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
1. Educational Objectives
2. Microbial Metabolism Overview
3. Bacterial Cell Wall Biosynthesis
4. Cytoplasmic Membrane
5. DNA Replication
6. Protein Synthesis
7. Competitive Antagonistic Anitbiotics
8. Summary
Educational Objectives
In 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 antibiotics
Specific 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 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:
A. Inhibition of cell wall synthesis
B. Alteration of cell membranes
C. Inhibition of protein synthesis
D. Inhibition of nucleic acid synthesis
E. 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. β-lactam antibiotics

3. 1. Penicillins
Penicillin G Oxacillin Ampicillin Amoxicillin Cloxaciillin
Penicillin V Nafcillin Ticarcillin Carbenicillin Dicloxacillin
Methicillin Piperacillin
2. Cephalosporins
First Generation Second Generation Third Generation Fourth Generation
Cefadroxil * Cefaclor * Cefdinir Cefepime
Cefazolin Cefamandole Cefoperaxone
Cefelixin * Cefonicid Cefotaxime
Cephalothin Ceforanide Ceftazidime
Cephaprin Cefotetan Ceftibuten
Cephradine * Cefoxitin Ceftizoxime
Cefuroxime Ceftriaxone
* Oral Agent
3. Monobactams
4. Thienamycins
5. β-lactamase inhibitors (e.g., clavulanic acid)
B. Cycloserine, Ethionamide, Isoniazid
C. Fosfomycin (Phosphonomycin)
D. Vancomycin
E. Bacitracin
F. 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 à -
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.

7. 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.

8. 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

9. 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, ß,
ß', 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 ß subunit in a non-covalent fashion.
Translation

10. 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 h -
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

11. (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.

12. 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

13. Macrolides:

14. 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

15. Lincomycin Clindamycin

16. Linezolid

17. Puromycin

18. Competitive Antagonistic Antibiotics
Inhibitors of metabolic pathways via competitive antagonism include:
Isoniazid - Inhibits mycolic acid synthesis
Sulfonamides - Inhibit folic acid biosynthesis

20. Trimethoprim - Inhibit folic acid biosynthesis
Summary
1. Antibiotics that are active against the cell wall of bacteria include the Þ -lactams, cycloserine,
ethionamide, isoniazid, phosphomycin, vancomycin, bacitracin and ristocetin.
2. The β-lactam antibiotics are related structurally in that they all contain a β-lactam ring. These
are 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, norfloxacin
and 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 the
aminoglycosides, nitrofurans, spectinomycin and the tetracyclines.
7. 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.

21. 8. Antibiotics that block translation by binding to the 50S ribosome include chloramphenicol,
erythromycin, clarithromycin, lincomycin, clindomycin, puromycin, fusidic acid and
quinopristin/dalfopristin.
9. 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.
10. Antibiotics that act by inhibiting folic acid biosynthesis include the sulfonamides and
trimethoprim.