Chemotherapy. Chemotherapeutic preparations. Antibiotics classification.

1. THEME: CHEMOTHERAPY. CHEMOTHERAPEUTIC PREPARATIONS.
ANTIBIOTICS. CLASSIFICATION. METHODS OF DETERMINATION OF
ANTIBIOTIC SENSITIVITY OF BACTERIA. THE MAIN PRINCIPLES OF
RATIONAL ANTIMICROBIAL THERAPY OF DISEASES
I. STUDENTS’ INDEPENDENT STUDY PROGRAMME
1. Concept of chemotherapeutic drugs:
a – main groups of chemotherapeutic drugs;
b – chemotherapeutic index, its value;
c – mechanisms of action of the main chemotherapeutic preparations;
2. Antibiotics and mechanisms of their action:
a – what does term "antibiotics" mean;
b – classification of antibiotics according their origin, action spectrum, mechanism of
action, chemical structure;
c – units of determination of antibiotics activity;
d – antimicrobial susceptibility testing (serial dilutions, standard disks, accelerated
methods, automated liquid diffusion method);
3. Main principles of a rational chemotherapy;
4. Side effects of antibiotics, complications of chemotherapy.
5. Resistance of microbes to antibiotics:
a – mechanisms, which cause drug resistance;
b – factor of a multiple resistance to drugs (R –, r–factors), transmission of them in
bacteria;
c – methods of warning of derivation of resistant causative agents.
Various chemical substances have a lethal action on pathogenic microorganisms. They are
widely used in medical practice for treating patients with infectious diseases and in some
cases for prophylaxis.
The basis of modem chemotherapy was founded by P. Ehrlich and D. Romanowsky, who
formulated the main scientific principles and the essence of chemotherapy. P. Ehrlich devised the
principles of synthesis of medicinal substances by chemical variations: methylene blue,
derivatives of arsenic–salvarsan ('"606"), neosalvarsan ("914"). By the further development of
chemistry new medicinal preparations could be obtained.
Extensive experimental and clinical tests of chemopreparations were carried out by E
Metchnikoff.
Chemopreparations should have a specific action, a maximal therapeutic effectiveness, and
a minimal toxicity for the body.
As a characteristic of the quality of a medicinal preparation, P. Ehrlich introduced the
chemotherapeutic index which is the ratio of the maximal tolerated dose to the minimal curative
dose:
maximal tolerated dose (DT—Dosis tolerata)
---------------------------------------------------------------- > 3
minimal curative dose (DC—Dosis curativa)
The chemotherapeutic index should not be less than 3.
Chemotherapeutic preparations include a number of compounds used in medicine
Arsenic preparations (novarsenol, myarsenol, aminarsone, osarsol, etc.) are administered
in syphilis, relapsing fever, trypanosomiasis, amoebiasis, balantidiasis, anthrax, sodoku, and
other diseases.

2. Bismuth preparations (basic bismuth nitrate, xeroform, basic bismuth salicylate,
bioquinol, bismoverol, bithiurol, pentabismol, etc.) are used against enterocolitis and syphilis
Antimony compounds (tartaric antimony potassium salt, stibenil, stibozan, surmine,
solusurmine, etc.) are used for treating patients with leishmaniasis and venereal
lymphogranulomatosis.
Mercury preparations (mercury salicylate, mercuric iodide, mercury cyanide, calomel,
unguentum hydrargyri cinereum containing metallic mercury, etc.) are prescribed for treating
patients with syphilis and are used as antiseptics in pyogenic diseases.
Acridine preparations (rivanol, tripaflavine, acriflavine, acricide, flavicide, etc.) are
recommended for pyogenic diseases and inflammatory processes of the pharynx and
nasopharynx
Antimalarial substances include more than 30 preparations, e g , chinine hydrochloride,
quinine sulphate, mepacrine (acrichine), rodochin (plasmocide), proguanyl (bigumal),
pyrimethamine (chloridine), resochine, quinocide sulphones and sulphonamides, sulphadiamine,
etc.
Alkaloid preparations (emetine, etc.) are used for treating patients with amoebiasis.
Sulphonamide preparations. The introduction into practice of compounds of the
sulphonamide group (streptocid, ethasole, norsulphazol, sulphazine, methylsulphazine,
sulphadimezin, urosulphan, phthalazole, sulgine, sulphacyi, soluble sulphacyl, disulphormin,
etc.) marked a revolution in the chemotherapy of bacterial infections.
Sulphonamide preparations are used for treating pyogenic diseases, tonsillitis, scarlet fever,
erysipelas, pneumonia, dysentery, anaerobic infections, gonorrhoea, cystitis, venereal
lymphogranulomatosis, psittacosis, ornithosis, trachoma, blennorrhoea in the newborn, etc.
There are several points of view concerning the mechanism of action of sulphonamides on
microbes.
Analogs of iso-nicotinic acid: PAS, tibone, phthivazide, isoniazid, saluzid, metazid,
larusan, etoxid, sulphonin, uglon, crisanol, etc., are used for treating tuberculosis patients. Of
these phthivazide which is a derivative of isonicotinic acid hydrazide has a good therapeutic
action.
Preparations of the nitrofurane series (furazolidone, furadantine, furaguanidine) are
used for treating intestinal infections.
The group of effective antibacterial agents includes quinoxidine, dioxidine, the derivatives
of nitrofurane– furagin, soluble furagin, solafur, etc.
Antibiotics (Fr. anti against, bios life) are chemical substances excreted by some micro-
organisms which inhibit the growth and development of other microbes (in recent years
several antibiotics have been obtained semisynthetically).
Antibiotics are obtained by special methods employed m the medical industry. For the
production of antibiotics strains of fungi, actmomycetes, and bacteria are used, which are seeded
in a nutrient substrate. After a definite growth period the antibiotic is extracted, purified and
concentrated, checked for inoculousness and potency of action.
According to the character of action, antibiotics are subdivided into bacteriostatic
(tetracyclines, chloramphenicol, and others) and bactericidal (penicillines, ristomycin, and
others). Each antibiotic is characterized by a specific antimicrobial spectrum of action (narrow
or broad spectrum).
The mechanism of action of antibiotics varies.
1) inhibition the synthesis of the bacterial cell (penicillins, cephalosporins, β-lactams)
2) inhibition protein synthesis ( tetracyclines, lincomycin, erythromycin, kanamycin,
neomycin, spectinomycin, sparsomycin, fucidine, streptomycin, chloramphenicol)
3) impairment the intactness of the cytoplasmic membrane (antifungal antibiotics
poliens)
4) suppressing the synthesis and function of nucleic acids (quinolons, rifampins,
antitumor antibiotics)

3. The activity of antibiotics is expressed in international units (IU). Thus, for example, 1 IU of
penicillin (Oxford unit) is the smallest amount of preparation inhibiting the growth of a standard
Staphylococcus aureus strain.
One unit of activity (AU) corresponds to the activity of 0.6 micrograms (µg) of. the
chemically pure crystalline sodium salt of benzylpenicillin. Consequently, in 1 mg of sodium salt
of benzylpenicillin there may be 1667 AU, and in 1 mg of potassium salt — 1600 AU. For
practical purposes both preparations are manufactured with an activity not less than 1550 AU.
The concentration of dry preparations as well as of solutions is expressed as the number of
micrograms of active substance in 1 g of preparation or in 1 mg of solution.
Antibiotics are classified according to the chemical structure, the molecular mechanism,
and the spectrum of activity exerted on the cells.
According to origin, antibiotics are subdivided into the following groups.
1) Antibiotics produced by fungi.
2) Antibiotics produced by actinomycetes.
3) Antibiotics produced by bacteria
Resistance of microbes to antibiotics. With the extensive use of antibiotics in medical
practice, many species of pathogenic micro-organisms became resistant to them.
Resistance may develop to one or simultaneously to more antibiotics (multiple resistance).
The molecular mechanism of the production of resistance to antibiotics is determined by
genes localized in the bacterial nucleoids or in the plasmids, the cytoplasmic transmissible
genetic structures.
Resistance to antibiotics occurs as the result of mutations or genetic recombination
(transfer r-genes and R-plasmid due conjugation, transduction and transformation).
Mechanisms of resistance are:
1) Change of permeability of the cytoplasmic membrane and cell wall for
antibiotics
2) Synthesis proteins which transport chemicals out of the cell
3) Forming of enzymes inactivating antibiotics
4) Appearance new metabolite ways for obtaining important for life compounds
Side effects of antibiotics.
1) Toxic actions (a neurotoxic action, affect the liver, a toxic effect on the
haematopoietic organs, etc)
2) Allergic reactions (a rash, contact dermatitis, angioneurotic oedema, anaphylactic
reactions or allergic asthma, anaphylactic shock)
3) Antimicrobial agents cause the formation of numerous variants of microbes with
weak pathogenicity (atypical strains, filterable forms, L-forms) which lead to the
formation of latent forms of infections
4) Antibacterial agents may induce disorders of the genetic apparatus of the macro-
organism's cells and cause chromosomal aberrations; some of them possess a
teratogenic effect leading to the development of foetal monstrosities if they are
taken in the first days of pregnancy.
5) Development of dysbiosis
ІI. Students’ Practical activities:
1. Examine the sensitivity of S.aureus and tE.coli to decamethoxine by serial dilutions
method in a liquid media.
Method of serial dilutions in a liquid medium (description of method).
• Meal peptone broth is poured by 2-ml portions into test tubes mounted in a tube
rack by ten in each row.
• Decamethoxine solution containing 1000 µg per ml is prepared.

4. • 2 ml of this solution is added into the first test tube. Transfer with a new sterile
measuring pipette 2 ml of the mixture from this tube into the next one, and so on
until the ninth tube is reached, from which 2 ml is poured off.
• The tenth tube do not contain preparation. It serves as a control of culture growth.
• Wash the 24-hour agar culture of the studied microorganism with isotonic sodium
chloride solution.
• Determine the density of the suspension by the turbidity standard, and dilute to a
concentration of 10000 microorganisms per ml.
• A sample of 0,2 ml of the obtained suspension is inoculated into all tubes of the
row beginning from the control one.
• The results of the experiment are read following incubation of the tube at 37 °C for
18-20 hrs.
• The minimal concentration of the preparation suppressing the growth of the given
microorganism is determined by the last test tube with a transparent broth in the
presence of an intensive growth in the control one.
Another approach to antimicrobial susceptibility testing is the determination of the
minimum inhibitory concentration (MIC) that will prevent microbial growth (fig. 3). The MIC
is the lowest concentration of antimicrobial agent that prevents the growth of a
microorganism in vitro.
MIC is not designed to determine whether the antibiotic is microbicidal. It is possible to
determine the minimal bactericidal concentration (MBC). The MBC is also known as the
minimal lethal concentration (MLC) (fig. 3). The minimal bactericidal concentration is the
lowest concentration of an antibiotic that will kill a defined proportion of viable organisms
in a bacterial suspension during a specified period of exposure..
To determine the minimal bactericidal concentration, it is necessary to plate the tube
suspensions showing no growth in tube dilution (MIC) tests onto an agar growth medium. This is
done to determine whether the bacteria are indeed killed or whether they survive exposure to the
antibiotic at the concentration being tested.
Registry the MIC and fill in table:
N test tube 1 2 3 4 5 6 7 8 9 10
Concentration
of decam-ne
(µg/ml)
500 250 125 62.5 31.3 15.6 7.8 3.9 1.95 Culture
control
E. coli
S.aureus
MIC of decamethoxine for E. coli is
MIC of decamethoxine for S.aureus is
Registry the MBC and fill in table (plate test):
N sector 1 2 3 4 5 6 7 8 9 10
Concentration
of decam-ne
(µg/ml)
500 250 125 62.5 31.3 15.6 7.8 3.9 1.95 Culture
control
E. coli
S.aureus
MBC of decamethoxine for E. coli is
MBC of decamethoxine for S.aureus is

5. 2. To determine sensitivity of microorganisms to antibiotics by standard disks method (by
Kirby-Bauer’s).
Disk method.
• On the surface of solidified and slightly dried sterile agar, pour 1 ml of suspension
of 24-hour culture of the causative agent.
• Spread uniformly over the agar surface the bacterial suspension, removing its
remainder with a Pasteur pipette.
• Disks with antibiotics (5-6 disks per plate) are placed onto the surface of the
inoculated plate at a distance of 25 mm from its centre.
• The plates are incubated at 37 °C for 16-18 hrs, after which the results of the test
are read by measuring the zones of growth retardation of microorganisms around
the disks, including the diameter of the disk itself (fig.4). The size of the zones
depends on the degree of sensitivity of the causative agent to a given antibiotic.
The diameter of retardation growth zone:
D < 10 mm for resistant bacteria ( R )
D < 15 mm for intermediate sensitive bacteria (I)
D < 20 mm for moderately susceptible strain (MS)
D > 20 mm for susceptible strain (S)
Write the results of antibiotic susceptible test by disk diffusion and made the conclusion
about sensitivity certain strain.

6. 2. To determine sensitivity of microorganisms to antibiotics by standard disks method (by
Kirby-Bauer’s).
Disk method.
• On the surface of solidified and slightly dried sterile agar, pour 1 ml of suspension
of 24-hour culture of the causative agent.
• Spread uniformly over the agar surface the bacterial suspension, removing its
remainder with a Pasteur pipette.
• Disks with antibiotics (5-6 disks per plate) are placed onto the surface of the
inoculated plate at a distance of 25 mm from its centre.
• The plates are incubated at 37 °C for 16-18 hrs, after which the results of the test
are read by measuring the zones of growth retardation of microorganisms around
the disks, including the diameter of the disk itself (fig.4). The size of the zones
depends on the degree of sensitivity of the causative agent to a given antibiotic.
The diameter of retardation growth zone:
D < 10 mm for resistant bacteria ( R )
D < 15 mm for intermediate sensitive bacteria (I)
D < 20 mm for moderately susceptible strain (MS)
D > 20 mm for susceptible strain (S)
Write the results of antibiotic susceptible test by disk diffusion and made the conclusion
about sensitivity certain strain.