antimicrobial resistance mechanisms

1. Antimicrobial resistance

2. Terms
Antibiotic: Chemical produced by a microorganism that kills or
inhibits the growth of another microorganism
Antimicrobial agent: Chemical that kills or inhibits the growth
of microorganisms

3. MIC = Minimal inhibitory concentration.
Lowest concentration of antimicrobial that inhibits growth of
bacteria.
Commonly used in clinical lab
MBC = Minimal bactericidal concentration.
Concentration of an antimicrobial that kills bacteria.
Used clinically only in special circumstances

4. • Susceptible: the antibiotic is effective against the bacteria
• Resistant:the bacteria can grow even if the drug is present
• Breakpoint = The MIC that is used to designate between
susceptible and resistant.
– Arbitrarily set by a committee
•

5. • Antimicrobial Resistance
• Relative or complete lack of effect of antimicrobial against a
previously susceptible microbe
• Increase in MIC

6. Antibiotic resistance is a type of drug resistance where a
microorganism is able to survive exposure to an antibiotic.
Resistant organism: One that will not be inhibited or killed by
an antibacterial agent at concentrations of the drug achievable
in the body after normal dosage.

7. Timeline of Antibiotic Resistance

8. Factors contributing for resistance
 Misuse of antibiotics
Use of antibiotics with no clinical indication (e.g, for viral infections)
Use of broad spectrum antibiotics when not indicated
Inappropriate choice of empiric antibiotics
 Overuse of antibiotics
 Addition of antibiotic to the feed of livestock
 Failure to follow infection control practices

9. Why resistance is important
• 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

11. Mechanisms of action of antibiotics

12. Figure 1: Sites of action and potential mechanisms of bacterial resistance to antimicrobial agents.
Mulvey M R , Simor A E CMAJ 2009;180:408-415
©2009 by Canadian Medical Association

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

14. Antibiotic resistance in bacteria
 Two types:
Intrinsic:
 Naturally occuring trait
 Species or genus specific
Acquired:
 Acquired resistance implies that a susceptible organism has developed resistance to
an agent to which it was previously susceptible
 can occur in two general ways: by mutation (s) in the existing DNA of the organims
or by acquisition of new DNA.
 Present in only certain strains of a species or of a genus

16. Antibiotics Resistance
• The four main mechanisms of antibiotic resistance are
• (1) enzymatic degradation of the drug
• (2) modification of the drug's target
• (3) reduced permeability of the drug
• (4) active export of the drug.
• Most drug resistance is the result of a genetic change in the
organism, caused either by a chromosomal mutation or the
acquisition of a plasmid or transposon.

17. Genetic Basis of Resistance
• Chromosomal mutations
– A single chromosomal mutation may result in the synthesis of an
altered protein
– change the target of the drug so that the drug does not bind or
– change the membrane so that the drug does not penetrate well into
the cell.

18. Genetic Basis of Resistance
Chromosomal mutations
Chromosomal mutations occur at a low frequency (perhaps 1 in 10
million organisms), and often affect only one drug or one family of
drugs.
 streptomycin resistance via alteration in a ribosomal protein
 the single aminoacid change in the enzyme dihydtropteroate synthetase
resulting in a lowered affinity for sulfonamides
 A series of mutations, for example, changes in penicillin binding proteins
(PBPs) in penicillin resistant pneumococci

19. Genetics of Resistance
 Resistance by acquisition of new DNA
– By Transformation
– Conjugation
– Transduction
Nature of elements involved in transferring DNA:
 Plasmids: plasmid mediated resistance much more efficient than the
resistance ass. with chromosomal mutation
 Transposons

20. 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.
• Many of the drug resistance encountered in clinical practice is
plasmid mediated

21. Plasmids
• Resistance plasmids (R plasmids, R factors) usually carry two
sets of genes.
– One set encodes the enzymes that degrade or modify drugs
– the other encodes the proteins that mediate conjugation

22. 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

23. 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.

25. Mechanisms of Resistance Gene Transfer
Transposons
Transposons are small pieces of DNA
that move from one site on the
bacterial chromosome to another or
from the bacterial chromosome to
plasmid DNA.
Transposons often carry drug
resistance genes.
Many R plasmids carry one or more
transposons.

26.  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.
Mechanisms of Resistance Gene Transfer
Transposons

27. 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

28. Resistance mechanisms
Enzymatic inactivation
Microbes can also express drug resistance genes that code
for enzymes that covalently
modify the antimicrobial drug, thereby reducing its antimicrobial
activity
Mutations, plasmid and/or transposones
Beta-lactam, Aminoglycoside, Chloramphenicol, Macrolids

29. Resistance mechanisms
Enzymatic inactivation
Beta-lactamases
hydrolyze the beta-ring of betalactams, thereby inactivating the
antibiotic activity of the beta-lactam molecule and conferring beta-
lactam resistance

30. Resistance mechanisms
• Enzymatic inactivation
• Aminoglycoside
– ACT N-acetyltransferase, which acetylates an NH2 group of the
aminoglycoside molecule
– APH O-phosphotransferase, which phosphorylates an OH group of the
aminoglycoside molecule
– ANT O-adenyltransferase, which adenylates an OH group of the
aminoglycoside molecule
• Chloramphenicol
– Acetyltransferases, which acetylate and thereby inactivate
chloramphenicol

32. Resistance mechanisms
Decreased Permeability
Decreased uptake(impermeability) and increased efflux of drug from
the microbial cell
 Decreased uptake of antimicrobial drugs and/or use of transmembrane efflux pumps prevents the
concentration of antimicrobial agent from increasing to toxic levels within the microbial cell
(↓uptake↓conc↓effect).

33. Resistance mechanisms
 Outer membrane may provide an extra barrier
against drug uptake (especially hydrophobic drugs)
in Gram negative bacteria, which is not present in
Gram positive bacteria
 This is one explanation why Gram negative bacteria
are less susceptible than Gram positive bacteria to
many antibiotics, including beta-lactams and
macrolids

34. Resistance mechanisms
Efflux Pumps
Activation of efflux pumps by mutation or plasmids
Quinolones, macrolids, tetracyclins
1. The major facilitator subfamily (MFS),
2. The resistance nodulation cell division subfamily (RND),
 Pseudomonas aeruginosa (MexAB)
3. The small multidrug regulator subfamily (SMR),
4. The adenosine triphosphate (ATP)-binding cassette (ABC) family
5. The multidrug and toxic effects (MATE) family.

35. Resistance mechanisms
Target Alteration
Expression of resistance genes that code for an altered
version of the substrate to which the antimicrobial agent
binds
GENE mutation → translated to altered
protein( substrate) → low binding affinity→ reduced
antibacterial activity → resistance developed

36. Resistance to β -lactams:
 Resistance due to β -lactamases: most prevalent
 Alteration in the pre-existing penicillin binding
proteins (PBPs)
 Acquisition of a novel PBP insensitive to beta β –
lactams: e.g, methicillin resistance in
Staphylococcus aureus (MRSA)
 Changes in the outer membrane proteins of Gram
negative organisms that prevent these compounds
from reaching their targets

37. Resistance to β -lactams
• β –lactamases
– TEM, SHV, KPC, VIM…
– Effect different betalactams
• Methicillin resistance
– If bacteria resistance to methicillin, bacteria accepted resistant against
penicillins and cephalosporins
– By mecA ans MecCgene PBP2a
– PBP2a shows low affinity to betalactams
– mecA gene PBP2a low affinity to betalactams cell wall synthesis continues

38. Aminoglycoside Resistance:
– Resistance to aminoglycosides is mediated by three main
mechanisms:
– modification of the drug by
• phosphorylating, adenylylating, and acetylating
enzymes,
– Mutations in the genes encoding one of the 30S ribosomal
proteins,
– Reduced permeability

39. Tetracycline resistance
• Most common antibiotic resistance encountered in
nature
• Mechanisms:
– Altered permeability due to chromosomal mutations
– Active efflux or Ribosomal protection (by production of a
protein) resulting from acquisition of exogenous DNA

40. Macrolids
Macrolide, Lincosamide and Streptogramin resistance:
 Resistance to erythromycins is primarily caused by a plasmid-
encoded enzyme that
 methylates the 23S ribosomal RNA, thereby blocking binding of the drug.
– Intrinsic resistance is due to low permeability of outermembrane
protein
– Drug inactivation and active efflux may also occur

41. Quinolone resistance
 Resistance to quinolones is primarily caused
by mutations in the gene encoding the
bacterial DNA gyrase
 Decreased permeability
 Efflux pumps

42. Glycopeptide resistance
 Alteration of target
Resistance to vancomycin is caused by a change in the D-ala-D-
ala part of the peptide in peptidoglycan to D-ala-D-lactate,
Vancomycin binds to d-Ala–d-Lac with markedly lower affinity
than it does to the normal dipeptide product

43. • Van genes mainly cause glycopeptide resistance
• Van A-G
• Van A is the most common, confers resistance to vancomycin
and teicoplanin
• VanC is chromosomal in E. casseliflavus and E.gallinorum