Powerpoint

1. Mkakosya, R.S.

2. At the end of this lecture students should be able to
 Explain why bacteria are used for metabolic studies
 Explain how microorganisms acquire energy and nutrients
 Explain the growth (culture) media
 Discuss the four main factors that affect microbial growth
 Differentiate among the various media used in culturing
microorganisms
 Know the significance of sterility in growing microorganisms
 Explain the bacterial growth patterns in broth and on agar
 Explain why bacteria die during the death phase
 Define the different terms (e.g. –cidal, -static, sepsis) associated
with the control of microbial growth
BSc N/M CUNIMA 2

3.  Vital life processes
 Bacteria mostly used for such studies
Inexpensive
Take up little space
Quick reproduction
Easily observable morphology, nutritional needs and metabolic
reactions
Available species to represent all forms nutritional types
 Each bacteria produce cells like itself
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4.  Nutrition required for cellular structure formation,
development, multiplication and vitality
 Carbon, oxygen, hydrogen, nitrogen, sulphur, phosphorous,
(macro elements)
 Micro elements include; manganese, potassium, calcium,
magnesium, iron zinc, cobalt, molybdenum, nickel and
copper
 Essential nutrients: materials not synthesized by
organisms
BSc N/M CUNIMA 4

5.  Phototrophs: Use light as energy source
 Chemotrophs:
Lithotrophs: Use inorganic chemicals for energy
Organotrophs: Use organic materials as energy source
 Autotrophs: Use CO2 as source of carbon
 Heterotrophs: Use organic compounds
 Photoautotrophs: Use light and CO2 (e.g cynanobacteria)
 Photoheterotphs: Use light and organic compounds (e.g.
nonsulfur bacteria)
 Chemoautotrophs: Chemical and CO2
 Chemoheterotrophs: Use chemical energy and organic
compounds for carbon
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6.  Optimal growth condition necessary
 Inability to sustain optimum condition limit growth and lead to
massive death of microorganisms
 Survivors of sub-optimum conditions lose some phenotypic
characteristics
 4 major factors affect bacterial growth
Physical factors
Chemical factors
Biological factors
Mechanical factors
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7.  Temperature
Heat
 Significantly affect growth
 Different species have maximum and minimum growth temperatures
 Generally optimum growth temperature 5-10C lower than the maximum but 20-
30C higher than the minimum
 Pathogenic microorganisms have a narrow temperature growth range
 Optimum growth temperature may not be appropriate for synthesis of some
essential components or bacterial products
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8.  Bacteria classified into 4 groups based on their temperature ranges
of growth
 Psychrophiles: 5-15C e.g. A. salmonicida
 Mesophiles: 30-45C most pathogenic bacteria
 Thermophiles: 50-60C e.g. B. stearothermophilus, Pyrolobus
fumarii
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9. BSc N/M CUNIMA 9

10.  Applied during
 Sterilization and disinfection
 Induce mutation
 Non-Ionizing
 UV light
 Infrared
 Ultrasonic vibration
 Ionizing
 Electromagnetic
 X-rays
 Gamma rays
 Particulate radiation
 Alpha
 Beta
 Cathod
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11.  Osmotic pressure
 Moisture
 Electrical effects
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12.  Oxygen
◦ Aerobic
◦ Anaerobic
◦ Falcutative anaerobic
◦ Microaerophilic
◦ Aerotolerant
 Carbon dioxide
 pH
 Redox potential
BSc N/M CUNIMA 12

13. Mkakosya, R.S.

14. BSc N/M CUNIMA 14

15. BSc N/M CUNIMA 15

16.  Aseptically prepared environment to grow microorganisms
 Some mo grow on simple defined media
 Many organisms require complex media
 Available in liquid and solid forms
 Solid media obtained by addition of agar into the media
 Some ingredients in media restrict growth of certain organisms while
permitting others e.g. antibiotics in fungal media
 Media for yeasts and moulds have lower pH than bacterial
BSc N/M CUNIMA 16

17.  Basal media: Simple synthetic media with a carbon and energy source
plus an inorganic source of nitrogen e.g. peptone water or nutrient broth
 Enriched media: Meets nutritional requirements of most bacteria e.g.
blood agar
 Selective media: Suppress unwanted microbes, or encourage desired
microbes
 Differential media: Distinguish colonies of specific microbes from others
 Enrichment media: Similar to selective media but designed to increase
the numbers of desired microorganisms to a detectable level without
stimulating the rest of the bacterial population
 Transport media: Devised to maintain the viability of desired pathogens
and avoid overgrowth of other contaminants
BSc N/M CUNIMA 17

18. Peptone: Consists of water soluble products from lean meat or other
protein material e.g. casein, fibrin, soya flour etc
Available as golden granular powder with low moisture content
Highly hygroscopic
Meat extract: Used as a substitute to fresh meat infusion
Yeast extract: Prepared from washed cells of brewer’s yeast
Contains amino acids, growth factors and inorganic salts
Comprehensive source of growth, can be substituted by meat extract
Blood: Aseptically collected blood should be used
Should be rendered non-coagulating by defibrination, heparinization or
adding citrate or oxalate
Blood so treated can be kept for 2 months but should not be allowed to
freeze
Serum: Used in some media
Can be filter-sterilized
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19. Water: Use glass distilled or demineralised water
Agar: Prepared from seaweed
1-1.5% w/v concentration enough to gel
Composed of long chain polysaccharide of D-galactopyranose
Has impurities such as inorganic salts and traces of long chain fatty acids
Dissolves at about 100C
Does not add to the nutritive properties of a medium
Can be decomposed by some marine bacteria
Carbohydrates: Used in the form of starch or sugars
Glucose (dextrose) only sugar used as nutrient
Ability to ferment sugar aid in identification
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20.  Bacteria and yeasts divide by binary fission
 Doubling of macromolecules
 Septum formation
 Constriction
 Generation time
 Growth consistent till stationary
 Broth
◦ turbidity
 Agar
◦ Colony
◦ Yeast colonies
◦ Mould colonies
 Planktonic (free) and sessile (attached) growth
BSc N/M CUNIMA 20

21. Bacterial growth curve
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22. A: The lag phase
 Cells adjust to new growth conditions and growth is unbalanced
 Length of the period depend on the extent of change
B: Exponential (log) Phase
 Cells divide at a constant rate depending on the composition of the growth medium
and the conditions of incubation
C: Stationary Phase
 Exponential growth cannot be continued forever in a batch culture (e.g. a closed
system such as a test tube or flask)
 Population growth is limited by factors such as
 Exhaustion of available nutrients
 Accumulation of inhibitory metabolites or end products
 Lack of "biological space".
D: Death Phase
BSc N/M CUNIMA 22

23.  Restricted growth
 Exposed to numerous factors
 Difficulty in accessing nutrients by organisms on the apex
 Nutrients underneath and on the sides get depleted
 Secondary metabolic activities not released
 Degeneration of apex colonies due to starvation
 Growth of other colonies exacerbate the scarcity of available nutrients
 Colonies underneath affected by the weight of the colonies above hence degeneration
takes place
 Toxic metabolites by organisms underneath spread through the agar
 Diffusion made more difficult by the drying of the media
 Colony: A cluster of organisms growing on the surface of or within a solid medium,
usually cultured from a single cell
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24. BSc N/M CUNIMA 24

25.  Volumetric
 Dry weight
 Turbidity
 Direct count
 Staining
 Titration
 Colony count
 Membrane filter colony count
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26. Mkakosya, R.S.

27. Germicide/Biocide A chemical agent that kills microorganisms
Antisepsis Refers to the destruction of microbial life on a living object
Disinfection
Refers to the killing of microbes on inanimate objects or
materials
Sterilization Kills or removes all forms of life, including bacterial endospores
Static Processes or chemical agents that inhibit microbial growth
Sanitization
Usually used by the food industry. Reduces microbes on eating
utensils to safe, acceptable levels for public health.
Pasteurization
A heating process that reduces the number of spoilage germs
and eliminates pathogens in milk and other heat sensitive foods
Clean
Refers to the removal of visible dirt and debris from tissues or
objects
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28.  Physical
 Chemical
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29.  Heat
 Filtration
 Radiation
 Refrigeration
 Desiccation
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30.  Most frequently used means to destroy microbes
 Economical and easily controlled
 Death occurs more rapidly as temperature increases.
 The nature of heat is also important:
◦ Moist heat penetrates better than dry heat
BSc N/M CUNIMA 30

31. Moist Heat
 Boiling: Kills in 10 min but some bacteria resistant
 Autoclaving: 121⁰C at 15psi (pounds per square inch) for at least 15 min
 Pasteurization:
◦ 63⁰C for 30 min (LTLT)
◦ 72 ⁰C for 15 sec (HTST)
◦ 140⁰ C for 15 sec (Ultra-High Temp)
◦ 149⁰C for 0.5 sec (UHT)
 Tyndallization steam for 30 minutes on each of three successive days.
Dry Heat
 Flaming of inoculating loops and the sterilization of glassware in hot air
drying ovens
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32. o Effect of heat on bacteria is determined by
o Temperature
o Type of the bacteria involved
o Number of bacterial cells
o Presence/absence of organic matters
o pH
o Growth phase
o Humidity
o Period
BSc N/M CUNIMA 8

33.  Slow down and inhibit the growth of most microbes
 Some spoilage germs and psychrophiles can continue to
replicate at cooler temperatures
BSc N/M CUNIMA 33

34.  Lag phase increased towards freezing
 Only psychrophiles continue growth at chilling temperatures
 Reaction to freezing range from virtually no effect to injury and cell death
 Most spores survive with nearly no effect
 Gram negative more sensitive to freezing than Gram positives
 Freezing may cause sublethal injury of bacteria which in turn lead to
underestimation of cell count if done from frozen specimen
 -2 to -10C very detrimental to bacterial cells
 Slow freezing causes maximum injury while minimum injury is observed during
rapid freezing
BSc N/M CUNIMA 10

35.  Commonly employed for substances that can not tolerate heat
 Membrane filters with pore sizes between 0.2-0.45 µm are used
 Remove particles from solutions that can't be autoclaved
 Membrane filtration of beer eliminates spoilage germs and
pasteurization is no longer needed
 Sub-micron filters also marketed for removal of protozoan cysts from
drinking water.
BSc N/M CUNIMA 35

36.  Effects of types of radiation depend on three important factors:
◦ Time (of exposure)
◦ Distance (from the source)
◦ Shielding (how penetrating is the radiation?)
Nonionizing radiation
 Microwaves and ultra violet radiation.
◦ The killing effect of microwaves are largely due to the heat that they generate.
◦ radiation is of short wavelength, between 220 and 300 nm and is not very penetrating
◦ Kill exposed microbes by causing damage to their DNA.
Ionizing radiation
 Includes gamma rays and X rays which are highly penetrating to cells
and tissues and have potent antimicrobial effects.
 Irradiation approved for sterilization of surgical supplies, vaccines and
drugs and in food industries
 Irradiation known to eliminate E. coli Listeria, Campylobacter and
Salmonella from meat.
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37.  A very useful means of food preservation and to control the growth of
spoilage germs and pathogens
 Foods that have a high water activity are most subject to spoilage
and typically must be refrigerated or frozen
 This process creates hypertonic conditions and causes water to
leave bacterial cells (plasmolysis)
 Lyophilization, a process in which liquids are quick-frozen and then
subjected to evacuation, which dries the material
BSc N/M CUNIMA 37

38.  Properties of an ideal antimicrobial
agent
 Fast-acting
 Acts against many microbes without
harming tissues (selective toxicity)
 Penetrating power (improves if dirt and
debris are first removed)
 Inexpensive
 Easy to prepare
 Chemically stable
 Inoffensive odor
 Not harmful to the environment
BSc N/M CUNIMA 38

39. Microbial Targets Chemical(s)
Vegetative bacterium:
Cell wall
Formaldehyde , Chlorine-releasing agents (CRAs),
Mercury, Phenols
Cytoplasmic coagulation
Chlorhexidine , Glutaraldehyde , Hexachlorophene ,
Mercurial compounds , Silver salts, QACS
Cell membrane: membrane
potential or electron transport
Hexachlorophene , Phenols, Parabens , Weak acids
used as food preservatives such as benzoic, sorbic
and proprionic acids
Leakage of cell components Phenols, Chlorhexidine , Alcohols , QACs
Nucleic acids Alkylating agents such as ethylene oxide gas
Bacterial endospores:
Spore core Glutaraldehyde , Formaldehyde
Spore cortex
CRAs, Glutaraldehyde , Nitrous acid , Nitrates/nitrates
act as food preservatives by preventing
germination of endosporesBSc N/M CUNIMA 39

40. Virus
Envelopes Alcohols, CRAs, QACs, Chlorhexidine
Viral nucleic acid CRAs
Capsid Glutaraldehyde, QACs, CRAs, Iodine, Phenols, Alcohols
Fungus
Cell membrane Chlorhexidine, Alcohols, QACS
Cell wall Glutaraldehyde
Nucleic acid Acridine dyes
BSc N/M CUNIMA 40

41. Mkakosya, R.S.

42.  Antimicrobial agent
◦ Substance with inhibitory properties against microorganisms
(includes antibiotics and synthetic compounds) but minimal effects
on mammalian cells
 Antibiotic
◦ Produced by microorganisms and acts on other microorganisms
 Semi-synthetic antibiotics
◦ Antibiotics chemically altered to improve properties
 Antimicrobial spectrum
◦ Range of activity of an antimicrobial against bacteria
◦ “Broad-spectrum” vs “narrow spectrum”
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43.  Bacteriostatic
◦ When growth of an organsim is inhibited by the antibacterial
 Bactericidal (viricidal, fungicidal)
◦ When the organism is killed by the antibacterial
 Additive
◦ Combined effect of antibacterials is equal to sum of individual agents
 Synergistic
◦ Combined effect is greater than that achieved with addition
 Antagonistic
◦ Drugs inhibit the action of each other
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44.  Bacterial cell wall synthesis inhibitors
◦ β-lactams: penicillins, cephalosporins,carbapenems
◦ glycopeptides
 Cell membrane synthesis inhibitors
 Protein synthesis inhibitors
◦ Aminoglycosides, macrolides, chloramphenicol, tetracyclines
 Nucleic acid synthesis inhibitors
◦ Quinolones,sulphonamides, trimethoprim, co-trimoxazole,
rifamycins, metronidazole
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45. 
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46.  Most have β-lactam ring
 Bactericidal
 Action
◦ Interfere with cross-linking of peptidoglycan by inhibiting
carboxypeptidase and transpeptidase reactions which form a link
between N-acetylglucosamine and N-acetylmuramic acid
◦ Cell wall weakened and lysis of microorganism occurs
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47. 47
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48.  Type of antibiotic derived from Penicillium fungi
 Originally discovered by accident in 1928. Alexander Fleming
 Given Orally or IM/IV
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49.  Pharmacokinetics
◦ Wide distribution, mainly renal excretion
 Toxicity
◦ Hypersensitivity reactions include anaphylaxis and skin rashes
◦ 10% of pen-allergic also allergic to cephalosporins
 Antibacterial resistance
◦ Alteration of target site eg PBP mutation in S. pneumoniae, mecA
of MRSA
◦ β-lactamases
◦ Cell membrane alterations reducing uptake or increasing loss from
the cell
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50.  Benzylpenicillin (also known as penicillin G (PenG) or BENPEN)
◦ Gram +ve orgs and Gram –ve cocci
◦ Streptococcal infections, gonorrhoea, meningococcal meningitis
 Phenoxymethylpenicillin is a narrow spectrum antibiotic also
commonly referred to as Penicillin V or Penicillin VK
 Flucloxacillin
◦ Active vs b-lactamase positive strains of staphylococcus
◦ S. aureus infections
 Amoxicillin/ampicillin
◦ More active against Enterococcus, Haemophilus and some Gram
–ve aerobes
◦ Urinary and respiratory tract infections
 Piperacillin
◦ Wider activity against coliforms and Pseudomonas aeruginosa
◦ Severe Gram –ve infections
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51. First generation “narrow spectrum” eg cephradine
 Second generation “expanded spectrum” eg cefuroxime
 Third generation “broad spectrum” eg ceftriaxone
 Fourth generation “extended spectum” eg cefpirome
◦ Same mechanism of action as penicillins, wider spectrum, resistant to many β-
lactamases, improved pharmocokinetics.
 Toxicity/SEs
◦ Hypersensitivity with rashes
 Resistance
◦ Similar to penicillins
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52.  Structure
◦ Similar to penicillins
◦ Broad spectrum antimicrobial spectrum of activity
 Pharmacokinetics
◦ Given iv once daily, renal excretion
 Toxicity/SEs
◦ Hypersensitivity with rashes, !0% cross reactivity with penicillins
 Resistance
◦ Hydrolysis by carbapenemases
◦ Reduced uptake by cell
 Examples
◦ Imipenem, meropenem, ertapenem
 Clinical application
◦ Severe gram –ve sepsis. Neutropenic sepsis
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53.  Pharmacokinetics
◦ Must be given iv, widely distributed, renal excretion
 Mechanism of action
◦ Interact with the terminal of pentapeptide side chains of peptidoglycan and thus
interferes with bridge formation between peptidoglycan chains, cause cell lysis
and death
 Only active against Gram positive bacteria. Used to treat infections
caused by oxacillin resistant staphylococci and other gram positive
b-lactam resistant bacteria
 Resistance
◦ Intrinsic, plasmid mediated
 Examples include: Dalbavancin, oritavancin, ramoplanin teicoplanin,
telavancin, vancomycin
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54.  Pharmacokinetics
◦ Poor absorption from gut, poor penetration into tissue and fluids
◦ Renal excretion, serum levels should be monitored
 Mechanism of action
◦ Bind irreversibly to the 30S ribosomal protein.
 Antimicrobial spectrum of activity
◦ Bactericidal, staphylococci and aerobic Gram –ves. Synergy with β-lactams
 Toxicity
◦ Hypersensitivity, ototoxicity, nephrotoxicity
 Resistance
◦ Mutation of binding site, decreased uptake into cell, increased expulsion from cell,
enzymatic modification of antibiotic
 Examples
◦ Amikacin, apramycin, arbekacin, astromicin, bekanamycin, dibekacin,
dihydrostreptomycin, framycetin, gentamicin, isepamicin, kanamycin, micronomicin,
neomycin, netilmicin, paromomycin, ribostamycin, sisomicin, streptoduocin, streptomycin,
tobramycin
 Use
◦ Severe sepsis caused by Gram negative bacteria
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55.  Pharmacokinetics
◦ Absorbed orally, iv, well distributed, excretion in bile
 Mechanism of action
◦ Bind to 50S ribosomal RNA unit, predominantly bacteriostatic
 Antimicrobial spectrum of activity
◦ Gram positive, Haemophilus, Bordetella, Neisseria, chlamydia, rickettsiae and
mycoplasmas
 Toxicity
◦ GI upset, rashes, hepatic damage (rare
 Resistance
◦ Alteration of RNA target or drug efflux
 Examples
◦ Erythromycin, azithromycin, clarithromycin
 Clinical application
◦ Strep and staph soft tissue infections, RTI
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56.  Pharmacokinetics
◦ Rapidly absorbed after oral administration, good tissue penetration inc. brain,
hepatic metabolism then renal excretion
 Mechanism of action
◦ Binds to 50S ribosomal subunit, Bacteriostatic
 Antimicrobial spectrum of activity
◦ Wide range of organisms including chlamdiae, mycoplasmas and rickettsiae
 Toxicity/SEs
◦ Dose related depressant effect on bone marrow, rarely aplasia, grey baby
syndrome
 Resistance
◦ Inactivation by an inducible acetylase enzyme, reduced permeability
 Clinical application
◦ Meningitis, typhoid fever
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57.  Pharmacokinetics
◦ Oral or iv, penetrate well into body fluids. Excretion via kidney and bile duct
 Mechanism of action
◦ Bind to 30S ribosomal subunit, bacteriostatic
 Antimicrobial spectrum of activity
◦ Broad spectrum: Gram +ve and Gram –ve, chlamydia, rickettsiae and
mycoplasmae
 Toxicity/SEs
◦ GI intolerance, deposition in developing bone and teeth, skin rashes
 Resistance
◦ Efflux from cell, Decreased penetration, alteration of target site
 Examples
◦ Tetracyline, doxycline
 Clinical application
◦ Important in treatment of infections by chlamydiae, rickettsiae and mycoplasmae
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58.  Pharmacokinetics
◦ Generally good absorption after oral administration, penetrates well into body
tissues and fluids, eliminated by renal excretion and liver metabolism
 Mechanism of action
◦ Inhibit the action of DNA gyrases which are important in “supercoiling” during
DNA synthesis, Bactericidal
 Antimicrobial spectrum of activity
◦ Act against gram –ves inc. Pseudomonas. Not good for streptococci or
anaerobes
 Toxicity/SEs
◦ GI disturbances, neurological, ruptured Achilles’ tendon
 Resistance
mutations in DNA gyrases, efflux from cell
 Examples
◦ ciprofloxacin
 Clinical application
◦ Severe sepsis caused by coliforms and other Gram –ve aerobic bacilli
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59.  Sulphonamides
◦ Act on folic acid synthesis as competitive inhibitor of p-aminobenzoic acid to
inhibit purine and thymidine synthesis. Now limited use because of toxicity and
resistance. Resistance via altered dihydropteroate synthetase enzyme
 Trimethoprim (diaminopyrimidine)
◦ Prevention of tetrahydrofolic acid synthesis, resistance via production of different
dihydrofolate reductase enzymes. Broad spectrum, used for UTI and RTIs
 Co-trimoxazole ( trimethoprim+ sulphamethoxazole)
◦ Synergistic antibacterial. Used for Pneumocystis jiroveci
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60.  Pharmacokinetics
◦ Well absorbed orally, widely distributed, metabolized in the liver and excreted via
bile
 Mechanism of action
◦ Binds to RNA polymerase and blocks synthesis of mRNA
 Antimicrobial spectrum of activity
◦ Active vs staph, strep,neisseria, legionella, mycobacteria. Coliforms
resistant
 Toxicity/SEs
◦ Skin rashes, LFT abnormalities, potent inducer of hepatic enzymes interfering
with other drugs eg warfarin
 Resistance
◦ Resistant mutants ( change in single amino acid at target site) occur when used
as single drug so often combined with other agents
 Clinical application
◦ Tuberculosis (part of triple/quadruple therapy), combination therapy,
chemoprophylaxis fro meningitis due to meningococcus or Hib
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61.  Pharmacokinetics
◦ Well absorbed orally, per rectum and good tissue distribution
 Mechanism of action
◦ Metabolized by nitroreductases to active intermediates which
result in DNA damage
 Antimicrobial spectrum of activity
◦ Active against anaerobes, Giardia, Trichomonas and other
parasites
 Toxicity
◦ Nausea, metallic taste, rarely peripheral neuropathy
 Resistance
◦ Rare but can occur due to decreased uptake
 Clinical applications
◦ Treatment of anaerobic infections, giardiasis, amoebiasis
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62.  Fucidin
◦ Active vs staphylococci, acts on ribosome, rapid resistance if used
alone, can cause hepatic damage, usually used in combination
 Nitrofurantoin
◦ Well absorbed, excreted in urine, for uncomplicated UTI
 Nalidixic acid
◦ Quinolone, used for simple UTI
 Polymixins
◦ Disrupt cell membrane, nephrotoxic, usually topical eg colistin
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63.  Linezolid (oxazolidinone)
◦ Excellent oral absorption, used in renal failure, inhibits protein
synthesies, acts on Gram positive (MRSA and VRE)
 Synercid (quinupristin and dalfopristin)
◦ For VRE
 Fluoroquinolones (moxifloxacin, levofloxacin)
◦ Better Gram +ve activity inc. pneumococci
 Tigecycline
◦ Use against multi-resistant, non-fermenting Gram –ves eg
Acinetobacter sp
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64.  Alteration of the target site
◦ Mutation of ribosome, topoisomerase, PBP
 Destruction/inactivation of the antibiotic
◦ B-lactamases, AG modifying enzymes
 Blockage of transport of the agent into the cell
 Metabolic bypass
◦ Eg dihydrofolate reductases
 Increased loss of drug from cell (efflux)
 Protection of target site by a bacterial protein
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65.  Via chromosomal mutation and then
duplication during cell division
 Plasmids
 Transposons “jumping genes”
 Bacteriophages
 Integrons
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66.  Prevalence of resistance is directly proportional to the
amount of antibiotic used
 Problems
◦ Use of antibiotics without prescriptions
◦ Uncontrolled use of antibiotics in agriculture
◦ Poor prescribing habits
◦ Absence of antibiotic policies
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67. antiviral drugs
 class of medicines particularly used for the treatment of viral infections
 focused on two different approaches
 Targeting the viruses themselves or the host cell factors
Antiviral drugs that directly target the viruses include the inhibitors of virus
attachment, inhibitors of virus entry, uncoating inhibitors, polymerase inhibitors,
protease inhibitors, inhibitors of nucleoside and nucleotide reverse transcriptase
and the inhibitors of integrase
 The inhibitors of protease (ritonavir, atazanavir and darunavir)
 viral DNA polymerase inhibitors: (acyclovir, tenofovir, valganciclovir and
valacyclovir)
 Inhibitors of integrase (raltegravir)
67
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68. Drugs with antiviral activities
Ribavirin:After intracellular phosphorylation, ribavirin triphosphate
interferes with the initial timeliness of virus translation
Lamivudine is a prescription nucleoside reverse transcriptase
inhibitor (NRTI) that is used in combination with other drugs as
antiviral treatment for human immunodeficiency virus type-1 (HIV-1)
monotherapy for hepatitis B virus (HBV)
Amantadine and rimantadine: Both drugs appear to suppress
influenza infection replication by blocking the particle channel of the
M2 protein virus
Interferon alpha: shown to be effective in the treatment of diseases
caused by human herpesvirus 8, papillomavirus (Kaposi’s sarcoma)
virus, hepatitis B and C virus 68
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69. Drugs with antiviral activities
 Remdesivir: nucleotide analogue metabolised intracellularly to adenosine triphosphate analogue inhibiting the viral
RNA polymerases
 acts as an inhibitor of RNA dependant RNA polymerase
 has broadspectrum antiviral activity against several virus family members including the coronaviruses for example,
Middle East respiratory syndrome coronavirus (MERSCoC) and SARSCoV, and filoviruses for example, Ebola
 Nitazoxanide:
 Virus inactivating agents
 Inhibitors of enzymes associated with virions
 DNA polymerases
 RNA polymerases
 Viral neuraminidase
69
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70. Antiviral drugs Mechanisms
BSc N/M CUNIMA 70

71. Antiviral drugs tested on covid 19
71
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72. ART (six main classes of HAART agents)
 Nucleoside/Nucleotide Reverse Transcriptase Inhibitors
 Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
 Protease inhibitors (PIs)
 Integrase Strand Transfer Inhibitors (INSTIs)
 Fusion inhibitors (FIs)
 Chemokine Receptor Antagonists (CCR5 Antagonists)
72
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73. Nucleoside/Nucleotide Reverse Transcriptase Inhibitors
 nucleoside or nucleotide analogs without hydroxyl at the 3’ end that are incorporated into the growing viral
DNA strand
 competitively bind to reverse transcriptase and cause premature DNA chain termination as they inhibit 3’ to
5’ phosphodiester bond formation.
 Examples include: abacavir, didanosine, lamivudine, stavudine, tenofovir, and zidovudine
Non-nucleoside Reverse Transcriptase Inhibitors (NNRTIs)
 bind to HIV reverse transcriptase at an allosteric, hydrophobic site causing a stereochemical change within
reverse transcriptase, thus inhibiting nucleoside binding and inhibition of DNA polymerase.
 Examples include delavirdine, efavirenz, nevirapine, and rilpivirine
Protease inhibitors (PIs)
 competitively inhibit the proteolytic cleavage of the gag/pol polyproteins in HIV-infected cells. These agents
result in immature, non-infectious virions.
 Generally used in patients who fail their initial HAART regimen and should be administered with boosting
agents such as ritonavir or cobicistat.
 Examples include atazanavir, darunavir, indinavir
73
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74.  Integrase Strand Transfer Inhibitors (INSTIs)
bind viral integrase and prevent viral DNA from being incorporated into the
host cell chromosome.
Examples include: dolutegravir, elvitegravir, raltegravir
 Fusion inhibitors (FIs)
bind to the envelope glycoprotein gp41 and prevent viral fusion to the CD4
T-cells.
Examples include enfuvirtide
 Chemokine Receptor Antagonists (CCR5 Antagonists)
selectively and reversibly block entry into the CD4 T-cells by preventing
interaction between CD4 cells and the gp120 subunit of the viral envelope
glycoprotein
Example: maraviroc
74
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75. ART mechanims
BSc N/M CUNIMA 75

76. ANTIFUNGAL THERAPEUTIC
AGENTS
ANTIFUNGAL DRUGS

77. ANTIFUNGAL THERAPEUTIC
77
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78. Choice & dose of an antifungal agent
 Depends on:
 Nature of the condition
 Whether there are underlying diseases
 Health of a patient
 Whether antifungal resistance has been identified
 The ideal antifungal agent should target a pathway or
process specific to the fungus
 Difficult because fungi are eukaryotic organisms
78
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79.  Limitations of antibiotics:
 Most have profound side effects
 A narrow antifungal spectrum
 Poor penetration of certain tissues
 Selection of resistant fungi
79
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80. CLASSES OF ANTIFUNGAL AGENTS
1. Polyenes, e.g. amphotericin B, nystatin
2. Azoles, e.g. fluconazole
3. Antimetabolites, e.g. flucytosine
4. Echinocandins, e.g. caspofungin
5. Allylamines, e.g. terbenafine
6. Miscelleanous, e.g. griseofulvin
80
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81. MAJOR SITES OF ACTION
 Cell wall (β-glucan)
 Cell membrane
(Ergosterol)
 Nucleus (DNA)
 Echinocandins
 Polyenes, azoles,
allylamines
 Flucytosine,
griseofulvin
81
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82. MAJOR SITES OF ACTION
82
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83. 1. POLYENE: AMPHOTERICIN B
 Produced by Streptomyces nodosus
 Binds to ergosterol in cell membranes
 Alters membrane fluidity
 Creating pores that cause cell leakage & eventually death
 Binds weakly to cholesterol, causing the toxicity effects in the
mammalian cell
 Fungicidal drug
 reserved for severe cases of systemic fungal disease
83
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84. POLYENE:
 Broad spectrum of activity against
 Yeasts e.g. Candida spp, C. neoformans
 Moulds e.g. Aspergillus spp
 Dimorphic fungi e.g. H. capsulatum, B. dermatitidis
 Response to drug is influenced by:
 Dose & route of administration
 Site of mycotic infection
 Immune status of patient
 Inherent susceptibility of pathogen
84
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85. 2. THE AZOLES
 Have a 5-membered azole ring & divided into:
 Imidazoles: have 2N in azole ring
:e.g. ketoconazole, miconazole, clotrimazole
 Triazoles: have 3N in azole ring
: e.g. fluconazole, itraconazole, voriconazole
 Can be used to treat a wide range of systemic and
localized infections
 Fungistatic drugs
85
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86. THE AZOLES CONT’
 Interfere with ergosterol biosynthesis
 Binds to cytochrome P450-dependent 14 α-demethylation of
lanosterol (precursor of ergosterol)
 Results in reduction in the amount of ergosterol which leads to
membrane instability, growth inhibition & cell death in some cases
86
BSc N/M CUNIMA

87. Fluconazole
 Triazole compound that is active against
 Yeasts e.g. Candida albicans, Crytococcus neoformans
 Dimorphic fungi e.g. Histoplasma capsulatum
 Ineffective against C. krusei, C. glabrata, Aspergillus spp
 Useful to treat mucosal & systemic candidiasis and
cryptococcal meningitis
87
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88. 3. ANTIMETABOLITE: FLUCYTOSINE
 Agent: 5-fluorocytosine- a fluorinated derivative of cytosine
(pyrimidine)
 Oral antifungal agent
 Mode of action
 Disrupts protein synthesis by inhibiting DNA synthesis
 Mainly used in conjunction with amphotericin B for
treatment of cryptococcus & candidiasis
 Many fungi are inherently resistant to flucytosine
88
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89. 4. ECHINOCANDINS
 New class of antifungal agent
 Synthetically modified lipopeptides
 Examples: caspofungin, micafungin, anidulafungin
 Perturb synthesis of cell wall polysaccharide β-glucan by
inhibiting 1,3-β-glucan synthase & disrupting the cell wall
 Fungicidal
 Highly active against Candida spp, Aspergillus spp &
Pneumocystis jiroveci
 Inactive against Zygomycetes, C. neoformans
89
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90. 5.ALLYLAMINES: TERBINAFINE
 Terbinafine: systemic (oral and topical)
 Naftiline: topical
 Inhibit squalene epoxidase and thus decrease ergosterol
synthesis
 Lipophilic, broad spectrum, few SEs
 High concentrations in fatty tissues, skin, hair and nails
90
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91. 6. GRISEOFULVIN
 Oral agent used vs dermatophytes
 Interacts with microtubules in cell and inhibits mitosis
 Often second line agent after terbinafine
 Mild SEs
91
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92. Antifungal drug resistance
 Primary (intrinsic) - present before exposure
to antifungal
 Secondary (acquired) - develops after
exposure to antifungal
92
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93. Primary antifungal resistance
 Amphotericin B (Aspergillus terreus, Candida lusitaniae,
Trichosporon beigelli, Scedosporium apiospermum)
 Fluconazole (Candida krusei, Candida glabrata,
Aspergillus spp.)
93
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94. Secondary antifungal resistance
 Predominantly seen with azole (esp. fluconazole) resistance among
Candida spp.
 Chronic mucosal candidiasis in AIDS patients esp. low CD4 counts,
multiple azole courses, prolonged heavy azole use (esp.
fluconazole, N.B. cross-resistance with itraconazole)
 Bloodstream candidiasis in critically ill or non-AIDS
immunosuppressed patients (1-3% of C. albicans resistant)
94
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95. Prevention
 Fungal infections remain serious and
underappreciated causes of illness and death.
 Environmental control may be difficult
 Observe and practice hygiene
 Taking treatment as prescribed
95
BSc N/M CUNIMA

96. Recommended Reading
• Mims Medical Microbiology
– Goering, Dockrell et al 4th edn p37-46
• Medical Microbiology
– Greenwood et al, 17th edn p20-22 and p80-94
96
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