26 1 Heat Sterilization Sterilization The killing or

26. 1 Heat Sterilization • Sterilization – The killing or removal of all viable organisms within a growth medium • Inhibition – Effectively limiting microbial growth • Decontamination – The treatment of an object to make it safe to handle • Disinfection – Directly targets the removal of all pathogens, not necessarily all microorganisms © 2012 Pearson Education, Inc.

26. 1 Heat Sterilization • Heat sterilization is the most widely used method of controlling microbial growth • High temperatures denature macromolecules – Amount of time required to reduce viability tenfold is called the decimal reduction time – Some bacteria produce resistant cells called endospores – Can survive heat that would rapidly kill vegetative cells © 2012 Pearson Education, Inc.

26. 1 Heat Sterilization • The autoclave is a sealed device that uses steam under pressure (Figure 26. 3) – Allows temperature of water to get above 100 C – Not the pressure that kills things, but the high temperature • Pasteurization is the process of using precisely controlled heat to reduce the microbial load in heat-sensitive liquids – Does not kill all organisms, so it is different than sterilization © 2012 Pearson Education, Inc.

Figure 26. 3 Chamber pressure gauge Steam exhaust valve Door Jacket chamber Thermometer and valve Air exits through vent Steam supply valve Steam enters here Autoclave time Temperature (C) Stop steam Begin pressure Flowing steam Sterilization time Temperature of object being of autoclave sterilized Total cycle time (min) © 2012 Pearson Education, Inc.

26. 2 Radiation Sterilization • Microwaves, UV, X-rays, gamma rays, and electrons can reduce microbial growth • UV has sufficient energy to cause modifications and breaks in DNA – UV is useful for decontamination of surfaces – Cannot penetrate solid, opaque, or light-absorbing surfaces © 2012 Pearson Education, Inc.

Figure 26. 4 © 2012 Pearson Education, Inc.

26. 2 Radiation Sterilization • Ionizing radiation – Electromagnetic radiation that produce ions and other reactive molecules – Generates electrons, hydroxyl radicals, and hydride radicals – Some microorganisms are more resistant to radiation than others © 2012 Pearson Education, Inc.

26. 2 Radiation Sterilization • Sources of radiation include cathode ray tubes, X-rays, and radioactive nuclides • Radiation is used for sterilization in the medical field and food industry – Radiation is approved by the WHO and is used in the USA for decontamination of foods particularly susceptible to microbial contamination • Hamburger, chicken, spices may all be irradiated © 2012 Pearson Education, Inc.

26. 3 Filter Sterilization • Filtration avoids the use of heat on sensitive liquids and gases – Pores of filter are too small for organisms to pass through – Pores allow liquid or gas to pass through • Depth filters – HEPA filters – Membrane filters – Function more like a sieve © 2012 Pearson Education, Inc.

Figure 26. 6 © 2012 Pearson Education, Inc.

26. 3 Filter Sterilization • Membrane filters (cont’d) – Filtration can be accomplished by syringe, pump, or vacuum – A type of membrane filter is the nucleation track (nucleopore) filter © 2012 Pearson Education, Inc.

Figure 26. 7 © 2012 Pearson Education, Inc.

Figure 26. 8 © 2012 Pearson Education, Inc.

26. 4 Chemical Growth Control • Antimicrobial agents can be classified as bacteriostatic, bacteriocidal, and bacteriolytic © 2012 Pearson Education, Inc.

Bacteriostatic Log cell number Figure 26. 9 Total cell count Viable cell count Bacteriocidal Log cell number Viable cell count Time Bacteriolytic Total cell count Viable cell count Time © 2012 Pearson Education, Inc. Total cell count

26. 4 Chemical Growth Control • Minimum inhibitory concentration (MIC) is the smallest amount of an agent needed to inhibit growth of a microorganism – Varies with the organism used, inoculum size, temp, p. H, etc. • Disc diffusion assay – Antimicrobial agent added to filter paper disc – MIC is reached at some distance • Zone of inhibition – Area of no growth around disc © 2012 Pearson Education, Inc.

Figure 26. 10 Minimum inhibitory concentration © 2012 Pearson Education, Inc.

Figure 26. 11 Nutrient agar plate Inoculate plate with a liquid culture of a test organism Discs containing antimicrobial agents are placed on surface Incubate for 24– 48 h Test organism shows susceptibility to some agents, indicated by inhibition of bacterial growth around discs (zones of inhibition) © 2012 Pearson Education, Inc.

26. 5 Chemical Antimicrobial Agents for External Use • These antimicrobial agents can be divided into two categories – Products used to control microorganisms in commercial and industrial applications • Examples: chemicals in foods, air-conditioning cooling towers, textile and paper products, fuel tanks – Products designed to prevent growth of human pathogens in inanimate environments and on external body surfaces • Sterilants, disinfectants, sanitizers, and antiseptics © 2012 Pearson Education, Inc.

III. Antimicrobial Agents Used In Vivo • Antimicrobial drugs are classified on the basis of – Molecular structure – Mechanism of action – Spectrum of antimicrobial activity © 2012 Pearson Education, Inc.

Figure 26. 12 Cell wall synthesis DNA gyrase Cycloserine Vancomycin Bacitracin Penicillins Cephalosporins Monobactams Carbapenems Nalidixic acid Ciprofloxacin Novobiocin Quinolones RNA elongation Actinomycin DNA-directed RNA polymerase Rifampin Streptovaricins Protein synthesis (50 S inhibitors) Erythromycin (macrolides) Chloramphenicol Clindamycin Lincomycin DNA Folic acid metabolism THF Trimethoprim Sulfonamides m. RNA Protein synthesis (30 S inhibitors) Ribosomes DHF 50 30 Cytoplasmic membrane structure and function Polymyxins Daptomycin Lipid biosynthesis PABA © 2012 Pearson Education, Inc. Platensimycin Cytoplasmic Cell wall membrane Tetracyclines Spectinomycin Streptomycin Gentamicin Kanamycin Amikacin Nitrofurans Protein synthesis (t. RNA) Mupirocin Puromycin

Figure 26. 13 Bacteria Eukaryotes Fungi Mycobacteria Gram-negative Bacteria Streptomycin Gram-positive Bacteria Chlamydia Penicillins Tobramycin Azoles Allylamines Cycloheximide Polyenes Polyoxins Nucleic acid analogs Echinocandins Obligately parasitic Bacteria Sulfonamides Cephalosporins Quinolones Tetracycline Isoniazid Polymyxins Vancomycin Daptomycin Platensimycin © 2012 Pearson Education, Inc. Rickettsia Viruses RNA viruses DNA viruses Nonnucleoside reverse transcriptase inhibitors Protease inhibitors Fusion inhibitors Nucleoside analogs Interferon

26. 6 Synthetic Antimicrobial Drugs • Paul Ehrlich studied selective toxicity in the early 1900 s – Selective toxicity is ability to inhibit or kill a pathogen without affecting the host © 2012 Pearson Education, Inc.

26. 6 Synthetic Antimicrobial Drugs • Sulfa drugs: discovered by Gerhard Domagk in the 1930 s – Inhibit growth of bacteria (sulfanilamide is the simplest; – Isoniazid is a growth analog effective only against Mycobacterium • Interferes with synthesis of mycolic acid © 2012 Pearson Education, Inc.

Figure 26. 16 Sulfanilamide Folic acid © 2012 Pearson Education, Inc. p-Aminobenzoic acid

26. 6 Synthetic Antimicrobial Drugs • Nucleic acid base analogs have been formed by the addition of bromine or fluorine • Quinolones are antibacterial compounds that interfere with DNA gyrase (e. g. , ciprofloxacin) © 2012 Pearson Education, Inc.

Figure 26. 17 © 2012 Pearson Education, Inc. Growth factor Analog Phenylalanine (an amino acid) p-Fluorophenylalanine Uracil (an RNA base) 5 -Fluorouracil Thymine (a DNA base) 5 -Bromouracil

26. 7 Naturally Occurring Antimicrobial Drugs: Antibiotics • Antibiotics are naturally produced antimicrobial agents – Less than 1% of known antibiotics are clinically useful • Can be modified to enhance efficacy (semisynthetic) • The susceptibility of microbes to different antibiotics varies greatly – Gram-positive and gram-negative bacteria vary in their sensitivity to antibiotics – Broad-spectrum antibiotics are effective against both groups of bacteria © 2012 Pearson Education, Inc.

26. 8 -Lactam Antibiotics: Penicillins and Cephalosporins • -Lactam antibiotics are one of the most important groups of antibiotics of all time – Include penicillins, cephalosporins, and cephamycins – Over half of all antibiotics used worldwide • Penicillins (Figure 26. 19) – Discovered by Alexander Fleming – Primarily effective against gram-positive bacteria – Some synthetic forms are effective against some gram-negative bacteria – Target cell wall synthesis © 2012 Pearson Education, Inc.

Figure 26. 19 N-Acyl group -Lactam ring Thiazolidine ring 6 -Aminopenicillanic acid N-Acyl group Designation NATURAL PENICILLIN Benzylpenicillin (penicillin G) Gram-positive activity -lactamase-sensitive SEMISYNTHETIC PENICILLINS Methicillin acid-stable, -lactamase-resistant Oxacillin acid-stable, -lactamase-resistant Ampicillin broadened spectrum of activity (especially against gram-negative Bacteria), acid-stable, -lactamase-sensitive Carbenicillin broadened spectrum of activity (especially against Pseudomonas aeruginosa), acid-stable but ineffective orally, -lactamase-sensitive © 2012 Pearson Education, Inc.

26. 8 -Lactam Antibiotics: Penicillins and Cephalosporins • Cephalosporins (Figure 26. 20) – Produced by fungus Cephalosporium – Same mode of action as the penicillins – Commonly used to treat gonorrhea © 2012 Pearson Education, Inc.

Figure 26. 20 Dihydrothiazine ring -Lactam ring © 2012 Pearson Education, Inc.

26. 9 Antibiotics from Prokaryotes • Many antibiotics effective against Bacteria are also produced by Bacteria – Aminoglycosides are antibiotics that contain amino sugars bonded by glycosidic linkage (Figure 26. 21) • Examples: kanamycin, neomycin, amikacin – Not commonly used today • Neurotoxicity and nephrotoxicity • Considered reserve antibiotics for when other antibiotics fail © 2012 Pearson Education, Inc.

Figure 26. 21 N-Acetyltransferase Streptomycin © 2012 Pearson Education, Inc. Kanamycin

26. 9 Antibiotics from Prokaryotes • Macrolides contain lactone rings bonded to sugars (Figure 26. 22) – Example: erythromycin – Broad-spectrum antibiotic that targets the 50 S subunit of ribosome • Tetracyclines contain four rings (Figure 26. 23) – Widespread medical use in humans and animals – Broad-spectrum inhibition of protein synthesis – Inhibits functioning of 30 S ribsomal subunit © 2012 Pearson Education, Inc.

Figure 26. 22 Macrolide ring Sugars © 2012 Pearson Education, Inc.

Figure 26. 23 Tetracycline analog R 1 R 2 R 3 R 4 Tetracycline H OH CH 3 H 7 -Chlortetracycline (aureomycin) H OH CH 3 Cl 5 -Oxytetracycline (terramycin) OH OH CH 3 H © 2012 Pearson Education, Inc.

26. 9 Antibiotics from Prokaryotes • Daptomycin (Figure 26. 24) – Also produced by Streptomyces – Used to treat gram-positive bacterial infections – Forms pores in cytoplasmic membrane • Platensimycin – New structural class of antibiotic (Figure 26. 25) – Broad-spectrum, effective against MRSA and vancomycin-resistant enterococci © 2012 Pearson Education, Inc.

Figure 26. 24 © 2012 Pearson Education, Inc.

Figure 26. 25 © 2012 Pearson Education, Inc.

26. 10 Antiviral Drugs • Most antiviral drugs also target host structures, resulting in toxicity • Most successful and commonly used antivirals are the nucleoside analogs (e. g. , AZT) – Block reverse transcriptase and production of viral DNA – Also called nucleoside reverse transcriptase inhibitors • Nonnucleoside reverse transcriptase inhibitors (NNRTI) bind directly to RT and inhibit reverse transcription © 2012 Pearson Education, Inc.

26. 10 Antiviral Drugs • Protease inhibitors inhibit the processing of large viral proteins into individual components • Fusion inhibitors prevent viruses from successfully fusing with the host cell • Two categories of drugs successfully limit influenza infection: – Adamantanes – Neuraminidase inhibitors • Interferons are small proteins that prevent viral multiplication by stimulating antiviral proteins in uninfected cells © 2012 Pearson Education, Inc.

26. 11 Antifungal Drugs • Fungi pose special problems for chemotherapy because they are eukaryotic (Figure 26. 26) – Much of the cellular machinery is the same as that of animals and humans – As a result, many antifungals are topical – A few drugs target unique metabolic processes unique to fungi © 2012 Pearson Education, Inc.

26. 12 Antimicrobial Drug Resistance • Antimicrobial drug resistance – The acquired ability of a microorganism to resist the effects of a chemotherapeutic agent to which it is normally sensitive © 2012 Pearson Education, Inc.

26. 12 Antimicrobial Drug Resistance • Most drug-resistant bacteria isolated from patients contain drug-resistance genes located on R plasmids • Evidence indicates that R plasmids predate the antibiotic era • The use of antibiotics in medicine, veterinary medicine, and agriculture selects for the spread of R plasmids (Figure 26. 28) – Many examples of overuse of antibiotics – Used far more often than necessary (e. g. , antibiotics used in agriculture as supplements to animal feed) © 2012 Pearson Education, Inc.

26. 12 Antimicrobial Drug Resistance • Almost all pathogenic microbes have acquired resistance to some chemotherapeutic agents (Figure 26. 29) • A few pathogens have developed resistance to all known antimicrobial agents – Methicillin-resistant S. aureus (MRSA) • Resistance can be minimized by using antibiotics correctly and only when needed • Resistance to a certain antibiotic can be lost if antibiotic is not used for several years © 2012 Pearson Education, Inc.

Figure 26. 29 Candida albicans Acinetobacter spp. Gram-negative Gram-positive/ acid-fast Fungus Enterococcus faecalis* Streptococcus pneumoniae Mycobacterium tuberculosis* Haemophilus ducreyi Salmonella typhi Haemophilus influenzae Neisseria gonorrhoeae Pseudomonas aeruginosa* Salmonella spp. Shigella dysenteriae Shigella spp. Other gram-negative rods Staphylococcus aureus Year © 2012 Pearson Education, Inc.

26. 13 The Search for New Antimicrobial Drugs • Long-term solution to antimicrobial resistance relies on the development of new antimicrobial compounds – Modification of current antimicrobial compounds is often productive – Automated chemistry methods (combinatorial chemistry) has sped up drug discovery – 7, 000 compounds must be screened to find a single useful clinical drug © 2012 Pearson Education, Inc.

26. 13 The Search for New Antimicrobial Drugs • Computers can now be used to design molecules to interact with specific microbial structures – Most successful example is saquinavir • Binds to active site of HIV protease • New methods of screening natural products are being used – Led to the discovery of platensimycin • Combinations of drugs can be used (e. g. , ampicillin and sulbactam) • Bacteriophage therapy © 2012 Pearson Education, Inc.