Microbial physiology, growth & growth control_Micro1.pptx

Powerpoint

1. MICROBIAL PHYSIOLOGY, GROWTH & CONTROLLING GROWTH 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 LEARNING OUTCOMES

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 BSc N/M CUNIMA INTRODUCTION

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 Nutritional requirements

5. Phototrophs : Use light as energy source Chemotrophs : Lithotrophs : Use inorganic chemicals for energy Organotrophs : Use organic materials as energy source Autotrophs : Use CO 2 as source of carbon Heterotrophs : Use organic compounds Photoautotrophs : Use light and CO 2 ( e.g cynanobacteria ) Photoheterotphs : Use light and organic compounds (e.g. nonsulfur bacteria) Chemoautotrophs : Chemical and CO 2 Chemoheterotrophs : Use chemical energy and organic compounds for carbon BSc N/M CUNIMA Microbial categories

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 BSc N/M CUNIMA FACTORS AFFECTING microbial GROWTH

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 BSc N/M CUNIMA PHYSICAL FACTORS

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 BSc N/M CUNIMA Thermal microbial grouping

9. BSc N/M CUNIMA Effects of temperature on growth

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 BSc N/M CUNIMA Radiation

11. Osmotic pressure Moisture Electrical effects BSc N/M CUNIMA OTHER PHYSICAL FACTORS

12. Oxygen Aerobic Anaerobic Falcutative anaerobic Microaerophilic Aerotolerant Carbon dioxide pH Redox potential BSc N/M CUNIMA CHEMICAL FACTORS

13. Growing the microorganisms Mkakosya , R.S.

14. BSc N/M CUNIMA CULTURE MEDIA

15. BSc N/M CUNIMA

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 CULTURE MEDIA

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 TYPES OF CULTURE MEDIA

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 BSc N/M CUNIMA COMMON INGREDIENTS OF CULTURE MEDIA

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 BSc N/M CUNIMA COMMON INGREDIENTS OF CULTURE MEDIA

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 Cultivation methods

21. Bacterial growth curve BSc N/M CUNIMA Bacterial Growth in Broth

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 Bacterial growth curve

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 BSc N/M CUNIMA GROWTH ON SOLID SURFACES

24. BSc N/M CUNIMA Colonial morphology

25. Volumetric Dry weight Turbidity Direct count Staining Titration Colony count Membrane filter colony count BSc N/M CUNIMA Measuring bacterial growth

26. Controlling microbial growth invitro 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 BSc N/M CUNIMA Terminology related to control of microorganisms

28. Physical Chemical BSc N/M CUNIMA Methods used in control of microbial growth

29. Heat Filtration Radiation Refrigeration Desiccation BSc N/M CUNIMA Physical Methods of microbial control

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 Heat Sterilization

31. Moist Heat Boiling : Kills in 10 min but some bacteria resistant Autoclaving : 121 ⁰ C at 15psi ( pounds per square inch ) for at least 15 min Pasteurization: 63 ⁰ C for 30 min (LTLT) 72 ⁰ C for 15 sec (HTST) 140 ⁰ C for 15 sec (Ultra-High Temp) 149 ⁰ C 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 BSc N/M CUNIMA Heat Sterilization cont’

32. Effect of heat on bacteria is determined by Temperature Type of the bacteria involved Number of bacterial cells Presence/absence of organic matters pH Growth phase Humidity Period BSc N/M CUNIMA 8 Effect of heating

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 Refrigeration

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 Effect of Chilling/freezing

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 Filtration

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. BSc N/M CUNIMA Radiation

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 Desiccation

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 Chemical Methods of Microbial Control

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 endospores BSc N/M CUNIMA A Summary of Mechanisms of Inactivation by Biocides

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

41. Antimicrobial agents: Mkakosya , R.S.

42. Definitions 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” BSc N/M CUNIMA

43. Definitions 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 BSc N/M CUNIMA

44. Antimicrobials agents according to mode of action 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 BSc N/M CUNIMA

45. Basic sites of antibiotic activity BSc N/M CUNIMA

46. Inhibitors of Cell wall synthesis 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 BSc N/M CUNIMA

47. Inhibitors of Cell wall synthesis β - lactams BSc N/M CUNIMA

48. Penicillins Type of antibiotic derived from Penicillium fungi Originally discovered by accident in 1928. Alexander Fleming Given Orally or IM/IV BSc N/M CUNIMA

49. Penicillins 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 BSc N/M CUNIMA

50. Types of penicillins 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 BSc N/M CUNIMA

51. Cephalosporins 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 BSc N/M CUNIMA

52. Carbapenems 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 BSc N/M CUNIMA

53. Cell wall inhibitors: glycopeptides 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 BSc N/M CUNIMA

54. Protein synthesis inhibitors: Aminoglycosides 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 negati ve bacteria BSc N/M CUNIMA

55. Protein synthesis inhibitors : Macrolides and lincosamides 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 BSc N/M CUNIMA

56. Protein synthesis inhibitors:Chloramphenicol 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 BSc N/M CUNIMA

57. Protein synthesis inhibitors : Tetracyclines 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 BSc N/M CUNIMA

58. Nucleic acid inhibitors : Fluoroquinolones 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 BSc N/M CUNIMA

59. Nucleic acid inhibitors 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 jirovec i BSc N/M CUNIMA

60. Nucleic acid inhibitors : Rifamycins-rifampicin 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 BSc N/M CUNIMA

61. Nucleic acid inhibitors : metronidazole 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 BSc N/M CUNIMA

62. Other antibiotics 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 BSc N/M CUNIMA

63. Newer antibiotics 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 BSc N/M CUNIMA

64. Mechanisms of resistance 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 BSc N/M CUNIMA

65. Genetics of resistance Via chromosomal mutation and then duplication during cell division Plasmids Transposons “jumping genes” Bacteriophages Integrons BSc N/M CUNIMA

66. Epidemiology of drug resistance 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 BSc N/M CUNIMA

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) BSc N/M CUNIMA

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 BSc N/M CUNIMA

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 BSc N/M CUNIMA

70. Antiviral drugs Mechanisms BSc N/M CUNIMA

71. Antiviral drugs tested on covid 19 BSc N/M CUNIMA

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 ) BSc N/M CUNIMA

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 BSc N/M CUNIMA

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 BSc N/M CUNIMA

75. ART mechanims BSc N/M CUNIMA

76. ANTIFUNGAL THERAPEUTIC AGENTS ANTIFUNGAL DRUGS

77. ANTIFUNGAL THERAPEUTIC BSc N/M CUNIMA

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 BSc N/M CUNIMA

79. Limitations of antibiotics: Most have profound side effects A narrow antifungal spectrum Poor penetration of certain tissues Selection of resistant fungi BSc N/M CUNIMA

80. CLASSES OF ANTIFUNGAL AGENTS Polyenes , e.g. amphotericin B, nystatin Azoles, e.g. fluconazole Antimetabolites, e.g. flucytosine Echinocandins , e.g. caspofungin Allylamines , e.g. terbenafine Miscelleanous , e.g. griseofulvin BSc N/M CUNIMA

81. MAJOR SITES OF ACTION Cell wall ( β - glucan ) Cell membrane ( Ergosterol ) Nucleus (DNA) Echinocandins Polyenes, azoles, allylamines Flucytosine, griseofulvin BSc N/M CUNIMA

82. MAJOR SITES OF ACTION BSc N/M CUNIMA

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 BSc N/M CUNIMA

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 BSc N/M CUNIMA

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 BSc N/M CUNIMA

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 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 BSc N/M CUNIMA

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 BSc N/M CUNIMA

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 BSc N/M CUNIMA

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 BSc N/M CUNIMA

91. 6. GRISEOFULVIN Oral agent used vs dermatophytes Interacts with microtubules in cell and inhibits mitosis Often second line agent after terbinafine Mild SEs BSc N/M CUNIMA

92. Antifungal drug resistance Primary (intrinsic) - present before exposure to antifungal Secondary (acquired) - develops after exposure to antifungal BSc N/M CUNIMA

93. Primary antifungal resistance Amphotericin B ( Aspergillus terreus , Candida lusitaniae , Trichosporon beigelli , Scedosporium apiospermum ) Fluconazole ( Candida krusei , Candida glabrata , Aspergillus spp.) BSc N/M CUNIMA

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 ) BSc N/M CUNIMA

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 BSc N/M CUNIMA

96. Recommended Reading Mims Medical Microbiology Goering, Dockrell et al 4 th edn p37-46 Medical Microbiology Greenwood et al , 17 th edn p20-22 and p80-94 BSc N/M CUNIMA