Touro Microbiology Lecture 11 Physical And Chemical Controls of Microorganisms.ppt

Physical/ Chemical Control of Microorganisms Introduction • Until late 19th century, patients undergoing even minor surgeries were at great risk of developing fatal infections • Physicians would not accept that their hands could pass diseases from one patient to the next • Did not understand airbo...

Physical/ Chemical Control of Microorganisms Introduction • Until late 19th century, patients undergoing even minor surgeries were at great risk of developing fatal infections • Physicians would not accept that their hands could pass diseases from one patient to the next • Did not understand airborne microbes could infect open wounds • Modern hospitals use strict procedures to avoid microbial contamination • Arthur Tilley/Getty Images 2 Approaches to Control 1 • Principles of Control • Sterilization: removal or destruction of all microorganisms and viruses • Sterile item is free of microbes including endospores, but does not consider prions • Disinfection: elimination of most or all pathogens • Some viable microbes may remain • Disinfectants: chemicals used on inanimate objects • Often called germicides • Bactericides kill bacteria • Fungicides kill fungi • Virucides inactivate viruses • Antiseptics: chemicals used on living tissues 3 Approaches to Control 2 Decontamination: reduces number of pathogens to a safe level • Washing, use of heat, or chemicals Sanitization: substantially reduces microbial population to meet accepted health standards that minimize spread of disease Preservation: process of delaying spoilage of perishable products • Choose conditions of storage to slow growth • Add bacteriostatic (growth-inhibiting) preservatives Pasteurization: brief heating to reduce number of spoilage organisms, destroy pathogens without changing characteristics of product 4 Approaches to Control 3 • Daily Life • Washing and scrubbing with soaps and detergents achieves routine control • Soap aids in mechanical removal of organisms • Beneficial skin microbiota reside deeper on underlying layers of skin, hair follicles • Not adversely affected by regular washing • Other control methods used in daily life include cooking foods, cleaning surfaces, and refrigeration 5 Approaches to Control 4 • Hospitals and Other Healthcare Facilities • Controlling microbes in healthcare settings is very important due to danger of healthcare-associated infections (HAIs) • Weakened patients more susceptible to infection; may undergo invasive procedures (surgery) • Pathogens more likely found in hospital setting • Feces, urine, respiratory droplets, bodily secretions 6 Approaches to Control 5 • Healthcare facilities must protect personnel. • The COVID-19 pandemic demonstrated that healthcare workers are at risk of contracting infectious disease from patients. • Practices called Standard Precautions are used in patient care to prevent infection of both patients and personnel. • If a patient might be infected with a highly transmissible or epidemiologically important pathogen, Transmission-Based Precautions are also used. 7 Approaches to Control • Hospitals and Other Healthcare Facilities • Special care must be taken to control microorganisms in operating rooms • Instruments must be sterilized to avoid introducing infection to deep tissues during surgery • Prions are a relatively new concern; difficult to destroy 8 Approaches to Control 6 • Microbiology Laboratories • Routinely work with microbial cultures • Use rigorous methods of control • Must eliminate microbial contamination to both experimental samples and environment • Careful treatment both before work (use sterile materials) and after work (sterilize cultures, waste) • Aseptic technique used to prevent contamination of samples, workers, environment 9 Selecting an Antimicrobial Procedure 1 • Selection of effective procedure is complicated • Ideal method does not exist; each has drawbacks • Choice depends on numerous factors • Type and number of microbes • Environmental conditions • Risk of infection • Composition of item to be treated 10 Types of Microbes Multiple highly resistant microbes • Bacterial endospores: most resistant; only extreme heat or chemical treatment destroys them • Protozoan cysts and oocysts: resistant to disinfectants; excreted in feces; causes diarrheal disease if ingested; destroyed by boiling • Mycobacterium species: waxy cell walls makes resistant to many chemical treatments • Pseudomonas species: resistant to and can actually grow in some disinfectants • Non-enveloped viruses: lack lipid envelope; more resistant to disinfectants 11 Number of Microbes • Only a fraction of population dies during given time interval so it takes more time to destroy a large population than a small one • Removing some organisms by washing reduces time needed to sterilize or disinfect a product • Scrubbing helps remove biofilms - important because organisms in a biofilm are resistant to disinfectants • Decimal reduction time (D value) is time required to kill 90% of population under specific conditions • Access the text alternative for slide images. 12 Environmental Conditions Dirt, grease, body fluids can interfere with heat penetration, action of chemicals • Important to thoroughly clean items Temperature and pH can influence effectiveness • For example, sodium hypochlorite (household bleach) solution can kill suspension of M. tuberculosis at 55 degrees Celsius in half the time as at 50 degrees Celsius • Even more effective at low pH 13 Risk for Infection Medical instruments categorized according to risk for transmitting infectious agents Critical items come into contact with body tissues • Must be sterile • Include needles and scalpels Semicritical instruments contact mucous membranes but do not penetrate body tissues • Must be free of microorganisms and viruses • Some endospores may remain but are often blocked by mucous membranes • Includes endoscopes and endotracheal tubes Non-critical instruments and surfaces contact unbroken skin only • Low risk of transmission • Countertops, stethoscopes, blood pressure cuffs 14 Composition of an Item Some sterilization and disinfection methods inappropriate for certain items • Heat can damage plastics and other materials • Irradiation provides alternative, but damages some types of plastic • Moist heat, liquid chemical disinfectants cannot be used to treat moisture-sensitive material 15 Physical Methods Used to Destroy or Remove Microorganisms and Viruses • Heat treatment useful for microbial control • Reliable, safe, relatively fast, inexpensive, nontoxic • Can be used to sterilize or disinfect • Filtration, irradiation, and high-pressure treatment can be used on materials that cannot withstand heat treatment. 16 Moist Heat 1 • Moist heat: irreversibly denatures proteins • Boiling destroys most microorganisms and viruses • Does not sterilize: endospores can survive • Pasteurization destroys heat-sensitive pathogens, spoilage organisms • High-temperature–short-time (HTST) method: Milk − 72 degrees Celsius for 15 seconds; ice cream − 82 degrees Celsius for 20 seconds • Ultra-high-temperature (UHT) method: shelf-stable boxed juice and milk; known as “ultra-pasteurization” • Milk − 140 degrees Celsius for a few seconds, then rapidly cooled 17 Sterilization Using Pressurized Steam Autoclave used to sterilize using pressurized steam • Increased pressure raises steam temperature; kills endospores • Sterilization typically at 121 degrees Celsius and 15 pound per square inch in 15 minutes Destroying prions involves immersion in 1 M NaOH during or before autoclaving • Access the text alternative for slide images. 18 Sterilization Using Pressurized Steam Autoclave used to sterilize using pressurized steam • To be effective, steam must enter item and displace air • Do not close containers tightly • Tape with a heat-sensitive indicator can confirm heating • Biological indicators can confirm lack of microbial growth after autoclaving • (a&b): ©Evans Roberts 19 Dry Heat • Incineration (destruction by burning) oxidizes cell components to ashes. • In microbiology laboratories, the wire loops continually reused to transfer bacterial cultures are sterilized by flaming or heating in a benchtop incinerator • Incineration is also used to destroy medical wastes and contaminated animal carcasses. • Hot air ovens kill microbes by destroying cell components and denaturing proteins. • Requires higher temperature and longer times than moist heat because dry heat takes longer to penetrate and is less efficient at killing microbes 20 Filtration • Membrane filtration in a liquid medium, retains bacteria while allowing the fluid to pass through. • Used extensively to remove organisms from heatsensitive fluids • Membrane filters or microfilters • Small pore size (0.2 micrometer) to remove bacteria • Vacuum used to move fluid through filter • • Source: CDC/Margaret Williams, PhD; Claressa Lucas, PhD; Tatiana Travis, BS Access the text alternative for slide images. 21 Filtration • Filtration of air • High-efficiency particulate air (HEPA) filters remove nearly all microbes over 0.3 micrometer from air • Specialized hospital rooms • Biological safety cabinets 22 Chemicals Methods Used to Destroy Microorganisms and Viruses 1 • Germicidal chemicals can disinfect and, in some cases, sterilize. • React irreversibly with proteins, DNA, cytoplasmic membranes, or viral envelopes. • Exact mechanisms of action are often poorly understood. • Less reliable than heat but useful for treating large surfaces and heat-sensitive items. • Some are sufficiently non-toxic to be used as antiseptics. 23 Chemicals Methods Used to Destroy Microorganisms and Viruses 2 • In the United States, the FDA is responsible for ensuring that chemicals used to treat medical devices work and that drug products, including antiseptics, are safe and effective. • Various active ingredients previously allowed in nonprescription antiseptic products must now be proven safe and effective • Antiseptic washes (antimicrobial soaps) • Antiseptic rubs (hand sanitizers) • Topical antiseptic products used by healthcare professionals to disinfect patients' skin in preparation for injections or surgery 24 Categories of Germicidal Potency • Germicidal Chemicals disinfect and, sometimes, sterilize. • Sterilants destroy all microbes • Heat-sensitive critical instruments • High-level disinfectants destroy viruses, vegetative cells • Do not reliably kill endospores • Semi-critical instruments • Intermediate-level disinfectants destroy vegetative bacteria, mycobacteria, fungi, and most viruses • Disinfect non-critical instruments • Low-level disinfectants destroy fungi, vegetative bacteria except mycobacteria, and enveloped viruses • Do not kill endospores, non-enveloped viruses • Disinfect furniture, floors, walls 25 Classes of Germicidal Chemicals 1 • Alcohols • 60 to 80% aqueous solutions of ethyl or isopropyl alcohol • Destroy vegetative bacteria and fungi • Not reliable against endospores, non-enveloped viruses • Denatures essential proteins, damages membranes • Proteins more soluble in water; pure alcohol less effective • Commonly used as antiseptic and disinfectant; nontoxic, inexpensive • Limitations • Evaporates quickly, limiting contact time • Can damage rubber, some plastics • Tincture: antimicrobial chemical dissolved in alcohol 26 Classes of Germicidal Chemicals 2 • Aldehydes • Includes glutaraldehyde, formaldehyde, and orthophthalaldehyde (OPA) • Inactivates proteins and nucleic acids • 2% alkaline glutaraldehyde common liquid sterilant • Immersion of medical items for 10 to 12 hours • Toxic; requires thorough rinsing after use • Formaldehyde used as gas or as formalin (37% solution) • Effective germicide that kills most microbes quickly • Used to kill bacteria and inactivate viruses for vaccines • Used to preserve specimens • Probable carcinogen 27 Classes of Germicidal Chemicals 3 • Biguanides • Chlorhexidine most effective • Extensive use as antiseptics • Stays on skin, mucous membranes • Relatively low toxicity • Destroys vegetative bacteria, fungi, some enveloped viruses • Common in many products: skin cream, prescription mouthwashes 28 Classes of Germicidal Chemicals 4 • Ethylene oxide • Gaseous sterilant for heat- or moisture-sensitive items • Destroys microbes, including endospores and viruses, by chemically modifying proteins and nucleic acids • Penetrates fabrics, equipment, implantable devices • Mattresses, electrical equipment, artificial hips • Used for many disposable laboratory items • Petri dishes, pipettes • Applied in special chamber resembling autoclave • Limitations: explosive, toxic, potentially carcinogenic • Must be eliminated by heated forced air for 8 to 12 hours 29 Classes of Germicidal Chemicals • Halogens: oxidizing agents which react with proteins, cellular components • Chlorine: Destroys all microorganisms, endospores and viruses • Caustic to skin and mucous membranes • 1:100 dilution of household bleach effective • Very low levels disinfect drinking water • Cryptosporidium oocysts, Giardia cysts survive • Can react with organic compounds in water • Disrupts germicidal activity • Chlorine dioxide (ClO2) used as disinfectant and sterilant • Jill Braaten/McGraw-Hill 30 Classes of Germicidal Chemicals 5 • Iodine: Kills vegetative cells, unreliable on endospores • Used as tincture (in alcohol) • Used as iodophor • Iodine slowly released from carrier molecule • Less irritating • Pseudomonas species can survive in stock solution 31 Metal Compounds Combine with sulfhydryl groups (–SH) of proteins High concentrations too toxic to be used medically Silver used in creams, bandages Antibiotics replaced silver nitrate eye drops once given at birth to prevent Neisseria gonorrhoeae infections 32 Peroxygens: powerful oxidizers used as sterilants Readily biodegradable, no residue Less toxic than ethylene oxide, glutaraldehyde Hydrogen peroxide: effectiveness depends on surface • Aerobic cells (humans) produce enzyme catalase • Breaks down H2O2 to O2  H2O • More effective on inanimate object • Doesn’t damage most materials, no residue • Hot solutions or vapor-phase used as sterilant Peracetic acid: more potent than H2O2 • Sterilizes in less than 1 hour • Effective in presence of organic compounds, no residue, used on wide range of materials 33 Phenolic Compounds (Phenolics) Phenol (carboic acid) one of earliest disinfectants • Has unpleasant odor, irritates skin Destroy cytoplasmic membranes, denature proteins Phenolics kill most vegetative bacteria; not reliable on all virus groups Wide activity range, reasonable cost, remain effective in presence of detergents and organic contaminants • Leave antimicrobial residue Hexachlorophene and triclosan have been widely used in medical and personal care products 34 Preservation of Perishable Products 2 • Low-Temperature Storage • Refrigeration inhibits growth of pathogens and spoilage organisms by slowing or stopping enzyme reactions • Psychrotrophs, psychrophilic organisms can still grow • Freezing preserves by stopping all microbial growth • Some microbial cells killed by ice crystal formation, but many survive and can grow once thawed 35 Antimicrobial Medications Importance of Antimicrobial Medications • Imagine a world without antimicrobial medications • Prognosis for people with common diseases (bacterial pneumonia, severe staphylococcal infections) was grim before availability of penicillin in 1940s • Physicians could identify cause of disease, but only treatment was usually bed rest • Misuse coupled with evolution of microbial resistance threatens these medications • Scientists scrambling to develop new medications • Source: James Gathany/CDC 37 History and Development of Antimicrobial Medications • Discovery of antimicrobial medications • Salvarsan (Paul Ehrlich, 1910) first documented case • Red dye Prontosil (Gerhard Domagk, 1932) used to treat streptococcal infections in animals • No effect in test tubes; enzymes in blood split to produce sulfanilamide, the first sulfa drug • Both are chemotherapeutic agents • Chemicals used to treat disease • Antimicrobial medications, antimicrobial drugs, or antimicrobials 38 Discovery of Antibiotics In 1928, Fleming identified mold Penicillium secreting compound toxic to Staphylococcus (penicillin) • Showed effective in killing many bacterial species • Unable to purify, he later abandoned research • Chain and Florey purified, tested compounds in 1941 on police officer with Staphylococcus aureus infection • Patient improved but supply of purified penicillin ran out and he later died • WWII spurred research and development; penicillin G, the first antibiotic (naturallyproduced antimicrobial) • • ©Biophoto Associates/Science Source Access the text alternative for slide images. 39 Discovery of Antibiotics Selman Waksman purified streptomycin from soil bacterium Streptomyces griseus • Researchers began screening hundreds of thousands of microbes for antibiotics • Pharmaceutical companies today examine soil samples from around world 40 Development of New Antimicrobial Medications Most antibiotics come from microorganisms that normally live in the soil including Streptomyces and Bacillus (bacteria), and Penicillium and Cephalosporium (fungi) Altering structure of antibiotics such as penicillin yields new medications (ampicillin, methicillin) • Development of new antimicrobial drugs is financially risky because FDA demands strict and expensive testing • Resistance will likely develop soon after drug is used • Drug may be held as last resort to avoid resistance • Access the text alternative for slide images. 41 Development of New Antimicrobial Medications Generating Antibiotic Incentives Now (GAIN) is U.S. law to encourage companies to develop new antimicrobials against certain pathogens • If promising, a drug may be designated a Qualified Infectious Disease Product (QIDP) • Receives high priority for review • Company has exclusive marketing rights for 5 years 42 Characteristics of Antimicrobial Medications • Selective toxicity: causes greater harm to microbes than to human host • Interfere with essential structures or properties in microbes but not human cells • Difficult with respect to antiviral medications because viruses rely on human cells for their replication • Toxicity is relative and expressed as therapeutic index • Calculated as the lowest dose toxic to patient divided by dose used for therapy • Penicillin G useful, has high therapeutic index; interferes with cell wall synthesis, a process not present in humans • Also has high therapeutic window, the range between the therapeutic dose and the toxic dose • Medications too toxic for systemic use may be used topically (applied to body surface) 43 Antimicrobial Action Bacteriostatic chemicals inhibit bacterial growth • Patient’s defenses must eliminate pathogen (sulfa drugs) Bactericidal chemicals kill bacteria 44 Spectrum of Activity Broad-spectrum antibiotics affect a wide range • Important for treating acute life-threatening diseases • Especially when no time to culture for identification • Problem: Disrupt microbiome that helps keep out other pathogens Narrow-spectrum antibiotics affect limited range • Requires identification and susceptibility of pathogen • Less disruptive to microbiome Patients may be started on broad-spectrum antimicrobial and later switched to narrow-spectrum once more is known about the pathogen 45 Effects of Antimicrobial Combinations Some medications interfere with others (antagonistic) • For example, bacteriostatic antimicrobials that prevent cell division interfere with bacteriocidal antimicrobials that kill only dividing cells Some medications enhance one another (synergistic) Other combinations are additive 46 Tissue Distribution, Metabolism, and Excretion of the Medication Antimicrobial behaviors differ in body • Only some can access the brain; only some can withstand stomach acid • If poorly absorbed from the intestinal tract must be administered by injection Half-life of medication is time it takes for serum concentration to decrease by 50% • Dictates frequency of doses required to maintain effective level in body • Penicillin V is taken 4 times a day; azithromycin is taken no more than once a day Patients with kidney or liver dysfunction excrete or metabolize medications more slowly; must adjust dosage to avoid toxic levels 47 Adverse Effects Antimicrobials have saved countless lives when properly prescribed and used •Allergic reactions • May be life-threatening; may wear bracelet alert •Toxic effects • Monitor those taking low therapeutic index drugs • Some side effects are life-threatening (chloramphenicol may cause aplastic anemia) •Dysbiosis: imbalance in the microbiome • For example, broad-spectrum antimicrobials may allow growth of Clostridium difficile without competition, resulting in diarrhea or colitis 48 Resistance to Antimicrobials Certain bacteria have intrinsic (innate) resistance • For example, Mycoplasma lack cell wall, resistant to penicillin that interferes with peptidoglycan synthesis • Outer membrane of Gram-negatives blocks many medications Bacteria may develop acquired resistance • Spontaneous mutations • Horizontal gene transfer 49 Mechanisms of Action of Antibacterial Medications • Antibacterial medications target specific bacterial processes and structures • Cell wall synthesis • Protein synthesis • Nucleic acid synthesis • Metabolic pathways • Cell membranes 50 Mechanisms of Action of Antibacterial Medications • Inhibit cell wall synthesis • Bacterial cell walls are unique, contain peptidoglycan • Include β-lactam antibiotics, glycopeptide antibiotics, and bacitracin • Access the text alternative for slide images. 51 Inhibit Cell Wall Synthesis • β-lactam antibiotics • All have β-lactam ring and high therapeutic index • Penicillins, cephalosporins, carbapenems, monobactams • Competitively inhibit penicillin-binding proteins (PBPs) that catalyze formation of peptide bridges between adjacent glycan strands; disrupt cell wall synthesis • Only effective against actively growing cells • Access the text alternative for slide images. 52 Inhibit Cell Wall Synthesis 1 • β-lactam antibiotics • Vary in activity • Peptidoglycan of Gram-positives exposed; outer membrane of Gram-negatives blocks • PBPs different in Gram-positives versus Gramnegatives • Some bacteria synthesize a β-lactamase, which breaks β-lactam ring; destroys activity of antibiotic • Penicillinase inactivates members of penicillin family • Extended-spectrum β-lactamases (ESBLs) inactivate a wide variety of β-lactam medications • Gram-negatives produce a more extensive array of β-lactamases than Gram-positives 53 Inhibit Cell Wall Synthesis 2 • β-lactam antibiotics • The penicillins are grouped by modifications in their side chain • Natural penicillins are from Penicillium chrysogenum • Narrow-spectrum, act against Grampositives and a few Gram-negatives; deactivated by penicillinases • Penicillinase-resistant penicillins developed in response to penicillinase from S. aureus strains • Some can produce altered PBPs to which β-lactam antibiotics do not bind well (methicillin-resistant S. aureus, or MRSA • Access the text alternative for slide images. 54 Inhibit Cell Wall Synthesis 3 • β-lactam antibiotics • Broad-spectrum penicillins act against Gram-positives and many Gramnegatives (ampicillin, amoxicillin) • Inactivated by many β-lactamases • Extended-spectrum penicillins have greater activity against Enterobacteriaceae, Pseudomonas species • Reduced activity against Gram-positives; destroyed by many β-lactamases • Penicillins + β-lactamase inhibitor includes inhibitor to protect penicillin (Augmentin) 55 Inhibit Cell Wall Synthesis 4 • β-lactam antibiotics • Cephalosporins • Structure makes resistant to some β-lactamases • Some have low affinity for PBPs of Gram-positives • Chemical modifications have led to five generations • Later generations more effective against Gram-negatives, resist β-lactamases • Fifth generation effective against MRSA • Others available with β-lactamase inhibitor • Zerbaxa was first medication approved under GAIN act 56 Inhibit Cell Wall Synthesis 5 • β-lactam antibiotics • Carbapenems • Effective against wide range of Gram-positive and Gramnegative bacteria • Not inactivated by extended-spectrum β-lactamases (ESBL) • Often reserved as last resort against ESBL-producing organisms • Inactivated by carbapenemases • Monobactam • Aztreonam used against the family Enterobacteriaceae 57 Inhibit Cell Wall Synthesis 6 • Glycopeptide antibiotics • Bind to amino acid side chain of NAM molecules; block peptidoglycan synthesis • Effective only against Gram-positives; does not cross outer membrane of Gram-negatives • Side effects give low therapeutic index • Vancomycin is most widely used glycopeptide • Poorly absorbed from intestinal tract, usually administered via IV except for intestinal infections • Often antibiotic of last resort to treat Gram-positives resistant to β-lactam antibiotics 58 Inhibit Cell Wall Synthesis 7 • Bacitracin: toxicity limits to topical applications • Interferes with transport of peptidoglycan precursors across membrane • Common in first-aid skin ointments 59 Mechanisms of Action of Antibacterial Medications • Inhibit protein synthesis • Generally bacteriostatic • Can exploit differences between prokaryotic and eukaryotic ribosomes • Prokaryotes have 70S, eukaryotes have 80S ribosomes • Mitochondria also have 70S ribosomes • May account for toxicity of some of these antibiotics • Access the text alternative for slide images. 60 Inhibit Protein Synthesis 1 • Aminoglycosides • Irreversibly bind to 30S ribosomal subunit, causing it to malfunction; bacteriocidal • Blocks initiation of translation; causes misreading of mRNA by ribosomes past initiation • Often toxic; generally used when alternatives unavailable • Generally ineffective against anaerobes, enterococci, and streptococci because cannot enter cells • Sometimes used synergistically with a penicillin that allows the aminoglycoside to enter cells • Inhaled form of tobramycin treats Pseudomonas lung infections in cystic fibrosis patients • Neomycin too toxic for systemic use; common in firstaid skin ointments 61 Inhibit Protein Synthesis 2 • Tetracyclines and glycylcyclines • Tetracyclines reversibly bind to 30S ribosomal subunit • Block tRNA attachment; prevent translation • Effective against certain Gram-positives and Gram-negatives • Some have longer half-life meaning less frequent doses • Resistance from decreased uptake or increased excretion • The Glycylcyclines are related to the tetracyclines • Wider activity • Effective against bacteria resistant to the tetracyclines • Relatively new, so acquired resistance is rare • Tigecycline is only one currently approved 62 Inhibit Protein Synthesis 3 • Macrolides • Reversibly bind to 50S subunit; prevent translation from continuing • Often antibiotic of choice for patients allergic to penicillin • Bacteriostatic against many Gram-positives and most common causes of atypical pneumonia • Outer membrane of Enterobacteriaceae blocks • Resistance occurs from modification of ribosomal RNA target, enzyme that modifies chemical, and decreased uptake 63 Inhibit Protein Synthesis 4 • Chloramphenicol • Binds to 50S ribosomal subunit; blocks translation • Active against wide range of bacteria • Used as last resort due to rare, but lethal side effect • May cause aplastic anemia, inability to form white, red blood cells 64 Inhibit Protein Synthesis 5 • Lincosamides • Bind 50S ribosomal subunit; block translation from continuing • Inhibit variety of Gram-negatives and Gram-positives • Useful against Bacteroides fragilis (resists antibiotics) • Increases risk of developing C. difficile infection; most strains are resistant to lincosamides • Oxazolidinones • Bind 50S ribosomal subunit; interfere with initiation of translation • Useful against variety of Gram-positives strains resistant to β-lactams, vancomycin 65 Inhibit Protein Synthesis 6 • Pleuromutilins • Bind to 50s ribosomal subunit; prevent formation of peptide bonds during translation • Active against many types of Gram-positives • Once used only in animals; a topical now approved in humans • Streptogramins • Bind to 50S ribosomal subunit; inhibit translation • Quinupristin and dalfopristin each are bacteriostatic, but bactericidal when given together • Effective against variety of Gram-positives, but often reserved for treating infections caused by strains that are resistant to other antimicrobials 66 Mechanisms of Action of Antibacterial Medications 1 • Inhibit nucleic acid synthesis • Fluoroquinolones • Inhibit topoisomerases, enzymes that maintain supercoiling of DNA; bactericidal against wide variety of bacteria • DNA gyrase breaks, rejoins strands to relieve strain from localized unwinding of DNA; function is essential • Resistance usually due to alteration in DNA gyrase target • Used extensively in the past; severe side effects limit their use • Rifamycins • Block prokaryotic RNA polymerase; prevents initiation of transcription • Rifampin is bactericidal against Gram-positives, some Gramnegatives, Mycobacterium • Resistance develops quickly due to mutation in RNA polymerase gene 67 Inhibit Nucleic Acid Synthesis Fidaxomicin • Binds to RNA polymerase; interferes with transcription • Relatively new; bactericidal • Not absorbed in intestinal tract; effective in treating C. difficile infections Metronidazole (Flagyl) • Anaerobic metabolism required to convert to active form • Active form binds DNA, interferes with synthesis, causes breaks • Used to treat bacterial vaginosis and C. difficile infection 68 Mechanisms of Action of Antibacterial Medications • Interfere with metabolic pathways • Folate inhibitors are most useful • Inhibit steps in synthesis of folate and ultimately synthesis of coenzyme required for nucleotide biosynthesis • Animals lack enzymes to synthesize folate; required in diet • Sulfonamides, trimethoprim inhibit different steps in synthesis • Access the text alternative for slide images. 69 Interfere with Metabolic Pathways Sulfonamides and related: called sulfa drugs • Inhibit many Gram-positives and Gram-negatives • Structurally similar to PABA, so enzyme binds chemical • Example of competitive inhibition • Human cells lack enzyme Trimethoprim inhibits enzyme in later step • Has little effect on enzyme’s counterpart in human cells • Combination of trimethoprim and sulfonamide has synergistic effect; co-trimoxazole (sulfamethoxazole and trimethoprim). 70 Mechanisms of Action of Antibacterial Medications 2 • Interfere with cell membrane integrity • A few antimicrobials damage bacterial membranes causing cells to leak, leading to cell death • Daptomycin inserts into cytoplasmic membrane • Used against Gram-positives resistant to other antibiotics • Ineffective against Gram-negatives; cannot penetrate outer membrane • Polymyxins bind to membranes of Gramnegatives • Generally limits usefulness to topical applications • Also bind to eukaryotic cells, though to a lesser extent • Newest glycopeptide antibiotics disrupt cell membranes (albavancin, oritavancin) 71 Mechanisms of Action of Antibacterial Medications 3 • Effective against Mycobacterium tuberculosis • Few antimicrobials effective against Mycobacterium • Waxy cell wall prevents entry of many chemicals; slow growth • First-line drugs are most effective, least toxic • Combination therapy decreases chance of development of resistant mutants • Second-line drugs given for strains resistant to first-line drugs • Less effective or have greater toxicity risk • Some target unique cell wall of mycobacteria • Isoniazid inhibits mycolic acid synthesis; ethambutol inhibits enzymes required for synthesis of other cell wall components; pyrazinamide interferes with protein synthesis 72 Antimicrobial Susceptibility Testing • Conventional disc diffusion method • Kirby-Bauer disc diffusion test routinely used to determine susceptibility of bacterial strain to antibiotics • Standard sample of strain uniformly spread on agar plate; discs containing different antibiotics placed on surface • Drugs diffuse outward, establish concentration gradients • Resulting zone of inhibition compared with specially prepared charts to determine whether strain is susceptible, intermediate, or resistant • Source: Gilda L. Jones/CDC 73 Resistance to Antimicrobial Medications • Increasing use, misuse selects for resistant microorganisms • Only 3% of Staphylococcus aureus originally resistant to penicillin G; now more than 90% are resistant • Antimicrobial resistance alarming • Impact on cost, complications, and outcomes of treatment • Dealing with problem requires understanding of mechanisms and spread of resistance 74 Resistance to Antimicrobial Medications • Mechanisms of acquired resistance • Medication-inactivating enzymes • Bacteria produce enzymes that interfere with drug • Penicillinase, extended spectrum βlactamases • Alteration in target molecule • Minor structural changes can prevent binding • PBPs (β-lactam antibiotics), ribosomal RNA (macrolides, lincosamides, streptogramins) • Access the text alternative for slide images. 75 Resistance to Antimicrobial Medications • Mechanisms of acquired resistance • Decreased uptake of the medication • Changes in porin proteins of outer membrane of Gram-negatives • Increased elimination of medication • Efflux pumps remove compounds from cell • Increased production or structural changes of pumps allows faster removal • Resistance to range of antimicrobials 76 Acquisition of Resistance 1 Spontaneous mutations • Mutations happen at low rate during replication, but can have significant effect • Just a single base-pair change in gene encoding a ribosomal protein yields resistance to streptomycin • In a population of 109 cells, at least one likely has that mutation; if streptomycin is added, only that cell and progeny will replicate, yielding resistant population • Spontaneous resistance to antibiotics with several different targets or multiple binding sites is less likely • Combination therapy of multiple antibiotics is often used; unlikely cells will simultaneously develop resistance 77 Acquisition of Resistance 2 Gene transfer • Genes encoding resistance can spread to different strains, species, even genera • Most commonly through conjugative transfer of R plasmids, which often carry several different resistance genes • Resistance genes on R plasmids originate from • Spontaneous mutations • Microbes that naturally produce the antibiotic • Gene coding for enzyme that modifies aminoglycoside likely originated from the Streptomyces species that produces the antibiotic 78 Examples of Emerging Resistance 1 Enterococci: part of normal intestinal microbiota • Common cause of healthcare-associated infections • Intrinsically less susceptible to many antimicrobials • PBPs have low affinity to many β-lactam antibiotics • Many have R plasmids • Some code for resistance to vancomycin, yield vancomycin-resistant enterococci (VRE), which is transferable to other organisms • Vancomycin often antibiotic of last resort 79 Examples of Emerging Resistance 3 Mycobacterium tuberculosis requires long treatment • Can become resistant to first-line antibiotics via mutation • Large numbers of cells found in active infection, so likely at least one cell has developed resistance • Combination therapy is therefore required • 6 months or more of treatment necessary due to slow rate of growth; many patients do not comply • Multi-drug-resistant tuberculosis (MDR-TB) resist two favored first-line antibiotics: isoniazid and rifampin • Directly observed therapy (DOT) can prevent emergence • Extensively drug-resistant tuberculosis (XDR-TB) of even greater concern, additionally resist three or more second-line anti-TB medications 80 Examples of Emerging Resistance 4 Neisseria gonorrhoeae was once very susceptible to penicillin • Some strains developed resistance through mutation; others acquired a plasmid that encoded production of penicillinase • Other treatments were used, but led to more resistance • Only certain cephalosporins are reliably effective • Newer combination therapy minimizes resistance • Intramuscular dose of ceftriaxone with oral dose of azithromycin 81 Examples of Emerging Resistance 5 Staphylococcus aureus: increasingly resistant • Common cause of healthcare-associated infections • Most strains resistant to penicillin, encode penicillinase • New strains emerged having PBPs with low affinity for most β-lactam antibiotics (ceftaroline binds new PBP) • Methicillin-resistant Staphylococcus aureus (MRSA) • Healthcare-associated (HA-MRSA) resistant to wide range of antibiotics, usually treated with vancomycin • Hospitals have reported resistant isolates; strict guidelines have halted spread of vancomycin-intermediate S. aureus (VISA) and vancomycin-resistant S. aureus (VRSA) strains • Community acquired (CA-MRSA) currently treatable 82 Examples of Emerging Resistance 6 Streptococcus pneumoniae • Historically susceptible; some recently acquired penicillin resistance • Produce PBPs with lower affinity, likely via DNAmediated transformation involving other Streptococcus species 83 Resistance to Antimicrobial Medications • Preventing resistance • Will require cooperation from everyone globally • Responsibilities of physicians and healthcare workers • Increase efforts to identify cause of infection • Only prescribe suitable antimicrobials when appropriate 84 Preventing Resistance 1 Responsibilities of patients • Carefully follow instructions even if inconvenient • Essential to maintain adequate blood levels of antibiotic; skipping a dose may reduce levels, allowing less-sensitive microbes a chance to grow and spread • Failure to complete treatment may not kill leastsensitive organisms, allowing subsequent spread The importance of an educated public • Antibiotics ineffective against viruses; cannot cure common cold! • Misuse selects for antibiotic-resistant bacteria in normal microbiota; they can eventually transfer R plasmids to pathogens 85 Preventing Resistance 2 Global impacts of the use of antimicrobial medications • Overuse is a worldwide concern • Antimicrobial antibiotics available without prescription in many parts of the world, may allow improper use • Antimicrobial antibiotics used in animal feeds at low levels to enhance growth; selects for antibioticresistant microbes • Resistant Salmonella strains linked to animals 86 Mechanisms of Action of Antiviral Medications 1 • Viruses difficult to target selectively • Rely on host cell’s metabolic machinery; lack cell walls, ribosomes, other structures targeted by antibiotics • Many viruses encode polymerases; potential targets of antiviral medications (antivirals) • Each is only effective against a specific type of virus • Greatest variety are directed at HIV • Antivirals targeting SARS-CoV-2 (causes COVID-19) in development • Antivirals are only effective against replicating viruses • Herpes and HIV infections remain latent in cells, so cannot be cured • For viruses such as HIV and HCV (hepatitis C virus) that evolve rapidly to develop resistance to single medications, combination therapy is used 87 Mechanisms of Action of Antiviral Medications 88 Prevent Viral Entry Some HIV medications prevent viral entry • Post-attachment inhibitors – Iblizumab a monoclonal antibody (mAb). •Binds to HIV receptor CD4 and prevents HIV particle from undergoing a change required for the virus to bind to a coreceptor • CCR5 antagonist - Maraviroc (MCV) blocks the HIV co-receptor CCR5 • Fusion inhibitor - Enfuvirtide (ENF) binds to an HIV protein that promotes fusion of the viral envelope with the cell membrane 89 Interfere with Viral Uncoating • Uncoating is the process by which the nucleic acid of a viral particle is released from the protein coat • Two medications—amantadine and rimantadine— interfere with uncoating of influenza A virus • Not currently used due to widespread resistance 90 Interfere with Nucleic Acid Synthesis 1 Many of the most effective antivirals target virally encoded enzymes used during replication of viral nucleic acid • Mostly limited to treating herpesviruses, HBV, HCV, HIV Nucleoside and nucleotide analogs • Structure similar to building blocks of DNA, RNA • When incorporated into growing nucleotide chain, many analogs act as chain terminators • Selective toxicity since virally encoded enzymes more likely to incorporate than host cell polymerases • More damage done to rapidly replicating viral genome 91 Interfere with Nucleic Acid Synthesis 2 • Nucleoside and nucleotide analogs • Acyclovir and others; little harm to uninfected cells since converted by virally encoded enzymes in infected cells • Herpesviruses (chickenpox, cold sores) • Sofosbuvir interferes specifically with HCV’s replicase • Highly effective against hepatitis C when used with another anti-HCV medication • Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) used to treat HBV, HIV • Often used in combination with other anti-retroviral medications to minimize development of resistance • Include zidovudine (anti-HIV), tenofovir (anti-HBV) • Some reserved for severe infections; significant side effects • Ganciclovir used to treat life- or sight-threatening cytomegalovirus (CMV) infections in immunocompromised 92 Interfere with Nucleic Acid Synthesis 3 • Non-nucleoside polymerase inhibitors • Inhibit viral polymerases by binding to site other than nucleotide-binding site • Dasabuvir is component of fixed-dose combination to treat HCV • Foscarnet used to treat resistant herpesviruses • Non-nucleoside reverse transcriptase inhibitors (NNRTIs) • Inhibit reverse transcriptase by binding to site other than nucleotide-binding site (doravirine, efavirenz, etravirine) • NS5A inhibitors • Relatively new option for treating HCV • Inhibit NS5A, HCV-encoded protein required for replication of viral genome (elbasvir, ombitasvir) 93 Mechanisms of Action of Antiviral Medications 2 • Prevent genome integration • Integrase inhibitors interfere with action of HIVencoded enzyme integrase • Prevent virus from inserting DNA copy of genome into host cell • Prevent assembly and release of viral particles • Protease inhibitors • During replication of some viruses, several proteins translated as a polyprotein that must be cleaved by a protease • Virus-specific • Include atazanavir (anti-HIV) and grazoprevir (anti-HCV) • Neuraminidase inhibitors • Enzyme encoded by influenza viruses, needed for release • Several available, ingested, inhaled, or injected 94 Mechanisms of Action of Antifungal Medications • Eukaryotic pathogens difficult to target • More closely resemble human cells than bacteria • Few targets for antifungals • Acquired resistance is a significant concern • Antifungal medications can interfere with • Fungal cytoplasmic membrane • Cell wall synthesis • Cell division • Nucleic acid synthesis • Protein synthesis 95 Mechanisms of Action of Antifungal Medications 1 • Interfere with cytoplasmic membrane synthesis and function • Most antifungal chemicals target ergosterol, which humans lack • Azoles inhibit ergosterol synthesis, membrane leaks • Newer less toxic triazoles used to treat systemic infections and nail infections • C. auris strains are resistant to at least one triazole; healthcare professionals are concerned about its spread • Polyenes produced by Streptomyces, bind to ergosterol, cause membrane to leak • Allylamines, tolnaftate, butenafine • Inhibit enzyme in ergosterol synthesis pathway • Most applied to skin for dermatophyte infections 96 Mechanisms of Action of Antifungal Medications 2 • Interfere with cell wall synthesis • Fungal cell walls have some components animals lack • Echinocandins interfere with synthesis of β-1, 3 glucan • Causes cells to burst • Caspofungin treats systemic Candida, invasive aspergillosis • Some C. auris strains are resistant • Interfere with cell division • Griseofulvin targets cell division, interferes with tubulin • Tubulin found in eukaryotic cells; selective toxicity may be due to greater uptake by fungal cells • Treats skin and nail infections 97 Mechanisms of Action of Antifungal Medications 3 • Interfere with nucleic acid synthesis • Common to all eukaryotes, generally poor chemical target • Flucytosine taken up by yeast cells, converted to active form, inhibits nucleic acid synthesis • Significant side effects • Interfere with protein synthesis • New antifungal inhibits protein synthesis by preventing enzyme to add amino acid to tRNA (tavaborole) • Used topically to treat nail infections 98 Mechanisms of Action of Antiprotozoan and Antihelminthic Medications • Relatively little research and development • Most parasitic diseases concentrated in poorer areas of the world; medications unaffordable • Most chemicals interfere with biosynthetic pathways of protozoan parasites or neuromuscular function of worms 99