
Translation during Bacterial Protein
Synthesis
The macrolides bind reversibly to
the 50S subunit.
They can inhibit elongation of the
protein by blocking the translocation
of the ribosome to the next codon on
mRNA

63. Macrolide resistance
Resistance
Intrinsic resistance- hydrophobic macrolides
have low permeability through outer membrane
(G- bacilli)
Acquired resistance
Ribosomal modification
Efflux pump
Enzyme inactivation

64. Clindamycin, lincomycin
Family of lincosamide antibiotics originally isolated from
Streptomyces lincolnensis
Mode of action: bind 50S ribosome subunit and blocks
protein elongation
Resistance is related to 23S ribosomal RNA Methylation
Active against staphylococci and G-ve anaerobic bacilli.
No activity against aerobic

65.  Replacement of a
sensitive pathway
› Acquisition of a
resistant enzyme
(sulfonamides,
trimethoprim)

66. Molecular Drug Susceptibility Testing
• Genotypic methods: the
drug target and nature of
the gene mutation are
known
• Usually molecular
amplification of target
DNA or RNA followed by
some means of detecting
mutation in the product.

67. Molecular methods of drug susceptibility testing
1. Sequencing
Universal and
reliable method
Expensive, time-
consuming and not
suitable for
everyday routine
testing
Applied as
reference method to
verify results of other
tests.

69. 2. PCR-based methods
PCR-Single Strand
Conformation
Polymorphism (PCR-
SSCP)
Mutations cause
alterations in
conformation of single-
strand DNA fragments
and it is registered in
non-denaturizing PAGE

70. Other molecular methods of drug susceptibility
testing:
Real-Time fluorescent PCR
Molecular combines amplification and
beacons detection: minimises amplicon
contamination

71. PCR+hybridization
Based on amplification of fragments of genes
responsible for drug resistance development
follwed by hybridization with oligonucleotide
probes immobilized on membranes;
Both commercial kits and in-house macro-
arrays have been reported to demonstrate
high sensitivity and specificity

72. Molecular tests for the detection of resistance to RIF and INH
GenoType® MTBDRplus test procedure

73. Reaction zones of GenoType® MTBDRplus (examples)

74.  Exposure to sub-optimal levels of
antimicrobial
 Exposure to microbes carrying resistance
genes

75.  Prescription not taken correctly
 Antibiotics for viral infections
 Antibiotics sold without medical supervision
 Spread of resistant microbes in hospitals due
to lack of hygiene

76.  Lack of quality control in manufacture or
outdated antimicrobial
 Inadequate surveillance or defective
susceptibility assays
 Poverty or war
 Use of antibiotics in foods

77.  Antibiotics are used in animal feeds and
sprayed on plants to prevent infection and
promote growth
 Multi drug-resistant Salmonella typhi has
been found in 4 states in 18 people who ate
beef fed antibiotics

78.  Infections resistant to
available antibiotics
 Increased cost of
treatment

80.  Methicillin-Resistant Staphylococcus aureus
 Most frequent nosocomial (hospital-acquired)
pathogen
 Usually resistant to several other antibiotics

81.  Speed development of new antibiotics
 Track resistance data nationwide
 Restrict antimicrobial use
 Direct observed dosing (TB)
 Use more narrow spectrum antibiotics
 Use antimicrobial cocktails

82. Ecology of Antimicrobial Resistance

84.  Antimicrobial peptides
› Broad spectrum antibiotics from plants and
animals
 Squalamine (sharks)
 Protegrin (pigs)
 Magainin (frogs)

85.  Antisense agents
› Complementary DNA or peptide nucleic
acids that binds to a pathogen's virulence
gene(s) and prevents transcription

86. DID U KNOW T HAT>>>>>>>>>
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