Antimicrobial Chemotherapeutic Agents

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Antimicrobial Chemotherapeutic Agents
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Drug Resistance
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• In the clinical context an organism is said to be
  resistant
• If it is not killed or inhibited by drug
  concentrations readily attainable in the patient
  this usually means blood and tissue
  concentrations.
• However, an organism resistant to these may of
  course be sensitive to the higher concentrations
  attainable in urine or by topical application.
• Even the broadest of broad-spectrum antibacterial
  drugs is ineffective against some bacterial
  genera, against some species of other genera, and
  usually against some strains of species that are
  in general sensitive to it.

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Resistance
Inherent (non specific)
Acquired
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Inherent (non specific) Resistance

• Certain bacteria are, and as far as we know
  always have been, more or less resistant to some
  antibiotics.
• For example, gram-negative bacteria, especially
  Ps. aeruginosa, are
  inherently resistant to a number of antibiotics
  that are very effective against gram-positive
  bacteria such as penicillin G, erythromycin,
  lincomycin.

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Acquired Resistance

• When a bacterial population adapts to the
  presence of an antibiotic, sensitive cells are
  gradually replaced by resistant cells as in the
  presence of antibiotic.
• Resistant cells continue to grow at the expense
  of sensitive cells.
• When a new antibiotic is introduced into clinical
  practice for the treatment of infections caused
  by bacteria that are not inherently resistant to
  the drug, the majority of infections respond to
  the new drug.

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• But following months or years of continuous use,
  resistant strains are reported.
• The degree of resistance and the speed with which
  it develops varies with
• ? The organism ? The drug.
• Generally, the development of acquired bacterial
  resistance is common and must be usually expected
  with some exceptions.

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• Streptococcus pyogenes has remained sensitive to
  Penicillin G after 40 year's exposure to the
  drug.
• Staph aureus develops slow or multisteps
  resistance to penicillin, chloramphenicol and
  tetracycline.
• While Mycobacterium tuberculosis and various
  organisms develops sudden or one step resistance
  to Streptomycin.

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Biochemical Mechanisms of Resistance
Production of drug-inactivating enzymes
Switch to alternative metabolic pathways
unaffected by the drug
?
?
Change in the antibiotic target site
?
Increased production of essential metabolite
?
. Reduction in cellular permeability to the
antibiotic
?
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Inactivation of Aminoglycosides
Inactivation of ß-lactams
Inactivation of Chloramphenicol
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Inactivation of Aminoglycosides

• Inactivation of aminoglycosides by plasmid
  controlled adenylating , phosphorylating or
  acetylating intracellular enzymes of drug
  resistant gram negative bacteria.
• Although the inactivating enzymes vary
  considerably in their substrate and
  specificities, known modifications are restricted
  to acetylation of amino groups and adenylylation
  or phosphorylation of hydroxyl groups.

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Inactivation of ß-lactams

• Inactivation of ß-lactams by ß -lactamases
  ( penicillinase,
  cephalosporinase ) into penicilloic or
  cephalosporoic acid.
• ß -lactamases are

Gram-positive
Gram-negative
Chromosomally
Plasmid
Constitutive
Inducible
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Penicilloic acid
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• The synthesis of gram positive ß-lactamase is
  induced by the antibiotic themselves and is
  released extracellularly and destroy antibiotic
  in the external environment.
• Most strains of gram-negative cells, by contrast,
  synthesize ß-lactamases constitutively. i.e.
  continuously and are not released into the
  external environment (cell-bound or
  intracellular).

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• ? Chromosomally-mediaied ß-lactamases of gram
  negative hydrolyze cephalosporins more rapidly
  than penicillins and are inhibited by cloxacillin
  but not by clavulanic acid.
• However, those of Aeromonas spp. and Klebsiella
  spp. are more active against penicillins and not
  inhibited by cloxacillin.

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1
2
R1 cl , R2 H (Cloxacillin)
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• Inducible types are found in microorganisms such
  as Pseudomonas spp., Proteus and other gram
  negative bacteria but infrequently in E. coli in
  which, as many enterobacter species, constitutive
  types could be isolated.

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• ? Different types of plasmid-mediated
  ß-lactamases were isolated.
• TEM type enzymes are present in almost all gram
  negative bacteria.
• These enzymes were first isolated from E. coli
  strains isolated, in Athens, from a young girl
  called Temoniera and was referred to as TEM
  enzyme.
• Electrophoretically different type was then
  isolated from Pseudomonas aeruginosa (TEM-2).

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E. coli OXA
Klebsiella spp. SHV
H. influenza ROB
Aeromonas spp. AER
Ps. aeruginosa LCR
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Inactivation of chloramphenicol

• Inactivation of chloramphenicol by
  chloramphenicol acetyl transferase (CAT).
• Usually they are a plasmid-mediated enzymes which
  are inducible type in gram-positive bacteria but
  constitutive in gram-negative bacteria.
• These enzymes acetylates the OH groups in the
  side chain of the drug.

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• Replacement of the terminal - OH group of this
  side chain, which is normally the first to be
  acetylated by an inert fluorine atom, yields a
  chloramphenicol derivative that is not
  susceptible to attack by CAT.

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Chromosomal Resistance to Aminoglycosides
Resistance to Erythromycin
Resistance to Sulfonamides Trimethoprim
Resistance- to some Penicillins
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Chromosomal Resistance to Aminoglycosides

• It is associated with the loss or alteration of a
  specific protein in the 30S subunit of the
  bacterial ribosome that serve as a binding site
  in the susceptible organisms.

Resistance to Erythromycin

• It is associated with alteration, of its receptor
  on the 5OS subunit of the ribosome.

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Resistance- to some Penicillins

• Resistance- to some penicillins due to loss or
  alteration of Penicillin Binding Proteins
  (PBPs).

Resistance to Sulfonamides Trimethoprim

• Occurs by alteration of the tetrahydropteroat
  synthetase and tetrahydrofolate reductase,
  respectively, that have a much higher
  affinity for PABA than these drugs.

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• Bacterial cells altering the permeability of
  their cell membrane making it difficult for
  antimicrobials to enter.
• This type of resistance is found in bacteria
  resistant to
• Polymyxins.
• Tetracyclines.
• Amikacin some aminoglycosides.
• Streptococci have a natural permeability barrier
  to aminoglycosides.
• This can be partly overcome by combination with
  cell wall active drug, e.g. (penicillin).

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• The organism develop an altered metabolic pathway
  that bypasses the reaction inhibited by the drug
  e.g. some sulfonamide-resistan
  t bacteria do not require extra-cellular PABA
  but, like mammalian cells, can utilize preformed
  folic acid.

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• That is competitively antagonized by the drug in
  sensitive cells e.g. resistance to sulfonamides
  may be associated with high level of bacterial
  synthesis of PABA.

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The origin of drug resistance
Non Genetic Origin
Genetic Origin
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Non Genetic Origin

• This involves metabolically inactive cells or
  loss of target sites.
• Most antimicrobial agents act effectively only on
  replicating cells.
• Mycobacteria survive for many years in tissue
  yet are restrained by the host's defenses and do
  not multiply.
• Such persisting organisms are resistant to
  treatment and cannot be eradicated by drugs.
• When they start to multiply they are fully
  susceptible to the drugs.

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• Loss of a particular target structure, often
  induced by the drug, may result in antimicrobial
  resistance.
• Exposure of some gram-positive bacteria to
  penicillin results in the formation of cell
  lacking cell wall
  (i.e. L-forms).
• These cells then are penicillin resistant, having
  lost the structural target site of the drug.
• When these organisms revert to their bacterial
  parent forms resuming cell wall production, they
  are again fully susceptible to penicillin.

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Genetic Origin

• Most drug-resistant microbes emerge as a result
  of genetic change and subsequent selection
  processes by antimicrobial drugs.

The mechanisms by which genetic change occur
are
Chromosomal Resistance
Extra Chromosomal Resistance
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Chromosomal Resistance

• This develops as a result of mutation in a gene
  locus that controls susceptibility to a given
  antimicrobial drug.
• The presence of the drug serves as a selecting
  mechanism to suppress susceptible organisms and
  favor the growth of drug resistant mutant.
• Spontaneous mutation occurs at a frequency of 10
  7 to 10 12.

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• Chromosomal mutants are most commonly resistant
  by virtue of a change in a structural receptor
  for a drug as in bacterial resistance to
  erythromycin, lincomycin, aminoglycosides and
  others by alteration of their target site in
  susceptible cells.
• Prevention of the emergence of resistant mutants
  is one of the main indications for the clinical
  use of combinations of drugs.

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• But provided that the mechanisms of action of the
  two drugs are unrelated.
• Therefore if both drugs are given in adequate
  dosage, the risk of the emergence of a resistant
  strain is very much less than if either is used
  alone.

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Extra Chromosomal Resistance
Plasmids
Transposons
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Plasmids

• Bacteria often contain extra chromosomal DNA
  units known as plasmids.
• Some of which alternate between being free and
  being integrated into the chromosome.
• R factors are a class of plasmids that carry
  genes for resistance to one and often several
  antimicrobial drugs and heavy metals.

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• Plasmid genes for antimicrobial resistance often
  control the formation of enzymes that inactivate
  the antimicrobial drugs such as ß-lactamases, CAT
  and enzymes that inactivates aminoglycosides or
  enzymes that determine the active transport of
  tetracyclines across the cell membrane, and for
  others.

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Transposons

• The drug resistance (R) genes are often part of
  highly mobile short DNA sequences known as
  transposons (Transposable elements or jumping
  genes) that is able to move, from one position to
  another, between one plasmid and another or
  between a plasmid and a
  portion of the bacterial chromosome within a
  bacterial cell.

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• Thus, transposons are able to insert themselves
  into many different genomic sites with no
  homology with them.
• Simple transposons (IS) only carry information
  concerned with the insertion function.
• Simple transposons (IS) (i.e. insertion
  sequences) have no known effects
  beyond transposition and inactivation of the gene
  (or operon) into which they may insert.

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• Complex or composite transposons (Tn) contain
  additional genetic material unrelated to
  transposition, such as drug-resistance genes.
• Such as penicillin, kanamycin, streptomycin,
  sulfonamides, tetracyclines, chloramphenicol ,and
  trimethoprim.

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Mechanisms of Transmission of Genetic Material
and Plasmids
Transduction
Transformation
Conjugation
Transposition
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Transduction

• This is the main mechanism for transmission of
  antibiotic resistance between gram-positive
  cocci, and occurs in other bacterial groups.
• Plasmid DNA is enclosed in a bacteriophage and
  transferred by the virus to another bacterium of
  the same species e.g. the plasmid carrying the
  gene for ß-lactamase production can be
  transferred from a penicillin-resistant to a
  susceptible staphylococcus if carried by a
  suitable bacteriophage.

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Transformation

• Naked DNA passes from one cell of a species to
  another cell, thus altering its genotype.
• This can occur through laboratory manipulation
  and perhaps spontaneously.

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Conjugation

• This is the commonest method by which, multi-drug
  resistance spreads among different genera of
  gram-negative bacteria.
• But also occurs among some gram-positive cocci.

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• The usual plasmid found in resistant
  gram-negative bacteria consists of two distinct
  but frequently linked elements
• One or more linked genes each conferring
  resistance to a specific antibacterial drug
  (resistance
  determinants).
• A resistance transfer factor (RTF) that enable
  the cell to conjugate with a sensitive bacterium
  and to transfer to It a copy of the entire
  plasmid.

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R-factor
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• The entire linked complex of RTF and resistance
  determinants is known as R-factor and takes the
  form of a double stranded circular molecule of
  DNA.
• A proportion of R-factor-bearing cells (R
  cells) possess hair-like structures that extend
  out from the bacterial surface known as pili.
• The pili, whose synthesis is under the control of
  the RTF component of the R-factor are
  essential to the conjugation phenomenon with R'
  bacteria and the transfer of an R- factor.

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Transposition

• A transfer of short DNA sequences (transposon)
  occur between a plasmid and another or between a
  plasmid and a portion of the bacterial chromosome
  within a bacterial cell.

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Transferable or infective drug resistance is
important for the following reasons

1. The transferable plasmids commonly determine
   resistance to several unrelated drugs.
2. Such plasmids are transferable not merely to
   related strains of the same species but to
   strains of other species and genera for example,
   antibiotic-resistant but harmless organism in
   human or animal intestine
   (E. coli) can confer antibiotic resistance,
   by plasmid transfer, on pathogenic but previously
   
   antibiotic-sensitive bacteria of other genera
   which the host happens to ingest (such as typhoid
   or dysentery bacilli).

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1. It is possible for multiple-resistant
   enterobacteria to develop in farm animals and be
   transmitted to man. Development of this
   resistance is due to the widespread use of
   antibiotics especially cheap types, as food
   supplements for young animal to accelerate their
   growth by partial suppression of their intestinal
   flora.

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Specific and Cross Resistance

• Specific resistance When the organism acquire
  resistance to a certain drug but Its
  susceptibility to other drugs is unaffected.
• Cross resistance Microorganisms resistant to a
  certain drug may also be resistant to other drugs
  that share a mechanism of action.

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• Such relationship exist mainly between agents
  that are closely related
• Polymyxin B and Polymyxin E.
• Erythromycin and Oleandomycin.
• Neomycin and kanamycin.
• However, it may also exist between unrelated
  chemicals ? Erythromycin-Lincomycin.

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• When the active nucleus of the chemicals is so
  similar, extensive cross-resistance is to be
  expected e.g. resistance to one of the
  tetracyclines imparts resistance to the other
  members of the group e.g. resistance to one
  sulfonamide cause resistance to hundreds of other
  sulfonamides.

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Antibiotic Policies

• Abuse of antibiotics, is avoided for many reasons
  including the following
• To prevent the emergence of antibiotic
  resistance.
• To reduce the cost of antibiotic use.
• To prevent antibiotic toxicity.

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General Principles of Optimal Antibacterial
Therapy

• Unless there is a valid reason for giving an
  antibiotic, the patient would probably be better
  off without it.

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Treatment of Known or Suspected Infection
Prevention of Bacterial Infection
Peri-operative Prophylaxis
Patients at Special Risk
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• In cases when immediate drug treatment is
  necessary.

2
It is bad treatment to use broad-spectrum
antibiotics when an infective condition can be
treated with a more specific
agent.
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It is essential to use bactericidal and not
bacteriostatic therapy.
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It is essential to use combination of
antimicrobial drugs in certain situations.
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For treatment of superficial infections it is
important to use either antiseptic or antibiotics
which are rarely or never used systemically.
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• Give enough, for long enough, and then stop
  treatment with the antibiotic.

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To reduce the spread of microbial resistance,
avoid the use of antibiotics as food supplement
for animals or for preservation of human food
stuffs, avoid liberation of antibiotic powders
and solutions into the environment.
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To reduce the emergence of antibiotic-resistant
strains, an antibiotic policy has to be
introduced for a hospital or area e.g.,
using antibiotics in rotation, keeping a
particular antibiotics and permitting their use
only on rare and special occasions, or insisting
on combined therapy.
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Drug combination

1. To provide broad coverage.
2. For initial (blind) therapy when the patient is
   seriously ill and results of cultures are
   pending.
3. To provide synergism when organisms are not
   effectively eradicated with a single agent alone
   e.g., in enterococcal endocarditis both
   penicillin and
   an aminoglycoside are given because their
   combined effect is greater than the sum of their
   independent activities.

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• To prevent emergence of resistance, as in the
  treatment of tuberculosis.
• Inappropriate use of combinations could result
  in
• Antagonism it occurs when a bactericidal agent is
  used with a bacteriostatic one as in penicillins
  plus tetracycline or Chloramphenicol.
• Sulphonamides do not antagonize penicillins,
  possibly because their bacteriostatic action is
  too low.
• An increase in the number or severity of adverse
  reactions.
• Increased coast.

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