- 1. Lecture 7- MicroMedical Microbiology Dr. Saleh M.Y. Tuesday;
13/10/2010 Bacteriology Review and OverviewGram +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 pneumonaeGram -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 controlBACTERIOLOGY1-
Introduction into antibiotics (Dr. Saleh M.Y./one lecture) 2-
Metabolism-Antibiotic Sensitivity (3-5lectures/ Pharmaceutical
Lecturer)Table of Contents 1. Educational Objectives2. Microbial
Metabolism Overview3. Bacterial Cell Wall Biosynthesis4.
Cytoplasmic Membrane5. DNA Replication6. Protein Synthesis7.
Competitive Antagonistic Anitbiotics8. SummaryEducational
ObjectivesIn general 1. To explore the relationship between
bacterial metabolism and susceptibility to anti-bacterial agents,
both physical and chemical
2. 2. To define the mode of action of antibioticsSpecific
educational objectives (terms and concepts upon which you will be
tested)1. Aminoglycoside antibiotics 2. Antibiotic mode of action
3. b-lactam antibiotics 4. Cell wall inhibitors 5. Competitive
antagonistic antibiotics 6. Macrolide antibiotics 7. Protein
synthesis inhibitors 8. Quinolone antibioticsLecture
Notes:Microbial Metabolism as Related to Sensitivity to
Antibiotics-OverviewMany 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: A. Inhibition of cell
wall synthesisB.Alteration of cell membranesC.Inhibition of protein
synthesisD. Inhibition of nucleic acid synthesisE.Antimetabolic
activity or competitive antagonismBacterial Cell Wall
BiosynthesisSince 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 3. 1. Penicillins Penicillin
GOxacillinAmpicillin AmoxicillinCloxaciillinPenicillin
VNafcillinTicarcillinCarbenicillinDicloxacillinMethicillinPiperacillin2.
Cephalosporins First Generation Second Generation Third
GenerationFourth Generation Cefadroxil * Cefaclor *Cefdinir
Cefepime CefazolinCefamandole Cefoperaxone Cefelixin *Cefonicid
Cefotaxime CephalothinCeforanideCeftazidime CephaprinCefotetan
Ceftibuten Cephradine * Cefoxitin CeftizoximeCefuroximeCeftriaxone*
Oral Agent 3. Monobactams4. Thienamycins 5. -lactamase inhibitors
(e.g., clavulanic acid)B. Cycloserine, Ethionamide, IsoniazidC.
Fosfomycin (Phosphonomycin)D. VancomycinE. BacitracinF. Ristocetin
4. 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. 5. 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 t -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. 6. The Cytoplasmic Membrane as the
Site of Antibiotic ActionThe 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 ActionThe 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. 7. 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
generationnorfloxacin, ciprofloxacin - second generation Protein
Synthesis as the Site of Antimicrobic ActionProtein synthesis is
the end result of two major processes, transcription and
translation. An antibiotic that inhibits either of these will
inhibit protein synthesis.Transcription 8. 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 s .
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 9. 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 thepeptidyl
donor site is released from its linkage to tRNA, and is joined with
a peptide bond to the h -amino group of the aminoacyl-tRNA in the
acceptor site to form a dipeptidyl-tRNA. The enzymecatalyzing 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 dipeptidyltRNA
occupies the A site. The final phase of the elongation cycle is
translocation, catalyzed byelongation 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 acceptorsite to the peptidyl donor
site 10. (3) themovement or translocation of the ribosome along
themRNA from the 5' towardthe 3' terminusbythelength ofthree
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, andtranslocation. 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 andremains 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
chaintermination signal (UAA, UAG, or UGA) is encountered at the A
site of the ribosome. Protein releasefactors bind to the terminator
codons triggering hydrolysis by the peptidyl transferase. The
polypeptideis released, and the messenger-ribosome-tRNA complex
dissociates. 11. 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 12. Macrolides: 13.
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 14. Lincomycin Clindamycin 15. Linezolid 16. Puromycin
17. Competitive Antagonistic AntibioticsInhibitors of metabolic
pathways via competitive antagonism include:Isoniazid - Inhibits
mycolic acid synthesis Sulfonamides - Inhibit folic acid
biosynthesis 18. Trimethoprim - Inhibit folic acid biosynthesis
Summary 1. Antibiotics that are active against the cell wall of
bacteria include the -lactams, cycloserine,ethionamide, isoniazid,
phosphomycin, vancomycin, bacitracin and ristocetin.2. The -lactam
antibiotics are related structurally in that they all contain a
-lactam ring. Theseare the penicillins, cephalosporins, monobactams
and thienamycins. They are all analogs of d-alanyl-d-alanine.3.
Antibiotics that are active against the bacterial cytoplasmic
membrane are polymyxin B and E(colistin).4. Antibiotics that are
active against bacterial DNA are the quinolones (nalidixic acid,
norfloxacinand ciprofloxacin), which inhibit DNA gyrase, and
metronidazole, which fragments DNA.5. Antibiotics that block
transcription in bacteria are actinomycin D and rifampin.6.
Antibiotics that block translation in bacteria by binding to the
30S ribosome are theaminoglycosides, nitrofurans, spectinomycin and
the tetracyclines.7. The aminoglycoside antibiotics are related
structurally in that they all contain a uniqueaminocyclitol ring
structure. These include amikacin, gentamycin, kanamycin,
neomycin,streptomycin and tobramycin. 19. 8. Antibiotics that block
translation by binding to the 50S ribosome include
chloramphenicol,erythromycin, clarithromycin, lincomycin,
clindomycin, puromycin, fusidic acid andquinopristin/dalfopristin.
9. The macrolide antibiotics are related structurally in that they
all contain a macrocyclic lactonering of 12-22 carbon atoms, to
which one or more sugars are attached. These includeerythromycin,
clarithromycin, azithrmycin and dirithromycin. 10. Antibiotics that
act by inhibiting folic acid biosynthesis include the sulfonamides
andtrimethoprim.