2. Sterilization, Disinfection and Antibacterial Agents Pin Lin ( 凌 斌 ), Ph.D. Departg ment of Microbiology & Immunology, NCKU ext 5632 lingpin@mail.ncku.edu.tw References: 1. Chapters 10 & 20 in Medical Microbiology (Murray, P. R. et al; 5 th edition) 2. 醫用微生物學 ( 王聖予 等編譯, 4th edition)

3. Outline Sterilization (Definition & Methods) Disinfection (Definition & Methods) Mechanisms of Antimicrobial Action Antibacterial Agents

4. What is Sterilization? Sterilization (in Microbiology) : 1.To completely remove all kinds of microbes (bacteria, mycobacteria, viruses, & fungi) by physical or chemical methods. 2.Effective to kill “bacterium spores” 3. Sterilant: material or method used to remove or kill all microbes

5. Methods of sterilization (I)

6. Methods of sterilization (II) Sterilize instruments

7. 1.Steam (Moist) & Dry Heat => the most common methods for most materials. Cons: NO good for heat-senstive, toxic or volatile chemicals 2.Filtration => remove bacteria and fungi from air or solutions eg: HEPA (High-Efficiency Particular Air) filters Cons: unable to remove viruses and some small bacteria (microplasma) 3.Ethylene oxide => the most common gas vapor sterilant Cons: (1) flammable & explosive (2) potential carcinogenic 4. Formaldehyde gas => carcinogenic Pros & Cons of Sterilants (I)

8. 1.Plasma gas => Hydrogen peroxide Reactive free radicals => No Toxic byproducts => may replace many applications for ethylene oxide Cons: NOT good for materials absorbing or reacting with H 2 O 2 2.Peracetic acid => an oxidizing agent w/ good activity => end products nontoxic 3.Glutaraldehyde => Not safe Pros & Cons of Sterilants (II) Microwave - or radio-freq energy

9. What is Disinfection? Disinfection (in Microbiology) : 1.To kill most of microbial forms except some resistant organisms or bacterium spores 2.Categorizing: High-level  sterilization Intermediate-level Low level 3. Disinfectant: a substance or method used to kill microbes on surfaces Not effective for all bacteria or spores

10. High-level disinfectants Used for items involved in invasive procedures but NOT withstand sterilization, e.g. Endoscopes, Surgical instruments

11. Intermediate-level disinfectants Used for cleaning surface or instruments without bacterial spores and highly resilient organism, eg. Laryngoscopes, Anesthesia breathing circuits…etc Low-level disinfectants Used to treat non-critical instruments and devices, not penetrating into mucosa surfaces or sterile tissues

12. Considerations of Disinfection Effectiveness of disinfectants is influenced by 1.Nature of the item to be disinfected 2.Number and resilience of the contaminants 3.Amount of organic material present 4.Type and concentration of disinfectant 5.Duration and temperature of exposure

13. Antisepsis & Antiseptic agents 1.Use of chemical agents on skin or living tissues to inhibit or eliminate microbes 2.Antiseptic agents are selected for their safety & efficacy

14. Outline Introduction Sterilization (Definition & Methods) Disinfection (Definition & Methods) Mechanisms of Antimicrobial Action

15. Physical methods (moist heat, dry heat, filtration, radiation) Moist heat Boiling: boiling for 10 min => Kill most vegetative forms of bacteria Longer time => Kill spores Addition of 2% Na 2 CO 3 or 0.1% NaOH => enhance destruction of spores and prevent rusting of the metal wares. Low temperature disinfection (Pasteurization): 62-65 o C for 30 min. or 71.5 o C for 15 sec. This is mainly used for disinfection of milk. Autoclave: 121-132 o C for 15 min or longer => Kill both the vegetative and spore forms of the bacteria. => Use Bacillius stearothermophilus spores to monitor the effectiveness of Autoclave

16. Flaming; incineration Radiation UV-light: UV-radiation causes damage to DNA. Ionizing radiation: less applicable. Filtration The pore size for filtering bacteria, yeasts, and fungi is in the range of 0.22-0.45 mm (filtration membranes are most popular for this purpose). HEPA filters Dry heat Dry oven: 160 o C for 2 hrs or 171 o C for 1 hr. (B. subtilis)

17. Chemical methods Alcohol : protein denaturant. 70% aqueous solution of ethyl alcohol and isopropyl alcohol are commonly used as skin disinfectants. Phenolics : Phenol and phenolic compounds (e.g. lysol) lyse the cell membrane and denature proteins at 1-2% (aqueous solution). Oxidizing agents : inactivate cells by oxidizing free sulfhydryl group, e.g., peracetic acid, iodine, iodophore, H 2 O 2 (3-6%), hypochlorite, and chlorine etc. Plasma gas sterilization: H 2 O 2 vapors treated with microwave or radio energy to produce reactive free radicals; no toxic byproducts. An efficient sterilization for dry surfaces.

18. Alkylating agents Formalin (37% aqueous solution of formaldehyde), glutaraldehyde Ethylene oxide gas (made nonexplosive by mixing with CO 2 or a fluorocarbon): a reliable disinfectant for dry surfaces. Detergents : surface-active agents that disrupt the cell membranes. Anionic detergents: e.g. soaps, and bile salts. Cationic detergents: e.g., the quaternary ammonium compounds, are highly bactericidal for both the gram-positive and negative bacteria in the absence of contaminating organic matter.

19. Outline Introduction Sterilization (Definition & Methods) Disinfection (Definition & Methods) Mechanisms of Antimicrobial Action Antibacterial Agents

20. The Discovery of Antibacterial Agents 1.In 1930s Gerhard Domagk discovered the anti-bacterial effect of prontosil (=> sulfanilamide) => 1939 Nobel Laureate 2.A. Fleming discovered that the mold Penicillium prevented the multiplication of staphyloocci. => The first antibiotic, Penicillin, was identified => 1945 Nobel Laureate 3.Streptomycin, tetracyclines & others were thereafter developed to treat infectious diseases. 4.Bacteria start developing resistance to these agents

21. Antibacterial agents 1. A useful chemotherapeutic agent should have in vivo effectiveness and selective toxicity. 2. Modes of action of the chemotherapeutic agents Inhibition of: cell wall synthesis protein synthesis nucleic acid synthesis (cell membrane function)

22. Sites of Action of Antibacterial Chemical Agents

23. Sites of antibiotic activity Disruption of cell wall

25. Peptidoglycan 1.A major component of cell wall 2.Forms a meshlike layer consisting: a polysaccharide polymer cross-linked by Peptide bonds 3.Cross-linking reaction is mediated by: transpeptidases DD-carboxypeptidases Targets of Penicillin

26. Outer wall of Gram-positive and Gram-negative species

27.  -lactam drugs: Drugs containing a  -lactam ring, e.g. penicillins and cephalosporins. Vancomycin: bactericidal for some gram-positive bacteria Inhibition of cell wall synthesis ( penicillins, cephalosporins, vancomycin, cycloserine, bacitracin) PBPs (penicillin-binding proteins): receptors for  -lactam drugs. There are 3-6 PBPs, some of which are transpeptidation enzymes.

29. Penicillins Produced by Penicillium chrysogenum Modifications: decrease acid lability; increase absorption; resistant to penicillinase; broader spectrum (e.g., ampicillin).  -lactamase inhibitors: bind  -lactamases irreversibly; combined use with some penicillins to increase effectiveness. Modifications of cephalosporins were to expand their spectra or increase their stability to  -lactamases.

30. Vancomycin A complex glycopeptide produced by Streptomyces orientalis Interacts with D-ala-D-ala termini of the pentapeptide side chains Inactive for gram-negative bacteria Some enterococci have acquired resistance to vancomycin The resistance genes are carried on plasmids

31. Polymyxins 1. Cyclic polypeptides (from Bacillus polymyxa) 2. Insert into bacteria outer membrane by interacting with LPS and phospholipids  increase cell permeability  bacterial cell death 3. Most Active for gram-negative bacteria, because Gram-pos bacteria have no outer member 4. Nephrotoxic 5. External treatment of localized infection and oral administration to sterilize the gut

32. Drug resistance of the microbes 1. Producing enzymes that degrade or modify the active drugs; 2. Decreasing drug entry; 3. Increasing drug efflux; 4. Increasing the amount of target enzyme; 5. Decreasing affinity of target for drug; 6. Developing an altered metabolic pathway that bypass the reaction inhibited by the drug.

33. How Bacteria Become Resistant to the  -Lactam Antibiotics? 1. To prevent the interaction between the antibiotic and the target PBP e.g. Gram-neg (Pseudomonas) => change porins on the pores => exclude antibiotic 2. To modify the binding of the antibiotic to the target PBP Modified PBP can result from mutation or acquisition of new PBP 3. Hydrolysis of the antibiotic by  -lactamases - They are in the same family of PBPs - Over 200 different  -lactamases: some are specific for penicillins others have a broad range of activity

34. Inhibition of protein synthesis Aminoglycosides (streptomycin, kanamycin, neomycin, gentamicin, tobramycin, amikacin, etc.): bind irreversibly to 30S ribosomal proteins and inhibit peptide formation; bactericidal. Gm and Tm are ototoxic. Tetracyclines: inhibit attachment of charged tRNA; bacteriostatic. Chloramphenicol: binds to peptidyl transferase of ribosome; toxic to bone marrow cells (aplastic anemia); bacteriostatic. Macrolides (erythromycins, clarithromycin, etc.): bind to 23S rRNA and block peptide elongation. Lincomycins (clindamycin): resembles macrolides in mode of action. Oxazolidinones (linezolid): blocks formation of imitiation complex. Active against staphylococci, streptococci and enterococci. No cross- resistance with the above antibiotics. Reserved for multidrug-resistant enterococci.

35. Resistance to aminoglycosides: 1.Mutation to ribosomal binding site; 2.Decreased uptake of antibiotic; 3.Enzymatic modification of the antibiotic.

36. Rifampin: inhibits RNA synthesis. Quinolones and fluoquinolones: blocking DNA gyrase. Metronidazol: effective to anaerobic bacterial infections. Reduction of its nitro group by bacterial nitroreductase produces cytotoxic compound that disrupts bacterial DNA. Inhibition of nucleic acid synthesis quinolones, rifampin, sulfonamides, trimethoprime, pyrimethamine Antimetabolites SulfonamidesSulfonamides: analogs of p-aminobenzoic acid (PABA) and inhibit synthesis of folic acid, which is an important precursor to the synthesis of nucleic acids. Trimethoprim: inhibits dihydrofolic acid reductase in synthesis of purines, methionine and glycine.

37. Environment Amount of pathogen State of bacterial metabolic activity Distribution of drug Location of organisms Interfering substances Antimicrobial activity in vivo Concentration of antibiotic Absorption Distribution Variability of concentration Factors affecting the effectiveness of antibiotics in vivo

38. Dangers of indiscriminate use of antibiotics 1. Development of drug resistance. 2. “Superinfection" resulting from changes in the normal flora of the body. 3. Masking serious infection without eradicating it. 4. Drug toxicity. 5. Widespread sensitization of the population with resulting hypersensitivity, anaphylaxis, rashes, fever, blood disorders, cholestatic hepatitis, and perhaps collagen-vascular diseases.

39. Genetic origin of drug resistance Chromosomal Extrachromosomal (e.g., R plasmids) Can be transferred by conjugation, transformation, and transduction.

41. A general rule in antimicrobial therapy give a sufficiently large amount of an effective drug as early as possible and continue treatment long enough to ensure eradication of infection, but give an antimicrobial drug only when it is indicated by rational choice.

42. Limitation of drug resistance 1. Maintain sufficiently high levels of the drug in the tissue to inhibit both the original population and first-step mutants. 2. Simultaneously administer two drugs that do not give cross-resistance. 3. Avoid exposure of microbes to a particular drug by limiting its use, especially in hospitals and in animal feeds. Cross-resistance: microbes resistant to a certain drug may also be resistant to other drugs that share a mechanism of action. (e.g., different aminoglycosides, macrolides, and lincomycins) Selection of antibiotics Diagnosis Antibiotic susceptibility tests

43. Antimicrobial drugs used in combination Indications Prompt treatment of patients suspected of having a serious microbial infection. To delay the emergence of mutants resistant to one drug in chronic infections. To treat mixed infections. To achieve bactericidal synergism or to provide bactericidal action. Disadvantages Relaxation of the effort to establish a diagnosis. Greater chance for adverse reactions. Unnecessary cost. Not necessarily effective than single drug treatment. Antagonism between drugs (rarely).

44. Effects of combined usage of two antibiotics Indifference (A + B=A or B) Addition (A + B=A + B) Synergism (A + B=A x B) Antagonism (A + B= 0 or less)

45. SUMMARY 1. Various antimicrobial agents act by interfering with: (1) cell wall synthesis, (2) plasma membrane integrity, (3) nucleic acid synthesis, (4) ribosomal function, and (5) metabolite synthesis. 2. Cell wall synthesis is inhibited by ß-lactams, such as penicillins and cephalosporins, which inhibit peptidoglycan polymerization, and by vancomycin, which combines with cell wall substrates. 3. Bacteria can evolve resistance to antibiotics. Resistance factors can be encoded on plasmids or on the chromosome. Resistance may (1) decreased entry of the drug, (2) changes in the receptor (target) of the drug, or (3) metabolic inactivation of the drug. 4. Combinations of antibiotics may act synergistically-producing an effect stronger than the sum of the effects of the two drugs alone or antagonistically, if one agent inhibits the effect of the other.

47. Basis of Antimicrobial Action Various antimicrobial agents act by interfering with (1) cell wall synthesis, (2) plasma membrane integrity, (3) nucleic acid synthesis, (4) ribosomal function, and (5) folate synthesis. Action of Specific Agents Cell wall synthesis is inhibited by ß-lactams, such as penicillins and cephalosporins, which inhibit peptidoglycan polymerization, and by vancomycin, which combines with cell wall substrates. Polymyxins disrupt the plasma membrane, causing leakage. The plasma membrane sterols of fungi are attacked by polyenes (amphotericin) and imidazoles. Quinolones bind to a bacterial complex of DNA and DNA gyrase, blocking DNA replication. Nitroimidazoles damage DNA. Rifampin blocks RNA synthesis by binding to DNA directed RNA polymerase. Aminoglycosides, tetracycline, chloramphenicol, erythromycin, and clindamycin all interfere with ribosome function. Sulfonamides and trimethoprim block the synthesis of the folate needed for DNA replication Bacterial Resistance Bacteria can evolve resistance to antibiotics. Resistance factors can be encoded on plasmids or on the chromosome. Resistance may involve decreased entry of the drug, changes in the receptor (target) of the drug, or metabolic inactivation of the drug. Effects of Combination Therapy Combinations of antibiotics may act synergistically-producing an effect stronger than the sum of the effects of the two drugs alone or antagonistically, if one agent inhibits the effect of the other. Adverse Effects of Antimicrobial Agents Many antibiotics are toxic to the host. Alterations of the normal intestinal flora caused by antibiotics may result in diarrhea or in superinfection with opportunistic pathogens.

48. B. stearothermophilus spores Back

49. Disinfection and Sterilization Disinfection: killing of most microbial forms. Disinfectant: a chemical substance used to kill microbes on surfaces but too toxic to be applied directly to tissue. Antisepsis: inhibit or eliminate microbes on skin or other living tissue Sterilization: removal of life of every kind by physical or chemical methods. Sterilant: an agent or method used to remove or kill all microbes. Septic: presence of pathogenic microbes in living tissue. Aseptic: absence of pathogenic microbes. Sterile: free of life of every kind. Bacteriostatic: inhibiting bacterial multiplication. Bacteriostatic action is reversible by removal or inactivation of agent. Bactericidal: killing bacteria.

50. Modes of action of antimicrobial agents 1. Damage to DNA Formation of pyrimidine dimer by UV irradiation Single- or double-strand DNA break by ionizing radiation DNA reactive chemicals, e.g. alkylating agents 2. Protein denaturation 3. Disruption of cell membrane or wall 4. Removal of free sulfhydryl groups Formation of disulfide bond by oxidizing agents Heavy metals combine with sulfhydryls 5. Chemical antagonism: interference with the normal reaction between an enzyme and its substrate.

51. Resistance to  -lactam antibiotics : 1.Prevention of interaction of drug and the target PBP; 2.Decrease binding of drug to PBP; Modified PBP can result from mutation or acquisition of new PBP 3. Hydrolysis of drug by producing b-lactamase (> 200 different kinds).