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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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2/28/2016 5:08:39 PM 3Dr. Medhat A. Eldaker

A)Cell Wall Inhibitors

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β- Lactams Glycopeptides Polypeptides

Cell wall inhibitors

Penicillins Cephalosporins CarbapenemsMonobactams

Aztreonam Imipenem

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Cell Wall Inhibitors

I. ββββ 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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I. ββββ Lactam antibiotics - Monobactam

• Aztreonam (Azactam):

- Resistant Gram-negative bacteria.

- Pseudomonas.

I. ββββ Lactam antibiotics - Carbapenem

• Imipenem (Tinam)

- Resistant Gram-negative & Gram-positive

bacteria.

- Pseudomonas.

Cell Wall Inhibitors

I. ββββ Lactam antibiotics - Cephalosporin

4th G3rd G2nd G1st G

+ve = -ve -ve > +ve+ve = -ve+ve> -ve

Pseudomonas √Pseudomonas X

CefipimeCefotaxime

Ceftriaxone

Cefuroxime

Cefaclor

Cephalexin

Cephdroxil

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II. Glycopeptides

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• Vancomycin

- Resistant Gram-positive bacteria.- MRSA.

A. Cycloserine

- TB

B. Bacitracin

- Diagnostic- Topical

III. Polypeptides

Cell Wall Inhibitors

Protein Synthesis Inhibitors

50 S30 S

ChloramphenicolMacrolideTetracyclinAminoglycoside

BothBactericidalBacteristaticBactericidal

Enteric feverGm +ve> -vevibrioGm –ve & Pseudo-

monas

ChloramphenicolErythromycin

Azythromycin

Clindamycin

Oxytetracyclin

Doxycyclin

Gentamicin

Amikacin

Tobramycin

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DNA Replication Inhibitors

I. Sulphonamides

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• Bactiristatic

• UTI-Chemoprophylaxis

- e.g Trimethoprim.

II. Quinolones

• Bactricidal

• Broad spectrum

- Ofloxacin - Ciprofloxacin

-Levofloxacin -Nitrofurantoin (UTI)

III. Rifampicin

• Bactricidal

• TB

Inhibition of precursor

Inhibition of RNA polymerase

Inhibition of DNA gyrase

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 mutationA) Genotypic Classification

1. Point mutation (micro lesion):

• Changes in a single nucleotide base pair by substitution, addition

or deletion (frame shift )

• Substitution of purine purine or pyrimidine by pyrimidine is

called Transition.

• Substitution of purine 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 codonresult 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