Microbiology

1. Ministry Of Higher Education
& Scientific Research
Alzaiem Alazhari University, Sudan
Faculty of Medical Laboratory Sciences
(MLS)
8 February 2023 1

2. Antimicrobial agents and
Chemotherapy
Prepared by:
Mohamed Ahmed Ibrahim Holie
AL-ZAIEM ALA-zhari University
Faculty of Medical Laboratory Sciences
Holie2330@yahoo.com
8 February 2023 2

3. Definitions of Antibiotics
• OLD: An antibiotic is a chemical substance
produced by various species of
microorganisms that is capable in small
concentrations of inhibiting the growth of
other microorganisms
• NEW: An antibiotic is a product produced by
a microorganism or a similar substance
produced wholly or partially by chemical
synthesis, which in low concentrations,
inhibits the growth of other microorganisms
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4. Disadvantages from taking
medication without doctor guide
 Emergency of Resistance.
 Drug toxicity.
 Cost.
 Drug dose.
 Interaction of the drug with other disease.
 Susceptibility of the pathogenes to the
drug.
 Time consuming.
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5. Antimicrobial agents:
 Chemical and therapeutic agents that acts
against bacteria, viruses, fungi and
parasite.
 Antibiotic ------ Natural product from
microorganism.
 Chemotherapy ------ Chemically synthetic
or semi synthetic agent.
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6. 6
 Antibacterials can be classified into:
a. By their activity level action:
1. Bacteriostatic: they arrest the growth &
replication of bacteria
e.g. Sulfonamides, Chloramphenicol
2. Bactericidal: kills the bacteria
e.g. Penicillins
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b. Spectrum of activity:
1. extended spectrum of antibiotic: active
against types of bacteria G- & G+
2. Narrow spectrum antibiotic: active against
single or limited # of M.O
3.broad spectrum: active against a wide
variety of microbial species
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8. 8
Combination of antimicrobials
Advantages:
1. Delay the development of resistance.
2. To broaden the spectrum of activity.
3. Potentiation of action of one another.
indifferent
Combination of antibiotics either synergism
antagonism
A 60% bacterial inhibition
A+B 80% synergism
A+C 40% antagonism
A+D 60% indifferent
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Prophylaxis antibiotics:
 Definition
A prophylaxis is a measure taken to maintain
health and prevent the spread of disease.
Antibiotic prophylaxis is refers to the use of
antibiotics to prevent infections as:
1.Treatment prior to surgical procedures as bowel
surgery, joint replacement to prevent infection.
2.prevention of tuberculosis or meningitis in
individuals who are close contact with infected
patients.
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3. Suppression of existing infection before it cause a
disease, e.g. animal bite.
4. Prevention of streptococcal infection in patients with
history of rheumatic heart disease.
5. prevention of opportunistic infection →infective
endocarditis.
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11. By their Mechanisms of Action Antibacterials
can be classified into:
 Inhibition of cell wall synthesis.
 Inhibition of Cell membrane synthesis.
 Inhibition of protein synthesis.
 Inhibition of Nucleic acid synthesis.
 Metabolic antagonism.
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12. Inhibition of cell wall synthesis:
 -Lactam containing antimicrobial:
 Penicillin.
 Ampicillin.
 Amoxicillin.
 Amoxicillin-clavulinic acid.
 Cephalosporins:
 1st generation (Cephalothin, Cephapirin,
Cephazolin, Cephalexin, Cephradine, and
Cefadroxil).
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13.  2nd generation: (Cefoxitin, Cefotetan,
Cefamandole, Cefuroxime, Cefonicid,
Cefaclor, Cefprozil, Cefmetazole).
 3rd generation: (Cefotaxime, Ceftizoxime,
Ceftriaxone, Ceftazidime, Cefoperazone,
Cefixime, Ceftibuten).
 4th generation: (Cefepim).
 Bacitracin.
 Vancomycin.
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14. 1. -Lactam containing Antibiotics
Penicillin:
 From Penicillium notatum.
 Discovered at 1928 and used at 1938.
 Active part is β-lactam ring.
 Inter through penicillin binding proteins.
 Resistant by:
 Production of β-lactamase.
 Lack or alteration of PBPs.
 Failure in activation of autolytic enzyme.
 Failure in synthesis of peptidoglycan (Mycoplasma).
 Side effect:
 Penicillin allergy.
 Types:
 Penicillin G Acid sensitive (IV and IM).
 Penicillin V Acid Resistant (Oral)
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15.  Ampicillin:
 Broad spectrum antimicrobial (G+ve and some G-ve).
 Acid tollerant.
 Amoxicillin:
 Have wide range of activity against G+ve G-ve and some
Anaerobe)
 Could be taken orally.
 Amoxicillin-Clavulinic acid (Augmentin or Amoclan):
 More penicillinase resistant antimicrobial
 Monobactam:
 IV or IM antimicrobial that active against G-ve bacteria and resist β-
lactamase (e.g.: Aztreonam).
 Carpenems:
 Active against G+ve and G-ve bacteria and anaerobes except
bacteroides.
 Could reach CSF.
 Side effects:
 Skin rash, diarrhoea, vomiting, and Hypersensitivity.
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16. Cephalosporins:
 Same as penicillin in action.
 Resistance:
 Poor permeation of bacteria (No entry).
 Lack or alteration of PBPs.
 β-lactamase production.
 Side effects:
 Hypersensitivity.
 Thrombocytopenia.
 1st generation:
 Active against G+ve cocci except enterococci,
moderate active against E.coli, Klebsiella, proteus
and some anaerobic.
 Cephalexin and Cephradin given orally.
 Could be used in UTI and RTI.
 Cephalexin used as post operative prophylaxis.
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17.  2nd generation:
 Active against G+ve and G-ve (except
pseudomonas) and some anaerobe.
 3rd generation:
 Less active against G+ve, but active
against G-ve and moderate action
against pseudomonas.
 Have the ability to reach the CSF.
 4th generation:
 Act against G-ve including enterobacter
and Citrobacter Pseudomonas and
moderate sensitive against G+ve.
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18. 2. Bacitracin:
 From B. subtilus.
 Inhibit early cell wall synthesis in cell
membrane (inhibit lipid carrier).
 Poor absorbance from GIT.
 Very active against G+ve bacteria.
 Used as ointment.
 Side effect:
 Kidney damage.
 Nitrogen retention.
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19. 3. Vancomycin:
 From S. orientalis
 Bind with peptide chain inhibiting
transpeptidation.
 Side effects:
 Skin rash.
 Kidney damage.
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20. Inhibition of cell membrane:
 Polymexin B:
 Disrupt plasma membrane structure.
 Bacteriocidal for G-ve and G+ve.
 High toxic used as Ointment.
 Side effects:
 Nephrotoxic.
 Neurotoxic.
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21. Inhibition of Protein synthesis:
 Inhibition of 30 S rRNA:
 Aminoglycoside.
 Tetracyclins
 Inhibition of 50 S rRNA:
 Chloramphenicol.
 Lincosamide (Lincomycin and clindamycin).
 Macrolides (Erythromycin, Clarithromycin).
 Fusidic acid Affect Elongation factor G.
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22. Aminoglycoside:
 Streptomycin, Neomycin, Kanamycin, Amikacin,
Gentamicin, and tobramycin.
 Bacteriostatic inhibit 30 S rRNA.
 Active at alkaline PH.
 Broad spectrum (G+ve and G-ve and Mycobacterium).
 Give synergistic effect with penicillin.
 In blood culture aminoglycoside inhibited by Na polyanithol
sulfonate.
 Resistance by:
 Deficiency in Ribosomal receptor.
 Lack of permeability to the drug (transportation is oxygen
dependent).
 Side effect:
 Nephrotoxic and Ototoxic.
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23. Doxycyclins and Tetracyclins:
 Inhibit G+ve, G-ve, Rickettsia, Chlamydia,
Mycoplasma, Brucella, Yersinea and Vibreo.
 Bacteriostatic in action.
 Have poor penetration to CSF.
 Side effects:
 Gastrointestinal upset (Nausea, Vomiting, and
Diarrhoea).
 Skin rash.
 Deposition in bone structures and teeth particularly in
fetus.
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24. Chloramphenicol:
 From S. vensuelae (Now synthesized).
 Wide distributed in all body fluid including CSF.
 Inactivated in liver and secreted in urine in inactive
form.
 Bacteriostatic in action.
 Resistant by production of Chloramphenicol acetyl
transferase.
 Side effects:
 GIT upset.
 Aplastic anaemia.
 Gray fetal syndrome. (Have no develop liver so No
inactivation).
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25. Lincosamide (Lincomycin and
Clindamycin):
 From S. licolnsis.
 Very active against bacteroides and
other anaerobes.
 Cause Pseudomembranous colitis.
 Acid stable.
 Cold not penetrate CSF.
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26. Macrolides (Erythromycin,
azithromycin and Clarythromycin):
 From S. erythrus.
 Resistance by alteration of rRNA receptor.
 Active at alkaline PH.
 Active at G+ve, G-ve, Chlamydia,
Mycoplasma and Legionella.
 Side effects:
 GIT upset.
 Fever.
 Affect liver cells causing hepatitis.
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27. Quinolones:
 Block DNA gyrase.
 E.g.: Nalidixic acid, Oxolinic acid,
Ciprofloxacin, Ofloxacin, Norfloxacin,
Pefloxacin, and Nitrofurantoin.
 Used in UTI.
 Active against G-ve, G+ve, Haemophilus,
Pseudomonas and Mycobacterium.
 Side effect:
 Nausea, headache, and dizziness.
 Skin rash.
 Impaired liver function.
Inhibition of Nucleic acid:
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28. Rifampin:
 Active against some G+ve, G-ve, Enteric
cocci, Mycobacterium, Chlamydia and pox
virus.
 Bind with RNA polymerase (inhibit RNA
synthesis).
 Could penetrate inside phagocytic cells.
 Side effects:
 Orange pigment in urine and sweat.
 Skin rash.
 Thrombocytopenia.
 Impaired liver function.
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29. Metabolic antagonistic:
 Bacteriostatic for some G+ve and G-ve bacteria.
 Inhibit folic acid synthesis.
 Synergistic effect obtained from Cotrimoxazole
(Trimethoprim and Sulphomethoxazole).
 Side effects:
 Hypersensitivity.
 Fever and skin rash.
 GIT disturbances.
 Depression of bone marrow.
 Liver and kidney function abnormality.
Sulphonamide and trimethoprim:
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30. Antimicrobial susceptibility
pattern:
 Kirby Bauer agar disk diffusion
methods:
 Aim:
 To perform standard sensitivity test on agar
media.
 Test requirements:
 Media: Muller & Hinton agar
and Broth media
 Antimicrobial disks: Multi or single disks
 STD turbidity tube: McForland tube (Bacl2 +
H2So4)
 Pure culture from organism under test.
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31. Standard MacForland tubes
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32. Disk dispenser
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33.  Test Procedure:
 Make a suspension by touching more than 10 colonies and emulsify
on M&H broth.
 Incubate for 30 min in water bath.
 Compare the turbidity with 0.5 McForland tube (contain 0.05 ml of
1.16% Bacl2 + 9.95 ml of 1% H2SO4).
 Streak on M&H agar using Ca++ alignment swab 3 times inverting the
plate 60 each time.
 For H influenzae, S. pneumoniae, and Neisseria we use M&H
supplemented with Heated sheep Blood and for Streptococci we use
Blood agar.
 Put the disk on the center of the plate allowing 1.5 cm distances from
the edge of the plate and 1 cm between each disks (the plate take
about 8 disks).
 Incubate at 37C for exactly 18 hours.
 Read the diameter of the disk using ruler and compare it with a STD.
 Report your results as HS, MS, SS, R.
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37. Antagonism
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38. Agar diffusion for fastidious
microorgnisms
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40. Same plate comparative
methods (Stoke)
 We culture control strain on the center
of the plate and tested organism on
the two edges. Put the disks between.
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41. Dilution methods:
 This used to detect antimicrobial susceptibility to
slow growing M.O and anaerobic.
 It could be carried out on Microtiter plate or test
tube.
 Also it used to detect MIC to antimicrobials
 We make serial dilution of antimicrobials and add
5 drops from 106 bacterial suspension and
incubate at 37 C, then read about turbidity
which indicate resistance and clearance which
indicate sensitivity, and detect the MIC.
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42. Tube dilution method
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43. Factors affect antimicrobial agar disk
diffusion method:
 Type of the medium.
 Depth of the medium (4 mm)
 Surface of the medium
 Supplement in the medium (mg and ca) and Blood
 Moisture of the medium
 pH of the medium
 Source and expiry date of antimicrobial agents
 Concentration of antimicrobials in the disk
 Distribution of disks on the surface of the medim.
 Nature of organism
 Inoculum size.
 Incubation temperature.
 Incubation condition.
 Incubation time.
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44. Microdilution methods
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46. Epsiloid test (E test):
 Used for detection of MIC in plate
procedure.
 We could detect more than one MIC at the
same time.
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47. E. Test
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49. Antimicrobial Resistant test:
 β lactamase test:
 Cefinase β lactamase disk:
 Using disks contain nitrocefin, we add suspension of
Microorganism and see the development of Pink-Brown color
within 10 min indicating β lactamase production.
 Acidimetric technique:
 Using citrate buffered penicillin and phenol red. Penicillin
destroyed by the enzyme to give penicilloic acid which
change the color from red into yellow.
 Iodometric technique:
 Using phosphate buffered penicillin and starch-iodine
complex, penicilloic acid prevent binding of starch with
penicillin which convert the color from blue to colorless.
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50. Errors of reading
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51. Yeast
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52. Beta lactamase detection
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