Download Antibiotics and resistance

2/28/2016
ANTIMICROBIAL AGENTS
Dr. Waleed Eldars
Lecturer of Medical Microbiology
and Immunology
Faculty of Medicine
Mansoura University
Anti-Microbial Agents
Definition: include antibiotics, anti-viral and
anti-fungal drugs.
Mechanism of action of clinically used
antibiotics:
• Inhibition of cell wall synthesis.
• Alteration of cell- membrane permeability.
• Inhibition of protein synthesis.
• Inhibition of nucleic acid synthesis.
• Other mechanisms of action.
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Dr. Medhat A. Eldaker
A)Cell Wall Inhibitors
Cell wall
inhibitors
β- Lactams
Penicillins
Glycopeptides
Cephalosporins
Dr. Medhat A. Eldaker
Polypeptides
Monobactams
Carbapenems
Aztreonam
Imipenem
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Cell Wall Inhibitors
I. β Lactam antibiotics
1. Penicillin
2. Monobactam
3. Carbapenem
4. Cephalosporins
Mechanisms of action:
• Penicillins and cephalosporins act through the
inhibition of the terminal cross- linking of the
peptidoglycan.
Resistance to penicillin:
• The organism produce penicillin destroying
enzyme ß- lactamases
• Absence of penicillin receptors.
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Cell Wall Inhibitors
I. β Lactam antibiotics - Monobactam
• Aztreonam (Azactam):
- Resistant Gram-negative bacteria.
- Pseudomonas.
I. β Lactam antibiotics - Carbapenem
• Imipenem (Tinam)
- Resistant Gram-negative & Gram-positive
bacteria.
- Pseudomonas.
I. β Lactam antibiotics - Cephalosporin
1st G
+ve> -ve
2nd G
+ve = -ve
3rd G
-ve > +ve
4th G
+ve = -ve
Pseudomonas X
Pseudomonas √
Cephalexin
Cephdroxil
Cefotaxime
Ceftriaxone
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Cefuroxime
Cefaclor
Cefipime
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Cell Wall Inhibitors
II. Glycopeptides
•
Vancomycin
- Resistant Gram-positive bacteria.
- MRSA.
III. Polypeptides
A. Cycloserine
- TB
B. Bacitracin
- Diagnostic
- Topical
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Protein Synthesis Inhibitors
30 S
50 S
Aminoglycoside
Tetracyclin
Macrolide
Chloramphenicol
Bactericidal
Bacteristatic
Bactericidal
Both
Gm –ve & Pseudomonas
vibrio
Gm +ve> -ve
Enteric fever
Gentamicin
Amikacin
Tobramycin
Oxytetracyclin
Doxycyclin
Erythromycin Chloramphenicol
Azythromycin
Clindamycin
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Dr. Medhat A. Eldaker
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DNA Replication Inhibitors
I. Sulphonamides
•
•
Bactiristatic
UTI-Chemoprophylaxis
- e.g Trimethoprim.
Inhibition of
precursor
II. Quinolones
• Bactricidal
• Broad spectrum
- Ofloxacin
-Levofloxacin
Inhibition of DNA
gyrase
- Ciprofloxacin
-Nitrofurantoin (UTI)
III. Rifampicin
•
•
Bactricidal
TB
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Inhibition of RNA
polymerase
Cytoplasmic membrane Inhibitors
Polyenes
• Bacteiristatic
- Polymyxin B topical
- Mitronidazole anaerobes
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Causes of failure of antimicrobial
chemotherapy:
• Clinical condition not suitable to antibiotic treatment (as
viral infection or mixed bacterial infection).
• Failure to use laboratory properly.
• Wrong choice of antibiotics.
• Inadequate doses of the antibiotics.
• Inadequate duration of treatment by the antibiotics.
• Wrong route of administration.
• Use of antagonistic antibiotic combination.
• Development of antimicrobial resistance.
Drug resistance
• It is the unresponsiveness of the organisms to the given
drug (antibiotics).
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Mechanisms of Drug resistance:
Microorganisms produce enzymes that destroy the drug: as β- lactamases
enzymes produced by Staphylococci to destroy the β-lactams.
Microorganisms decrease their permeability to the drug.
Microorganisms develop an altered structural target for the drug: As
alteration of the receptor protein that give attachment to the drug.
Microorganisms develop an altered metabolic pathway that bypasses the
reactions inhibited by the drug.
Microorganisms develop an altered enzyme that can still perform its
metabolic function but is much less affected by the drug.
Origin of drug resistance:
The origin of drug resistance may be:
• Non- genetic: in this case microorganism may loss
the specific target structure for the drug for several
generations.
Genetic origin:
• Chromosomal resistance: this results after
spontaneous mutation in a locus that controls
susceptibility to antimicrobial drug (change
receptors, permeability).
• Extra chromosomal resistance (plasmids):
• Plasmids genes for antimicrobial resistance often control
the formation of enzymes capable of destroying the drug.
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Microbial Genetics
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Microbial Genetics
• Gene (the unit of heredity): a segment of DNA
that carries in its nucleotide sequence
information for a specific property.
• Genotype: is the genetic make-up of a cell which
reflects the actual sequence of nucleotides in
the chromosome.
• Phenotype: is a set of observable characteristics
of an organism (structural and physiologic).
• Chromosome: the chromosome consists mainly
of a polymer called deoxyribonucleic acid (DNA).
This polymer is built up of subunits called
nucleotides. The sequence of nucleotides in
chromosomal DNA encodes all the information
needed to specify the structure and behavior of
a given bacterium.
• Plasmid: is an extra-chromosomal piece of DNA
usually much smaller than the chromosome and
can replicate independently.
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Nucleic Acids Structure
• A nucleic acid is a long chain of repeated subunits of nucleotides
linked together by 3‘- 5‘phosphor-diester bonds.
• Each nucleotide has 3 parts:
(1) Sugar:
• Deoxyribose: nucleotides containing the deoxyribose are deoxyribo
nucleotides which form deoxyribo nucleic acid (DNA).
• Ribose: nucleotides containing the sugar ribose are ribonucleotides
which form ribonucleic acid (RNA).
(2) Nitrogen bases:
• Purines: adenine (A) and guanine (G).
• Pyrimidines: cytosine (C) and thymine (T) /or uracil
(U) in RNA.
(3) Phosphate group:
• Is linked to the 5‘ position of the sugar.
• The removal of a phosphate group from the
nucleotide give a nucleoside.
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DNA STRUCTURE
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DNA Replication
• DNA replication is the process of producing two identical rep
• This biological process occurs in all living organisms and is the
basis for biological inheritance.
• Each strand of the original DNA molecule serves as a template
for the production of the complementary strand, a process
referred to as semiconservative replication.
Cellular proofreading and error-checking mechanisms ensure
near perfect fidelity for DNA replication.
• Important enzymes: DNA polymerase and Topoisomerase
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Gene Expression
• The genetic information in DNA is expressed by copying DNA into
RNA and the RNA is translated into a protein. Genetic information
flows from DNA to mRNA to proteins.
• 1) Transcription: RNA synthesis
• Transcription is the process of synthesis of RNA (transcript) from a
DNA template.
• The RNA carries the gene's massage specifying the protein and so
called messenger RNA (mRNA).
• Carried out via RNA polymerase
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2) Translation (protein synthesis)
• Translation is the synthesis of a polypeptide on mRNA. It takes
place on the ribosomes.
• A protein consists of one or more polypeptides; a polypeptide
is a chain of amino acids covalently linked by peptide bonds.
• Requires the interaction of mRNA, tRNA and rRNA.
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Mutation
Definition:
• A mutation is a stable, heritable change in the sequence of
nucleotides in the DNA.
• Transcription of altered DNA produces altered RNA and
altered mRNA gives a different polypeptide with different
biological function and result in the appearance of altered
phenotype.
Classification of mutation
A) Genotypic Classification
1. Point mutation (micro lesion):
• Changes in a single nucleotide base pair by substitution, addition
or deletion (frame shift )
• Substitution of purine
called Transition.
• Substitution of purine
purine or pyrimidine by pyrimidine is
pyrimidines is called transversion.
2. Multisite Mutation ( macro lesion ):
• Change of multiple nucleotide it includes :•
•
•
•
Deletion: Missing of nucleotides
Insertion: addition of novel base pairs.
Rearrangements: all the base pairs are present but the order changes.
Frame shift: may be point (addition or deletion of one bp) also
multisite (addition or deletion of more than one base pairs).
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B)Phenotypic classification :
1) Same sense mutation:
• Means the change of one base by mutation in amino acid codon
result in another codon for the same amino acid for example
leucine.
UUA
UUG
Leucine Mutation
leucine
2. Missense mutation:
• Means the change in amino acid codon result in codon of another
amino acid
For example :
TTG
GTG
Tryptophan
Glycine
• The effect of missense depends on the location of the changed
amino acid in the polypeptide chain.
3) Nonsense mutation:
• Means that base pair substitution that change a codon into one of
the 3 chain termination codons ( UAG , UAA, UGA)
• The effect of nonsense mutation depends on where the chain is
terminated i.e. the truncated protein may have no activity, some
activity or full activity.
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C) According to inducibility
1- Spontaneous mutation: occurs spontaneously due to error in reading by the
polymerase enzymes.
2- Induced Mutations: mutation by damaging DNA by physical and chemical agents
(mutagens).
• a- Physical mutagens:
• Ultraviolet rays. pyrimidine dimers T-Tcovalent linking of two adjacent
pyramidine on the same strand) like T-T (68%) C-T (13%), T-C (19%) and C-C
(3%).
• Ionizing radiation. Causes DNA strand break either single or double strands
• b- Chemical mutagenesis:
• Chemicals that mimic normal DNA bases ( Base analogs ) These analogs are
structurally related to bases but differ in pairing manner
• Chemical that react with DNA bases ( base modifiers ) These chemical react
directly with the nucleotide bases , alter the chemical structure
• Alkylating agents: adding methyl or ethyl group to the oxygen of bases e.g
: Nitrosoguanidine (NTG)
• Chemicals that bind DNA bases (Intercalators). Acridine dyes and Acridine–like
derivatives (proflavin, ethidium bromide) have the same dimensions as the
normal bases so can slide between two adjacent base pairs (intercalating)
causing frame shift
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Effects of Mutation:• Have no effect on the expression of a gene
means silent mutation.
• Changes the level of gene expression (increase or
decrease).
• Produce a related but structurally different
protein.
• Mutation may proceed to carcinogenesis.
• Deletion mutation of the virulence gene in
bacteria can be used as a reference strain for
vaccine (low virulent or avirulent strain).
Gene Transfer
New DNA may enter a bacterium naturally through transformation,
conjugation or transduction.
1. Transformation:
• It is the transfer of pure or naked DNA from one cell to another.
• In transformation, the donor cell becomes lysed first, and one strand
of this donor DNA is taken up into the recipient cell from its
environment.
• The donor DNA may genetically transform the recipient cell by
recombining with a region of the chromosome.
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2. Conjugation:
• Conjugation occurs mainly in Gram negative bacteria.
• It is the matting of two bacterial cells during which DNA is
transferred from the donor (male) to the recipient cell (female).
• The mating process is controlled by F plasmid.
• The mating process is mediated by the sex pilus.
3. Transduction:
• The transfer of DNA (chromosomal or plasmid) from one bacterial
cell to another by means of a bacterial virus (bacteriophage).
• May be general or specific.
Conjugation
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Bacterial Plasmids
• In addition to the chromosome, many bacteria
contain one or more plasmids.
• A plasmid is an extra chromosomal piece of DNA
usually much smaller than the chromosome and
which can replicate independently (autonomous
replication).
• Plasmids are widely used in recombinant DNA
technology.
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Classification of plasmids:
According to:
Size of plasmid:
• Small: 1-10 kb.
• Large: > 50 kb.
• Mega: > 400 kb.
Copy number:
• Few copy number plasmids: have a copy number of 1 or 2 per
bacterial cell.
• Multicopy plasmids: have a copy number of 10-30 per bacterial
cell.
Shape:
• Circular: most plasmids are circular.
• Linear.
Genetic content and function of plasmids:
a)Drug- resistance (R) plasmids:
• Carry antibiotic resistance genes which encode enzymes that
inactivate particular antibiotics making the bacterial cell
resistant to the relevant antibiotic.
• R-plasmids carry antibiotic resistance genes for many antibiotics
like β- lactams, aminoglycosides and tetracyclins.
b)Virulence (Vi) Plasmids:
• Carry genes which encode for virulence factors involved in the
pathogenic characters of bacteria e.g. toxin production.
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C)Plasmids encoding for other functions:
• Resistance to heavy metal ions e.g. mercury.
• Resistance to intercalating agents as acridines.
• Protection against radiation damage by UV.
• Resistance to certain bacteriophages.
Molecular Diagnosis of Microorganisms
1)PCR
2)Probe
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Polymerase Chain Reaction (PCR)
• PCR is a technique for making millions of copies of a
specific target sequence of nucleotides in DNA.
Requirments:
• The sample dsDNA
• The primers: small pieces of ssDNA each about 20-30
nucleotides in length.
• Deoxyribonucleoside triphosphates of all four types
• DNA polymerase (Taq polymerase).
Steps of PCR:
• The above mentioned reaction mixture
undergoes a series of changes in temperature
in a thermocycler device. This cycle is
repeated about 20-35 times. Each cycle
includes:
• Denaturation step: initial heating to about 94ºC
denatures the dsDNA fragment to two single
strands.
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• Annealing step: transient cooling to 45- 60ºC
allow the primers to bind (anneal) to their
complementary sites on the sample DNA.
• Extension step: a change to 72ºC then permits
the DNA polymerase enzyme to start DNA
amplification from the 3‘ end of each primer.
When the temperature cycle is repeated, the
newly synthesized strands act as templates
and so on.
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Uses of PCR:
• Diagnosis of microbial diseases: by detecting specific
sequences of their DNA. This is especially useful for
those pathogens that:
• Cannot be cultured or cultured with difficulty e.g.
HBV, HCV and HIV.
• Those that grow very slowly e.g. M. tuberculosis.
• Detection of genes coding for virulence factors e.g.
detection of toxigenic strains of bacteria.
• Determination of antibiotic resistance.
• In forensic medicine.
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Probe
Def: A piece of single stranded DNA or RNA (or PNA) (peptide
nucleic acid) which is complementary to the sequence of
interest (to be detected) and labeled by detectable material at
its 5’ phosphate end.
Principle:
• Because of the specificity of base pairing the sequence of
interest (DNA, RNA unknown in the sample to be diagnosed)
can be detected by the binding (base – pairing) of the probe
label.
• The binding is specific if wanted DNA is present which is
complementary to the probe.
• If not present the probe and its label will not bound and
washed out
Types of label of the probes
• Radioactive isotopes p32 detected by Autoradiography.
• Fluorescence Rhodamine detected by UV radiation.
Importance and Uses:- Nearly the same uses of PCR
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Thank you
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