Micro sheet#11( corrections are added!) - Nouran Hatim Mustafa

Inhibitors of Protein Synthesis

 Aminoglycosides
• contains numerous antimicrobial agents and individual other antimicrobial agents like: tetracycline,Chloramphenicol, microlides represented by erythromycin, Clindmycin and others.
• Aminoglycosides are amino sugars linked through glycosidic bonds to
an aminocyclitol ring.
• These antimicrobial agents were discovered in the 40’s,and the 1st of these antimicrobial agents to be utilized in human applications was Streptomycin followed by Neomycin & Kanamycin.
• Streptomycin was the antibiotic commonly used in treatment of bacterial infections and mycobacteria.
It is one of the combination drugs used to treat TB >>Tuberculosis .
But because of toxicity associated with these agents, their use has been almost neglected.
(they don’t have any place or room for treatment of infections becoz of the toxicity associated with these antimicrobial agents.)
**the major side effects associated with Aminoglycosides are:
1- Kidney function impairment that may lead to complete renal failure.
2- Ototoxicity: toxicity to the auditory nerve which can lead to deafness.
So as a consequence, these functions should be monitored closely when an individual is put on these antimicrobial agents. However, this is possible especially when using them to treat adults ,but the use of these antimicrobial agents to treat children may be associated with the mentioned effects(points1+2).

- Many causes of deafness among children is due to amino glycosides .
(their parents discover that their children can’t hear after 2-3years)
Kidney impairment can also affect children.

-This was followed by the development of more effective, less toxic agents; these are:
Tobramycin, Gentamicin, Netilmicin, Amikacin



Among all of these, netilmicin is the least ototoxic >>it is less commonly associated with ototoxicity.
These drugs act by binding to 30s subunit of the bacterial ribosome , and the consequences are:
1- Production of aberrant proteins as a result of misreading of mRNA.
2- Interruption of protein synthesis by causing the premature release of ribosome.
So>> misreading of codons + incomplete production of incomplete proteins.

**A cell that cannot synthesize proteins for its own function will die. becoz its incompatible with the viability of the cell>>the cell has to produce enzymes and proteins that are either excretory or functional components of the bacteria, so these 2 effects (points: 1+2) are lethal to bacteria.
These antibiotics are bactericidal becoz binding to the 30s subunit is irreversible.
However, entry of amino glycosides into the cell is oxygen-dependent, that’s why amino glycosides cannot be used to treat anaerobes.
amino glycosides are oxygen-dependent agents, their passage across the cellular envelope requires oxygen .
on the other hand, anaerobes cannot survive in the presence of oxygen>>that’s why these agents do not affect anaerobes or other organisms like: facultative anaerobes such as :streptococci , so these organisms are naturally resistant to amino glycosides since the entrance of amino glycosides to the bacterial cell is oxygen-dependent ,and these organisms do not utilize oxygen.

Similar to other antimicrobial agents, the resistance to amino glycosides can develop among bacteria.

Mechanisms of resistance:
a. Mutation of the protein in 30s subunit in ribosome lead to inability of the drug to bind. (so the drug can no more bind to 30s subunit becoz of the mutation of the binding site).
b. Decreased the uptake of antibiotic into the bacterial cell especially anaerobes as well as streptococci.
c. Enzymatic modification of the antibiotic (the most common).
The plasmid-mediated conjugation is responsible for this mechanism with synthesis of enzymes that can phosphorylate, adenylate & acetylate the drug (especially the amino & hydroxyl groups of the amino glycosides) results in resistance of the organism to these antimicrobial agents.

Amino glycosides are usually used in combination with other antibiotics especially for the use of treatment or gram –ve bacterial infections, like: pseudomonas or other highly resistant organisms.

Tetracyclines
 Bind to 30s subunit.
 Similar in their mechanism to Amino glycosides by being able to bind to 30s subunit.
 They are broad spectrum antimicrobial agents.
 They are bacteriostatic.
 They affect wide variety of organisms, They are active against gram –ve, gram +ve and odd organisms like Mycoplasma and Chlamydia, which are resistant to other antimicrobial agents.
 They inhibit protein synthesis by blocking the binding of aminoacyl tRNA(initiation complex) to 30s subunit of ribosome ( is called mRNA complex)>> as a consequence, initiation of protein synthesis is blocked becoz of the binding to aminoacyl tRNA.
 These agents can cause staining of teeth and that’s why they are contraindicated for pregnant Women and children below the age of 8 years becoz they cause brownish fluorescent discoloration of teeth.
 In addition, they accumulate in bones, so bones are stained as well.

 (why children< 8 yrs and pregnant women?
becoz it accumulates in teeth during osteogenesis (development of bones), while in adults growth stops so no accumulation occurs

 Derivatives of tetracycline:

1- Chlorotetracyclin: result when atom replaced by CL.
2- deoxycycline: result when atom replaced by OH.
3- Minocyclin: the most active form of tetracycline.
prolonged use of tetracycline especially for propionibacterium acne (bacteria that causes acne حب الشباب ) can lead to overgrowth of Candida especially in the oral cavity and the vagina causing candidiasis (vaginal or oral candidiasis) which cause oral thrush , that’s lead to the killing of normal flora which is responsible for infection prongs usually non pathogen.
Candida under normal condition does not cause disease but if the normal flora is widely altered, it can cause disease and that’s how candidiasis develops.
Acne treatment requires months of administration of tetracycline (3 to 6 or more) to eradicate the infection and this is commonly associated with the development of much membrane candidiaisis (whether oral or vaginal candidiasis).

 Organisms can develop resistance to tetracycline and this can result from:
1- Decrease Penetration of the antibiotic into the bacterial cell.
2- Active efflux of the antibiotic out of the cell, after the antibiotic enters the cell, it will be excluded ,so it cant continue acting in the cell.(which is the most common)
3- Mutation of the chromosome encoding the outer membrane porin protein (OMPF) which is the binding site of the antibiotic.

chloramphenicol

 is of historical significance:
no more used to treat infections becoz of its high toxicity
 broad spectrum antimicrobial agent:
binds to peptidine transferase component of 50s subunit which blocks peptide elongation.
Peptidine transferase is responsible for transfer of amino acids and that’s how the chain of protein elongates as the consequence of inhibition of peptidine transferase >>elongation of nascent peptide chain is blocked
 can disrupt protein synthesis in the bone marrow and this can lead to blood discrasia such as aplastic anemia
although it develops in one out of 24000 ,the activity of this drug has been limited to few applications >>that’s why it is not commonly used >>to avoid bone marrow suppression in which patient becomes gray(very toxic) that’s why its called gray patient syndrome as a consequence of toxicity associated with chloramphenicol use.
Chloramphenicol was used to treat typhoid fever.
Becoz of the resistance among salmonella to chloramphenicol, it has no place or room in treatment of most infections now ,so it is rarely used in clinical practice.
 blood discrasia: suppression of bone marrow
 pancytopenia: all components of blood are inhibited from being produced in the bone marrow .
 But sometimes abnormality of synthesis of certain components of cells derived from bone marrow resistance to chloramphenicol which is observed in bacteria producing chloramphenicol acetyltransferase which is plasmid encoded >>It catalyses the acetylation of 3-hydroxyl group of chloramphenicol ,the product is incapable of binding to the 50s subunit.
 Less commonly chromosomal mutations alter the outer membrane porin proteins causing decreased permeability of the drug.

Microlides

 are represented with erythromycin
 newer microlides:
1) clarithromycin: addition of CL.
2) azithromycin: derivative of erythromycin.

Erythromycin has limited application, the newer ones has broader application that’s why this group of antibiotics has important application in treatment of many disease.
 They have broad spectrum of activity, being active against gram +ve and gram –ve.
 They are bacteriostatic.
 2nd drug of choice to streptococcal infections: in individuals who are allergic to penicillin we give them erythromycin instead of amphoicillin.
 Drug of choice to treat diphtheria: (infection with diphtheria and protozoal infection >>the causative agent of whooping cough.
 Drug of choice for legionnaires disease caused by Legionella pneumophila.
 Treatment of some other odd organisms such as: mycoplasma and clameidia.
 It has important clinical application
 It has application in treatment of certain infection diseases.
 Similar to chloramphenicol, it binds to 50s ribosome (which is reversible and that’s why it bacteriostatic) which blocks polypeptide elongation.
 Resistance seems from methylation of the 23s ribosome RNA which prevents binding of the antibiotic.
 Other mechanisms include destruction of the lactone ring of the drug by an erythromycin esterase and the active efflux from the bacterial cell.
 The mechanism of resistance to erythromycin is multiple, the bacteria has diverse, different mechanisms to resist action of antimicrobial agents.

Clindmycin and Lincomycin

 Active against aerobic, particularly gram +ve Not gram –ve bacteria.
 They block protein elongation by binding to 50s ribosome.
 The exact mechanism of action: is the inhibition of peptidyltransferase by interfering with the binding of the a.a-acyl-tRNA complex>>similar to tetracycline
 Methylation of the 23s ribosomal RNA is the source of bacterial resistance (cross resistance with erythromycin).
So bacteria that are resistant to erythromycin are resistant to clindmycin becoz they have the same mechanism of action which is methylation of the 23s ribosomal RNA.
 Clindmycin used to treat streptococcal infections. however, the use of clindmycin has been associated with the development of antibiotic –associated diarrhea which can develop into pseudo membrane colitis and this is treated by vancomycin.

• Inhibitors of nucleic acid synthesis:

 They inhibit DNA synthesis, RNA synthesis( or transcription) or metabolic pathways that lead to synthesis of purines and pyrimidines referred to as anti-metabolites.

QUINOLONES:

 The prototype of quinolones is nalidixic acid which was available in 1964 as urinary antiseptic only used for treatment of urinary tract infection , has no effect on other organism.(single application)
 Modification of this drug (Nalidixic acid) results in new antibiotics with broad spectrum activity against both gram-ve and gram +ve, infections of GI tract, respiratory tract and blood stream.
 These new drugs are: Norfloxacin , ciprofloxacin , ofloxacin ,levofloxacin , and spar floxacin.
 Just modification of side chain results in production of new drugs with new pharmacological properties with improved activity, the spectrum could be narrow or broad depending on modification.
 All these act by inhibiting bacterial gyrases (topoisomerase) very important in replication of DNA >>they allow the utilization of DNA , then they seal and supercoil the DNA.
>>So the drug inhibits the enzyme by binding to alpha- subunit of enzyme.
 Resistance develops rapidly in pseudomonas, oxacillin-resistant staphylococci and entrococci.
 Resistance is chromosomal mutation of alpha- subunit or decrease uptake.
 So quinolones used to treat many gram +ve and gram-ve bacterial infections.
 The only is the development resistance rapidly.

Rifampin and Rifbutin:

 Usually used to treat tuberculosis so they are active against myco-Bacterium tuberculosis.
 But they can be used in other infection like prosilosis>> can be treated by rifampin.
 Rifampin has been recommended for prophylactic prevent
Spread of streptococcal infection (meningitis).
 These antibiotic bind to transcriptase inhibiting initiation of RNA Synthesis (so transcription is inhibited >>RNA synthesis is inhibited).
 Bactericidal to M.tuberulosis and very active against gram+ ve cocci.

 Resistance develops rapidly due to mutation in the chromosomal gene encoding the Beta-subunit of RNA polymerase.
 Decreased uptake by gram-ve bacteria.
(So the problem is with the development of resistance).

Metronidazole:

 Has no activity against aerobes or facultative anaerobes.
 Used with other antibiotics for prevention of oral cavity infections.
 Its nitro group is reduced by bacterial nitroreductase producing cytotoxic compounds that disrupt DNA, so it acts by damaging the DNA backbone \as a consequence of reduction of the drug : the reduced form causes breaks in DNA.
 Resistance is due to decreased uptake or elimination of cytotoxic compounds before inter acting with DNA so the only application of metronidezale is on: anerobic infection and amoeba.
 It’s the (flagyl).

Anti metabolites

Sulfonamides:
Structural analogues of P- aminobenzoic acid thereby preventing folic acid synthesis.
It interferes with the ATP –dependent condensation of petridine with PABA to yield dihydropetroic acid which is a substance converted to folic acid.
So sulfonamides are structurally similar to PABA and when they are present in an environment of bacteria,they are utilized instead of PABA to produce this drug .
So when they are incorporated in this reaction (ATP –dependent condensation of petridine with PABA), they (sulfonamides)have high affinity for the enzyme (dihydropetroate synthetase)>>so they inhibit the action of the drug.
Sulfonamides inhibit dihydropetroate synthetase action so the conversion of PABA to dihydropetroate is blocked.

**as you can see in slide #46:
Sulfonamides can be incorporated in place of PABA in dihydropetroate becoz of the analogues structure between Sulfonamides & PABA.


Trimethoprim

Inhibits dihydrofolic acid rductase which reduces dihydrofolate to tetrahydrofolate a stage in the sequence leading to the synthesis of purines (thymidine).>>so thymine synthesis is inhibited.

Sulfonamides, 5 parts and trimethoprim, 1 part
(Co- Trimoxazole) are synergistic and bacteridical but each alone is bacteriostatic.
So the type of activity is changed from bacteriostatic to bacteridical>>the net effect of using them in combination is more than the sum of using both separately.
* Sulfonamides>>inhibit dihydropetroate synthetase.
* Trimethoprim>>inhibit dihydrofolic acid rductase.
Both enzymes are inhibited leading to inhibition of purines synthesis.
During synthesis of purine:
1st : we need to synthesize folic acid which is a precursor of purine .
2nd: then folic acid is reduced producing another component before being converted to purine.
So we have 2 drugs acting on the same criteria,so combination of these 2 drugs has synergistic & bactericidal effect.

Resistance is common due to permeability barriers or decreased affinity of the enzyme and the ability to use exogenous thymidine (enterococci).

***NOTE:
- bacteriostatic: stops the replication of the organism.(when removed, the bacteria resumes its normal function).
- Bactericidal: kills the bacteria.


Measuring antibiotic sensitivity

1) Disk diffusion (the most common): organisms are streaked on a plate then streak is followed by 15 min. of incubation so that organisms establish itself on the media then we place this ring of filter paper integrated with antibiotics (single or multiple) then incubated for 18-24 hours. After incubation we test the organisms:
A. Organisms that are susceptible to antibiotic will be inhibited from growth and that’s why a clear zone of inhibition is seen around this antibiotic (susceptible).
B. Organisms are resistant to antibiotics >>no zone of inhibition will be seen unaffected by the antibiotic, antibiotic diffuses in agar and kills bacteria >>clear zone (resistant).
C. Antibiotic could be active in minimal activity >>very small zone of inhibition (intermediate).
2) Dilution method: determine MIC:-
We look for visual (naked eye) loss of turbidity.
Clear -preparation.
The bubles that show clear (no turbidity) appearance are regarded as MIC.
**For different antibiotic we have different MIC.

3) E-test: a new method has been developed to test for MIC.
Strip of antimicrobial agent which has a gradient of concentrations of antibiotic, so when these gradient of concentrations are put on a plate that has been already streaked with an organism >>an eclipse is formed (شكل بيضاوي) >>becoz of different concentration (less in an area, more in onother) so diameter differs according to concentration >>the diameter is most at highest concentration.
So MIC: is the concentration-immediately- above the line of interception between antimicrobial agent and growth.
So MIC is determined by 2 methods: dilution & E-test.

Note: dr. read slides# 51+52

Picture at slide #53: antibiotic selection for resistant bacteria:-
We have resistant and susceptible bacteria….. if we use an antimicrobial agent ,it will kill only susceptible and this will allow proliferation and propagation of the resistant >> becoz use of an antimicrobial agent will select for their(resistant ones) survival ,so they increase in concentration.

Note: dr. read slides #54+55

Here are some explanations and notes about some slides:
In slide 55: (explanation of some points):
-prescription not taken correctly>> explanation: when you take a drug for several days ,then you feel better, so you stop taking that drug>>this is wrong.
- Antibiotics for viral infections>> Note: this point is the most common one.

In slide 56:
-Use of antibiotics in food.
Explanation:
The Use of antibiotics in food is the origin of VRE ( vancomycin resistance enterococci),which is a major problem in hospitals becoz of incorporation of vancomycin in animal feed(العلف) .
-VRE was originated in 1988 in Europe &in 1989 in America. so organisms develop resistance>>so these organisms could be transferred to human bodies in food and affect them.

In slide 57:
-This slide shows the cost of treatment.
-Treatment of primary infections is not coasty.
-Treatment of secondary &tertiary infections is coasty>>due to resistance development.

In slide 58:
-This slide shows the antibiotic abuse.
-100% of common cold treatment with antibiotics leads to resistance.

In slide 59:
Multi-drug resistance
-there are agents resistant to everything!
-TB patients>>25% …chance of living.
>>75%...of them die.

In slide 60:
-restrict antimicrobial use>> Note: this point is the most important one.




**NOTE:
-The answers of the questions asked during the lecture are written as notes in the sheet.

*******



 DONE BY: nouran Hatim Mustafa.
• date of lecture:11-10-2010
 lec.#11
 2nd lecture of 4rth week.

I've found these mistakes in my sheet:
Page7>>line 12>> the only (problem) is the development (of) resistance rapidly
Page8>>when talking about Sulfonamides>>line 13>> sulfonamides inhibit the action of the (Enzyme) NOT the (drug)
Page 10>> line 4>> the (tubes) NOT the (bubles) that show clear…..etc.