Lecture 7- Micro Medical Microbiology Dr. Saleh M.Y. Tuesday; 13/10/2010 Bacteriology Review and Overview Gram +ve cocci: (1) Streptococci, ( St. pyogenes Group A, St. agalactia-B, St. mutnas and St. viridians) (2) Staphylococci (S. aureus, S. epidermidis, S. saprophyticus) (4) Streptococcus pneumonae Gram -ve cocci: (5) Nessireia (N. meningitidis, N. gonorhheae) (6) Sites of infection and names of the Diseases (7) Toxins/mechanisms (8) Pathogenicity (9) Diagnosis (clinical and laboratory diagnosis) (10) Antibiotics/mechnisms/treatment and control BACTERIOLOGY 1- Introduction into antibiotics (Dr. Saleh M.Y./one lecture) 2- Metabolism-Antibiotic Sensitivity (3-5lectures/ Pharmaceutical Lecturer) Table of Contents 1. Educational Objectives 2. Microbial Metabolism Overview 3. Bacterial Cell Wall Biosynthesis 4. Cytoplasmic Membrane 5. DNA Replication 6. Protein Synthesis
1. Lecture 7- Micro
Medical Microbiology
Dr. Saleh M.Y.
Tuesday; 13/10/2010
Bacteriology
Review and Overview
Gram +ve cocci:
(1) Streptococci, ( St. pyogenes Group A, St. agalactia-B, St.
mutnas and St. viridians)
(2) Staphylococci (S. aureus, S. epidermidis, S.
saprophyticus)
(4) Streptococcus pneumonae
Gram -ve cocci:
(5) Nessireia (N. meningitidis, N. gonorhheae)
(6) Sites of infection and names of the Diseases
(7) Toxins/mechanisms
(8) Pathogenicity
(9) Diagnosis (clinical and laboratory diagnosis)
(10) Antibiotics/mechnisms/treatment and control
BACTERIOLOGY
1- Introduction into antibiotics (Dr. Saleh M.Y./one lecture)
2- Metabolism-Antibiotic Sensitivity (3-5lectures/ Pharmaceutical
Lecturer)
Table of Contents
Educational Objectives
Microbial Metabolism Overview
Bacterial Cell Wall Biosynthesis
Cytoplasmic Membrane
DNA Replication
Protein Synthesis
Competitive Antagonistic Anitbiotics
Summary
Educational Objectives
In general
To explore the relationship between bacterial metabolism and
susceptibility to anti-bacterial agents, both physical and
chemical
To define the mode of action of antibiotics
Specific educational objectives (terms and concepts upon which you
will be tested)
Aminoglycoside antibiotics
Antibiotic mode of action
b-lactam antibiotics
Cell wall inhibitors
Competitive antagonistic antibiotics
Macrolide antibiotics
Protein synthesis inhibitors
Quinolone antibiotics
Lecture Notes:
Microbial Metabolism as Related to Sensitivity to
Antibiotics-Overview
Many metabolic activities of the bacterial cell differ
significantly from those in the human cell. At least theoretically
these differences can be exploited in the development of
chemotherapeutic agents. Ideally, an antimicrobial agent should
have its maximal effect on the bacterial cell and have little or no
effect on the human cell. In reality there is almost always some
effect on the human be it induction of hypersensitivity or liver or
kidney toxicity. Despite some adverse reactions in the human,
effective antibiotics have been developed that have one ore more of
these modes of action on the bacterial cell:
Inhibition of cell wall synthesis
Alteration of cell membranes
Inhibition of protein synthesis
Inhibition of nucleic acid synthesis
Antimetabolic activity or competitive antagonism
Bacterial Cell Wall Biosynthesis
Since bacteria have a cell wall made up of repeating units of
peptidoglycan and human cells lack this feature, it would seem that
the bacterial cell wall presents an ideal target for chemotherapy.
Indeed, this has been the case; the following antibiotics have been
developed as inhibitors of cell wall synthesis:
A. -lactam antibiotics
1. Penicillins
Penicillin GOxacillinAmpicillinAmoxicillinCloxaciillinPenicillin
VNafcillinTicarcillinCarbenicillinDicloxacillinMethicillinPiperacillin
2. Cephalosporins First Generation Second Generation Third
GenerationFourth Generation
Cefadroxil * Cefaclor *CefdinirCefepime Cefazolin
CefamandoleCefoperaxone Cefelixin * CefonicidCefotaxime Cephalothin
CeforanideCeftazidime Cephaprin CefotetanCeftibuten Cephradine *
CefoxitinCeftizoxime CefuroximeCeftriaxone
* Oral Agent
3. Monobactams
4. Thienamycins
5. -lactamase inhibitors (e.g., clavulanic acid)
B. Cycloserine, Ethionamide, Isoniazid
C. Fosfomycin (Phosphonomycin)
D. Vancomycin
E. Bacitracin
F. Ristocetin
G. Fosphomycin (Phosphonomycin)
The biosynthesis of peptidoglycan consists of three stages, each of
which occurs at a different site in the cell.
Stage 1 occurs in the cytoplasm. In this stage the recurring units
of the backbone structure of murein, N-acetylglucosamine and
N-acetyl-muramylpentapeptide are synthesized in the form of their
uracil diphosphate (UDP) derivatives. The only antibiotic that
affects this stage of cell wall metabolism is D-cycloserine.
D-cycloserine is a structural analog of D-alanine; it binds to the
substrate binding site of two enzymes, thus being extremely
effective in preventing D-alanine from being incorporated into the
N-acetylmuramylpeptide.
Structural relationship between cycloserine (left) and D-ala-nine
(right).
Stage 2 of peptidoglycan synthesis occurs on the inner surface of
the cytoplasmic membrane where N-cetylmuramylpeptide is transferred
from UDP to a carrier lipid and is then modified to form a complete
nascent peptidoglycan subunit. The nature of the modification
depends upon the organism. This stage terminates with translocation
of the completed subunit to the exterior of the cytoplasmic
membrane. The only antibiotic that affects this stage of cell wall
synthesis is bacitracin. Bacitracin is an inhibitor of the lipid
phosphatase.
Bacitracin A. One of a group of polypeptide antibiotics containing
a thiazoline ring structure.
Stage 3 occurs in the periplasmic space (in gram-negative bacteria)
and in the growing peptidoglycan of the cell wall. This is a
complex metabolic sequence which offers multiple targets for
chemotherapeutic agents. The earliest acting of these are
vancomycin and ristocetin. They act by binding to the
D-alanyl-D-alanine peptide termini of the nascent
peptidoglycan-lipid carrier. This inhibits the enzyme
transglycosylase.
Stage 3 of biosynthesis continues with transpeptidation and the
binding of soluble uncrosslinked, nascent peptidoglycan to the
preexisting, crosslinked, insoluble cell wall peptidoglycan matrix.
The -lactam antibiotics are structural analogs of the
D-alanyl-D-alanine end of the peptidoglycan strand. In the cell
wall there are as many as seven enzymes (depending on the bacterial
species) which bind peptidoglycan units via their
D-alanyl-D-alanine residues. The -lactams fill these substrate
binding sites and thus prevent the binding of D-alanyl-D-alanine
residues. Enzymes binding -lactam antibiotics are known as
penicillin-binding proteins.
The Cytoplasmic Membrane as the Site of Antibiotic Action
The cytoplasmic membrane of bacteria is only affected by two
clinically-used antibiotics. These are polymyxin B and polymyxin E
(colistin). They act by competitively replacing Mg2+ and Ca2+ from
negatively charged phosphate groups on membrane lipids. The result
is disruption of the membrane.
DNA Replication as the Site of Antimicrobic Action
The major group of antibacterial agents that act by blocking DNA
synthesis/activity is the quinolone group.
Metronidazole represents as an antibiotic active against DNA in a
different way. This antibiotic, upon being partially reduced,
causes the fragmentation of DNA in an, as yet, undefined way. The
antibiotic is only effective against anaerobic bacteria and some
parasites.
The quinolones all act by blocking the A subunit of DNA gyrase and
inducing the formation of a relaxation complex analogue.
DNA gyrase introduces negative superhelical turns into duplex DNA,
using the energy of ATP. This is the crucial enzyme that maintains
the negative superhelical tension of the bacterial
chromosome.
The sign-inversion mechanism for DNA gyrase.
The quinolones include:
nalidixic acid - first generation
norfloxacin, ciprofloxacin - second generation
Protein Synthesis as the Site of Antimicrobic Action
Protein synthesis is the end result of two major processes,
transcription and translation. An antibiotic that inhibits either
of these will inhibit protein synthesis.
Transcription
During transcription, the genetic information in DNA is transferred
to a complementary sequence of RNA nucleotides by the DNA-dependent
RNA polymerase. This enzyme is composed of 5 subunits, , ', a, a'
and . Antibiotics that either alter the structure of the template
DNA or inhibit the RNA polymerase will interfere with the synthesis
of RNA, and consequently with protein synthesis.
Actinomycin D binds to guanine in DNA, distorting the DNA, and thus
blocking transcription.
Rifampin (Rifampicin or Rifamycin) inhibits protein synthesis by
selective inhibiting the DNA-dependent RNA polymerase. It does this
by binding to the subunit in a non-covalent fashion.
Translation
In bacterial cells, the translation of mRNA into protein can be
divided into three major phases: initiation, elongation, and
termination of the peptide chain. Protein synthesis starts with the
association of mRNA, a 30S ribosomal subunit, and
formyl-methionyl-transfer RNA (fMet-tRNA) to form a 30S initiation
complex. The formation of this complex also requires guanosine
triphosphate (GTP) and the participation of three protein
initiation factors. The codon AUG is the initiation signal in mRNA
and is recognized by the anticodon of fMet-tRNA. A 50S ribosomal
subunit is subsequently added to form a 70S initiation complex, and
the bound GTP is hydrolyzed.
In the elongation phase of protein synthesis, amino acids are added
one at a time to a growing polypeptide in a sequence dictated by
mRNA. It is this phase that is most susceptible to inhibition by a
number of antibiotics. For many of these the ribosome is the target
site. There are two binding sites on the ribosome, the P (peptidyl
or donor site) and the A (aminoacyl) site. At the end of the
initiation stage, the fMet-tRNA molecule is empty. In the first
step of the elongation cycle, an aminoacyl-tRNA is inserted into
the vacant A site on the ribosome. The particular species inserted
depends on the mRNA codon that is positioned in the A site. Protein
elongation factors and GTP are required for polypeptide chain
elongation.
In the next step of the elongation phase, the formylmethionyl
residue of the fMet-tRNA located at the peptidyl donor site is
released from its linkage to tRNA, and is joined with a peptide
bond to the -amino group of the aminoacyl-tRNA in the acceptor site
to form a dipeptidyl-tRNA. The enzyme catalyzing this peptide
formation is peptidyl transferase, which is part of the 50S
ribosomal subunit.
Following the formation of a peptide bond, an uncharged tRNA
occupies the P site, whereas a dipeptidyl tRNA occupies the A site.
The final phase of the elongation cycle is translocation, catalyzed
by elongation factor EF-G and requiring GTP. It consists of three
movements:
(1) the removal of the discharged tRNA from the P site
(2) the movement of fMet-aminoacyl-tRNA from the acceptor site to
the peptidyl donor site
(3) the movement or translocation of the ribosome along the mRNA
from the 5' toward the 3' terminus by the length of three
nucleotides.
After translocation, the stage is prepared for the binding of the
next aminoacyl residue to the fMet-aminoacyl-tRNA, each addition
requiring aminoacyl-tRNA binding, peptide bond formation, and
translocation. Peptidyl-tRNAa replace the fMet-tRNA in the second
and in all subsequent cycles.
The polypeptide chain grows from the amino terminal toward the
carboxyl terminal amino acid and remains linked to tRNA and bound
to the mRNA-ribosome complex during elongation of the chain. When
completed it is released during chain termination. Termination is
triggered when a chain termination signal (UAA, UAG, or UGA) is
encountered at the A site of the ribosome. Protein release factors
bind to the terminator codons triggering hydrolysis by the peptidyl
transferase. The polypeptide is released, and the
messenger-ribosome-tRNA complex dissociates.
Several medically important antibiotics owe their selective
antimicrobial action to a specific attack on the 70S ribosome of
bacteria, with mammalian 80S ribosomes left unaffected. Those that
act on the 30S ribosome are:
Amikacin
Gentamycin
Kanamycin
Neomycin
Streptomycin
Tobramycin
Macrolides:
Azithromycin Dirithromycin Clarithromycin Erythromycin
Antibiotics that act on the 50S portion of the ribosome
include:
Chloramphenicol
Clindamycin
Furadantin
Fusidic acid
Lincomycin
Nitrofuran
Puromycin
Quinopristin/Dalfopristin
Spectinomycin
Tetracycline
Trimethoprim - Inhibit folic acid biosynthesis
Summary
Antibiotics that are active against the cell wall of bacteria
include the -lactams, cycloserine, ethionamide, isoniazid,
phosphomycin, vancomycin, bacitracin and ristocetin.
The -lactam antibiotics are related structurally in that they all
contain a -lactam ring. These are the penicillins, cephalosporins,
monobactams and thienamycins. They are all analogs of
d-alanyl-d-alanine.
Antibiotics that are active against the bacterial cytoplasmic
membrane are polymyxin B and E (colistin).
Antibiotics that are active against bacterial DNA are the
quinolones (nalidixic acid, norfloxacin and ciprofloxacin), which
inhibit DNA gyrase, and metronidazole, which fragments DNA.
Antibiotics that block transcription in bacteria are actinomycin D
and rifampin.
Antibiotics that block translation in bacteria by binding to the
30S ribosome are the aminoglycosides, nitrofurans, spectinomycin
and the tetracyclines.
The aminoglycoside antibiotics are related structurally in that
they all contain a unique aminocyclitol ring structure. These
include amikacin, gentamycin, kanamycin, neomycin, streptomycin and
tobramycin.
Antibiotics that block translation by binding to the 50S ribosome
include chloramphenicol, erythromycin, clarithromycin, lincomycin,
clindomycin, puromycin, fusidic acid and
quinopristin/dalfopristin.
The macrolide antibiotics are related structurally in that they all
contain a macrocyclic lactone ring of 12-22 carbon atoms, to which
one or more sugars are attached. These include erythromycin,
clarithromycin, azithrmycin and dirithromycin.
Antibiotics that act by inhibiting folic acid biosynthesis include
the sulfonamides and trimethoprim.