Chapter 12. Drugs, Microbes, Host. The Elements of Chemotherapy
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1 Chapter 12 Drugs, Microbes, Host The Elements of Chemotherapy
2 Principles of antimicrobial therapy Administer a drug to an infected person that destroys the infective agent without harming the host s cells Antibiotic originally referred to any compound produced by one microbial species that could kill or inhibit the growth of other microbes. Today antibiotic is also used for synthetic chemotherapeutic agents, such as sulfonamides. Many natural and synthetic compounds affect microbial growth, but their utility in a clinical setting is dictated by certain key characteristics.
3 The ideal antimicrobial drug
4 Terminology
5 The golden age of antibiotic discovery The modern antibiotic revolution began in 1928 with the discovery of penicillin by Alexander Fleming. A contaminating mold had inhibited the growth of Staphylococcus aureus colonies on a plate. The mold was identified as Penicillium notatum. Penicillin was purified in the early 1940s by Howard Florey and Ernst Chain. Has saved millions of lives since!
6 4 days 9 days
7 Selected microbial sources of antibiotics Antibiotics are common metabolic products of aerobic bacteria and fungi. By inhibiting the other microbes in the same habitat, antibiotic producers have less competition for nutrients and space
8 Classification of Antibiotics Bactericidal antibiotics kill target organisms. Many drugs only affect growing cells. Inhibitors of cell wall synthesis Only effective if organism is building new cell wall Example: penicillin Bacteriostatic antibiotics prevent growth of organisms. Cannot kill organism Immune system removes intruding microbe
9 Interactions between drug and microbe Antimicrobial drugs should be selectively toxic drugs should kill or inhibit microbial cells without simultaneously damaging host tissues As the characteristics of the infectious agent become more similar to the vertebrate host cell, complete selective toxicity becomes more difficult to achieve and more side effects are seen
10 Spectrum (range of activity) of an antimicrobial drug Narrow-spectrum effective on a small range of microbes Target a specific cell component that is found only in certain microbes Broad-spectrum greatest range of activity Target cell components common to most pathogens (ribosomes) Synergistic effect the effects of a combination of antibiotics are greater than the sum of the effects of the individual antibiotics
11 Mechanisms of drug action The following aspects of a microbe s physiology are classic targets: Cell wall Cell membrane DNA synthesis RNA synthesis Protein synthesis Metabolism
12 Also look at Table 12.4
13 Antimicrobial drugs that affect the bacterial cell wall Most bacterial cell walls contain peptidoglycan Penicillins and cephalosporins block synthesis of peptidoglycan, causing the cell wall to lyse Active on young, growing cells Penicillins that do not penetrate the outer membrane and are less effective against Gram-negative bacteria Broad spectrum penicillins and cephalosporins can cross the cell walls of Gram-negative bacteria
14 Antimicrobial drugs that disrupt cell membrane function A cell with a damaged membrane dies from disruption in metabolism or lysis Polymyxin interact with phospholipids and cause leakage, particularly in Gram-negative bacteria Polymyxin produced by Bacillus polymyxa Polymyxin only used topically Amphotericin B and nystatin form complexes with sterols on fungal membranes which causes leakage
15 Drugs that affect nucleic acid synthesis May block synthesis of nucleotides, inhibit replication, or stop transcription Chloroquine binds and cross-links the double helix; quinolones inhibit DNA helicases Antiviral drugs that are analogs of purines and pyrimidines insert in viral nucleic acid, preventing replication
16 Drugs that affect metabolic pathways Sulfonamides and trimethoprim block enzymes required for tetrahydrofolate synthesis needed for DNA and RNA synthesis Competitive inhibition drug competes with normal substrate for enzyme s active site
17 Drugs that affect metabolic pathways
18 Antibiotics that inhibit transcription are bactericidal and most active against growing bacteria Rifampin RNA synthesis (transcription) inhibitors Binds to the beta subunit of RNA polymerase Prevents the elongation step of transcription Actinomycin D Prevents the initiation step of transcription Binds to DNA from any source Thus, more toxic
19 Drugs that block protein synthesis Ribosomes of eukaryotes differ in size and structure from prokaryotes. Antibiotics usually have a selective action against prokaryotes.
20 Protein synthesis (translation) inhibitors Drugs that affect the 30S subunit Aminoglycosides cause the translational misreading of mrna Are bactericidal Include streptomycin and gentamycin Tetracyclines: block the binding of charged trnas to the A site of the ribosome Are bacteriostatic Include doxycycline Drugs that affect the 50S subunit Chloramphenicol: inhibit peptidyl transferase
21 Antiviral chemotherapeutic agents Selective toxicity is almost impossible due to obligate intracellular parasitic nature of viruses Block penetration into host cell Block replication, transcription, or translation of viral genetic material Nucleotide analogs Acyclovir herpesviruses Ribavirin a guanine analog RSV, hemorrhagic fevers AZT thymine analog HIV Prevent maturation of viral particles Protease inhibitors HIV
22 Drugs for treating influenza Amantadine, rimantidine restricted almost exclusively to influenza A viral infections; prevent fusion of virus with cell membrane Relenza and tamiflu slightly broader spectrum; blocks neuraminidase in influenza A and B
23 Antiherpes Drugs Many antiviral agents mimic the structure of nucleotides and compete for sites on replicating DNA Acyclovir (Zovirax), Valacyclovir (Valtrex), Famiciclovir (Famvir), Peniciclovir (Denavir) Oral and topical treatments for oral and genital herpes, chickenpox, and shingles
24 Drugs for treating HIV infection Reverse transcriptase inhibitors The reverse transcriptase enzyme is unique to retroviruses. Zidovudine (ZDV or AZT) is an analog of thymine. Protease inhibitors HIV makes long, nonfunctional polypeptide chains that are cleaved by HIV protease to make the mature proteins. Viracept and Lopinavir block the HIV protease. Entry inhibitors CCR5 inhibitors block HIV from binding to receptor. Fusion inhibitors prevent HIV membrane fusing with T-cell membrane.
25 Interferons (INF) Human-based glycoprotein produced primarily by fibroblasts and leukocytes Therapeutic benefits include: Reduces healing time and some complications of infections Prevents or reduces symptoms of cold and papillomavirus Slows the progress of certain cancers, leukemias, and lymphomas Treatment of hepatitis C, genital warts, Kaposi s sarcoma
26 Agents to treat fungal infections Fungal cells are eukaryotic; a drug that is toxic to fungal cells also toxic to human cells Superficial mycoses: treated topically Deep mycoses: treated systemically Five antifungal drug groups: 1) Macrolide polyene Amphotericin B mimic lipids, most versatile and effective, topical and systemic treatments Nystatin topical treatment
27 Agents to treat fungal infections Five antifungal drug groups: 2) Griseofulvin stubborn cases of dermatophyte infections, nephrotoxic 3) Synthetic azoles broad-spectrum; ketoconazole, clotrimazole, miconazole 4) Flucytosine analog of cytosine; cutaneous mycoses or in combination with amphotericin B for systemic mycoses 5) Echinocandins damage cell walls; capsofungin
28 Antiparasitic chemotherapy Antimalarial drugs quinine, chloroquinine, primaquine, mefloquine Antiprotozoan drugs metronidazole (Flagyl), quinicrine, sulfonamides, tetracyclines Antihelminthic drugs immobilize, disintegrate, or inhibit metabolism Mebendazole, thiabendazole broad-spectrum inhibit function of microtubules, interferes with glucose utilization and disables them Pyrantel, piperazine paralyze muscles Niclosamide destroys scolex
29 Subgroups and uses of Penicillins Penicillins G and V most important natural forms Penicillin is the drug of choice for Gram-positive cocci (streptococci) and some Gram-negative bacteria (meningococci and syphilis spirochete) Semisynthetic penicillins ampicillin, carbenicillin, and amoxicillin have broader spectra Gram-negative infections Penicillinase-resistant methicillin, nafcillin, cloxacillin Primary problems allergies and resistant strains of bacteria
30 Characteristics of selected Penicillins
31 Cephalosporins Account for one-third of all antibiotics administered Synthetically altered beta-lactam structure Relatively broad-spectrum, resistant to most penicillinases, and cause fewer allergic reactions Some are given orally; many must be administered parenterally Generic names have root cef, ceph, or kef
32 Cephalosporins 4 generations exist: each group more effective against Gram-negatives than the one before with improved dosing schedule and fewer side effects First generation cephalothin, cefazolin most effective against Gram-positive cocci and few Gram-negative Second generation cefaclor, cefonacid more effective against Gram-negative bacteria Third generation cephalexin, ceftriaxone broad-spectrum activity against enteric bacteria with beta-lactamases Fourth generation cefepime widest range; both Gram- negative and Gram-positive
33 Additional Beta-lactam drugs Carbapenems Imipenem broad-spectrum drug for infections with aerobic and anaerobic pathogens; low dose, administered orally with few side effects Monobactams Aztreonam narrow-spectrum drug for infections by Gramnegative aerobic bacilli; may be used by people allergic to penicillin
34 Non Beta-lactam cell wall inhibitors Vancomycin narrow-spectrum, most effective in treatment of Staphylococcal infections in cases of penicillin and methicillin resistance or if patient is allergic to penicillin; toxic and hard to administer; restricted use Bacitracin narrow-spectrum produced by a strain of Bacillus subtilis; used topically in ointment Isoniazid (INH) works by interfering with mycolic acid synthesis; used to treat infections with Mycobacterium tuberculosis
35 Antibiotics that damage bacterial cell membranes Polymixins, narrow-spectrum peptide antibiotics with a unique fatty acid component Treat drug resistant Pseudomonas aeruginosa and severe UTI
36 Drugs that act on DNA or RNA Fluoroquinolones work by binding to DNA gyrase and topoisomerase IV Broad spectrum effectiveness Concerns have arisen regarding the overuse of quinoline drugs CDC is recommending careful monitoring of their use to prevent ciprofloxacin-resistant bacteria
37 Drugs that interfere with protein synthesis Aminoglycosides Products of various species of soil actinomycetes in genera Streptomyces and Micromonospora Broad-spectrum, inhibit protein synthesis, especially useful against aerobic gram-negative rods and certain gram-positive bacteria Streptomycin bubonic plague, tularemia, TB Gentamicin less toxic, used against Gram-negative rods Newer tobramycin and amikacin against Gram-negative bacteria
38 Tetracycline antibiotics Broad-spectrum, block protein synthesis by binding ribosomes Treatment for STDs, Rocky Mountain spotted fever, Lyme disease, typhus, acne, and protozoa Generic tetracycline is low in cost but limited by its side effects
39 Chloramphenicol Potent broad-spectrum drug with unique nitrobenzene structure Blocks peptide bond formation and protein synthesis Entirely synthesized through chemical processes Very toxic, restricted uses, can cause irreversible damage to bone marrow Typhoid fever, brain abscesses, rickettsial, and chlamydial infections
40 Macrolides and related antibiotics Erythromycin large lactone ring with sugars; attaches to ribosomal 50s subunit Broad-spectrum, fairly low toxicity Taken orally for Mycoplasma pneumoniae, legionellosis, Chlamydia, pertussis, diphtheria and as a prophylactic prior to intestinal surgery For penicillin-resistant gonococci, syphilis, acne Newer semi-synthetic macrolides clarithromycin, azithromycin
41 Drugs that block metabolic pathways Most are synthetic; most important are sulfonamides, or sulfa drugs - first antimicrobic drugs Narrow-spectrum; block the synthesis of folic acid by bacteria Sulfisoxazole shigellosis, UTI, protozoan infections Silver sulfadiazine burns, eye infections Trimethoprim given in combination with sulfamethoxazole UTI, PCP
42 Newly developed classes of antimicrobials Formulated from pre-existing drug classes Three new drug types: Fosfomycin trimethamine a phosphoric acid effective as alternate treatment for UTIs; inhibits cell wall synthesis Synercid effective against Staphylococcus and Enterococcus that cause endocarditis and surgical infections; used when bacteria is resistant to other drugs; inhibits protein synthesis Daptomycin directed mainly against gram-positive; disrupts membrane function
43 Newly developed classes of antimicrobials Ketolides telitromycin (Ketek), new drug with different ring structure from Erythromycin; used for infection when resistant to macrolides Oxazolidinones linezolid (Zyvox); synthetic antimicrobial that blocks the interaction of mrna and ribosome Used to treat methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Enterococcus (VRE)
44 Challenges of drug resistance Antibiotics are considered secondary metabolites because they often have no apparent primary use in the producing organism. Not essential for survival But enhance ability to survive competition Microbes prevent self-destruction by means of various antibiotic resistance mechanisms. Example: make enzymes to disable antibiotics Genes encoding some of these drug-resistance mechanisms have been transferred to pathogens.
45 Antibiotic resistance is a growing problem worldwide Antibiotics are overused Overprescribed; used in farm animal feed This exerts selective pressure for drugresistant strains Streptococcus pneumoniae Resistant to multiple drugs
46 There are four basic forms of antibiotic resistance
47 Antibiotic resistance mechanisms 1) Modify the target so that it no longer binds the antibiotic. Mutations in ribosomal proteins confer resistance to streptomycin. 2) Destroy the antibiotic before it gets into cell. - The beta-lactamase enzyme specifically destroys penicillins.
48 Antibiotic resistance mechanisms 3) Add modifying groups that inactivate the antibiotic. Three classes of enzymes are used to modify and inactivate the aminoglycoside antibiotics. 4) Pump the antibiotic out of the cell. Specific and nonspecific transport proteins
49 Mechanisms of drug resistance
50 How does drug resistance develop? 1) De novo antibiotic resistance develops through spontaneous mutations. 2) Can be acquired via horizontal gene transfer: DNA fragment Plasmid donor Plasmid Conjugation Resistance gene Transduction Transformation Transfer of free DNA Conjugation Plasmid transfer Transformation Dead bacterium Gene goes to plasmid or to chromosome Virus 3) Transposons Resistance gene Bacterium Receiving Resistance genes Transduction Transfer by viral delivery
51 Natural selection and drug resistance Large populations of microbes likely to include drug resistant cells due to prior mutations or transfer of plasmids no growth advantage until exposed to drug If exposed, sensitive cells are inhibited or destroyed while resistance cells will survive and proliferate. Eventually population will be resistant natural selection Non-drug-resistant cell Cells dying Drug-resistant cell Exposure To drug Continued Exposure To drug (a) Microbial cells involve in an infection (b) Most sensitive cells are eliminated by drug early :resistant cells survive and row (late)row (late) (c) Most new cells are resistant, infection will no longer respond to drug
52 Strategies to limit drug resistance
53 Interactions between drug and host Estimate that 5% of all persons taking antimicrobials will experience a serious adverse reaction to the drug side effects Major side effects: Direct damage to tissue due to toxicity of drug Allergic reactions Disruption in the balance of normal flora- superinfections possible
54 Infection Superinfections Drug Superinfection Circulating drug (a) (b) (c) Intestine Intestine Intestine Potential pathogen resistant to drug but held in check by other microbes Drug destroys beneficial flora Pathogen overgrows Normal flora important to maintain intestinal balance
55 Major adverse reactions to common drugs
56 Considerations in selecting an antimicrobial drug Identify the microorganism causing the infection Test the microorganism s susceptibility (sensitivity) to various drugs in vitro when indicated The overall medical condition of the patient
57 Identifying the agent Identification of infectious agent should be attempted as soon as possible Specimens should be taken before antimicrobials are initiated
58 Measuring drug susceptibility One critical decision a clinician must make when treating an infection is which antibiotic to prescribe for the patient. There are several factors to consider, including: The relative effectiveness of different antibiotics on the organism causing the infection The average attainable tissue levels of each drug
59 Minimal Inhibitory Concentration (MIC) The MIC is the lowest concentration that prevents growth Varies for different bacterial species Test by diluting antibiotic - Lowest concentration with no growth: MIC
60 MIC strip test (E-test) The time required to evaluate antibiotic effectiveness can be reduced by using a strip test that avoids the need for dilutions. The MIC is the point at which the elliptical zone of inhibition intersects with the strip.
61 Kirby-Bauer disk susceptibility test Clinical labs can receive up to 100 or more isolates in one day, so individual MIC determinations are impractical. The Kirby-Bauer assay tests strain sensitivity to multiple antibiotics. Uses a series of round filter paper disks impregnated with different antibiotics. A dispenser delivers up to 12 disks simultaneously to the surface of an agar plate covered by a bacterial lawn. During incubation, the drugs diffuse away from the disks into the surrounding agar and inhibit growth of the lawn. Size of cleared zones reflects relative sensitivity
62
63 Standardization of Kirby-Bauer test To make the test reproducible and easier: Size of the agar plate: 150 mm Media composition: Mueller-Hinton agar The number of organisms spread on the agar plate Size of the disks: 6 mm Concentrations of antibiotics in the disks Incubation temperature: 37 o C
64 Comparing MICs for common drugs and pathogens Minimum inhibitory concentration (MIC) smallest concentration of drug that visibly inhibits growth
65 The MIC and Therapeutic Index In vitro activity of a drug is not always correlated with in vivo effect If therapy fails, a different drug, combination of drugs, or different administration must be considered Best to chose a drug with highest level of selectivity but lowest level toxicity measured by therapeutic index Therapeutic index: the ratio of the dose of the drug that is toxic to humans as compared to its minimum effective dose High index is desirable Toxic dose= 200 ug/ml TI= 2 MIC = 100 ug/ml Toxic dose= 10 ug/ml TI= 0.1 MIC = 100 ug/ml
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