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Mechanisms of Antimicrobial Action and Resistance Alan L. Goldin, M.D./Ph.D. Sections in Medical Microbiology & Immunology Chapter 10 Mechanisms Pages of action 69-84 Chapter 11 Resistance Pages 85-93 Useful reference, but recommendations change about drugs of choice Information on Antibiotics The Medical Letter Bi-weekly publication Independent evaluation of new drugs 100 Main Street, New Rochelle, NY 10801 (800) 211-2769 http://www.medletter.com/ Choice of Antibacterial Drugs (annual issue) http://medlet-best.securesites.com/restrictedtg/t57.pdf Handbook of Antimicrobial Therapy Every other year (small handbook) Mechanisms of Action Antibacterial drugs can be classified in many ways – mechanism of action will be used in these lectures Biochemical mechanism of action is crucial to understanding the mechanism of selective toxicity Mechanisms of Action Antimetabolites (sulfonamides) Affect nucleic acids (quinolones, rifampin) Inhibit cell wall synthesis (penicillin) Act on ribosomes - Reversible (tetracycline, chloramphenicol) - Irreversible (aminoglycosides) Disrupt cell walls (nystatin, polymyxin) Pharmacology Route of administration (iv, oral) Route of elimination (kidney, liver) Half-life, which is affected by diseases (liver or kidney disease) and other drugs Interactions with other drugs Dosing schedule, particularly compliance Side effects and idiosyncratic responses Resistance The most important problem in therapeutic use of antibacterial drugs Biochemical mechanisms of resistance Genetics Societal and physician behaviors Approaches to retard the development of resistance Definitions Antimicrobial Inhibits Antibacterial Inhibits growth of micro-organisms growth of bacteria Antibiotic Inhibits growth of micro-organisms Made by other micro-organisms Usually extended to include synthetic drugs Bacteriostatic versus Bactericidal Bacteriostatic Reversible inhibition of growth When the antibiotic is removed, almost all of the bacteria can replicate Bactericidal Irreversible inhibition of growth When the antibiotic is removed, almost none of the bacteria (10-7 to 10-3) can replicate Minimal Inhibitory Concentration MIC Lowest concentration of antibiotic that prevents visible growth Broth or tube dilution method Serial 2-fold dilutions of the antibiotic Accurate but time-consuming Disk sensitivity test Rapid, but must be related to results from the tube dilution method MIC Tube Dilution Method for Determination of MIC 128 64 32 16 8 4 2 μg Antibiotic per ml 1 0.5 Disk Sensitivity Test 0 Time Disk Sensitivity Test 24 Hours Zone of Inhibition (mm in diameter) Correlation of Distance from Disk and Antibiotic Concentration Concentration (μg per ml) 128 64 32 16 8 4 2 1 0.5 Tetracycline Amikacin 4 8 12 16 20 24 28 32 Distance from Disk (mm) Minimal Bactericidal Concentration MBC Lowest concentration of antibiotic that reduces the number of viable cells by at least 1000-fold Performed in conjunction with MIC by the tube dilution method Aliquots from the tubes at and above the MIC are plated onto agar media The antibiotic is diluted, so that the remaining viable cells grow and form colonies The MBC of a truly bactericidal agent is equal to or just slightly above its MIC Tube Dilution Method for Determination of MBC 128 64 32 16 8 4 2 μg Antibiotic per ml 1 0.5 MIC Tube Dilution Method for Determination of MBC 128 64 32 16 8 4 2 μg Antibiotic per ml 1 0.5 MIC Tube Dilution Method for Determination of MBC 128 64 32 16 8 4 2 μg Antibiotic per ml 1 0.5 64 32 MIC 128 MBC Tube Dilution Method for Determination of MBC 16 8 4 2 μg Antibiotic per ml 1 0.5 Attainable Level of Antibiotic Concentration that can be reached in the target tissue without toxic side effects If the attainable level of an antibiotic is greater than the MIC for at least 90% of the isolates, that species is considered susceptible to that antibiotic For serious infections, those odds may provide inadequate guidance for treatment Trough Levels of Antibiotics Levels of antibiotics reach minimal levels (troughs) at roughly predictable times after administration The troughs may be at or below the MIC This may or may not be a problem because of two mitigating factors Post Antibiotic Effect, a prolonged period before bacteria resume growth Synergism between host defenses and subMIC levels of antibiotics Trough Levels of Antibiotics Trough levels may increase the frequency of drug-resistant bacteria Frequency of developing resistance is greatly increased at levels just above the MIC Development of resistance to ciprofloxacin is 10,000 times more frequent at 2 times the MIC compared to 8 times the MIC Choice of Drugs Starts with Susceptibility Susceptibility by itself does not assure therapeutic success Lack of susceptibility guarantees therapeutic failure There are many other considerations in the choice of antibacterial drugs Toxicity and side-effects Interactions with other drugs Pharmacology of the drug Antimetabolites Sulfonamides Prontosil NH2 O H2N N N S NH2 - O A red dye that cured streptococcal and staphylococcal infections in mice (1933) Ineffective against bacteria in laboratory media Confirmed the dogma that clinically effective treatment could not be achieved with drugs acting directly on bacteria The first Sulfonamide Sulfanilamide O H2N S NH2 O The active component of Prontosil A product of cleavage at the diazo bond, which occurs naturally in the body Effective against bacteria in both patients and laboratory media Sulfonamides and PABA Are Analogs O H2N S O NHR H2N C O- O- Sulfonamides PABA Sulfonamide antagonizes para-Aminobenzoic acid Competition for uptake by bacteria PABA is 1,000-fold more effective Small amounts of PABA negate large amount of sulfonamides This competition is not a clinical problem, because we don’t get PABA in out diets, and it is rapidly excreted Sulfonamides and PABA Are Analogs O H2N S O NHR H2N C O- O- Sulfonamides PABA Sulfonamides competitively inhibit the condensation of PABA with dihydropteridine to form dihydropteroic acid This is the first step in the biosynthesis of tetrahydrofolic acid Metabolic competition is roughly equivalent Site of Action of Sulfonamides Dihydropteridine + para-Aminobenzoic acid (PABA) SULFONAMIDES INHIBIT Dihydropteroic acid + Glutamic acid Dihydrofolic acid (DHF) NADPH NADP Tetrahydrofolic acid (THF) Selective Toxicity of Sulfonamides We lack dihydropteroic acid synthase We require folic acid in our diet Bacteria must synthesize folic acid using dihydropteroic acid synthase They cannot use an external source Sulfonamides are still effective even when folic acid is present Consequences of Inhibition by Sulfonamides Sulfonamide block Tetrahydrofolic acid deficit Tetrahydrofolic acid cofactor deficits Thymidine Purines Methionine DNA DNA RNA Protein Effect of Sulfonamides Depends on the Environment Bactericidal in blood and urine Blood and urine have large amounts of methionine and purines, so protein and RNA synthesis continue Selectively blocking DNA synthesis is lethal Bacteriostatic if protein and RNA synthesis are also blocked Adding a bacteriostatic antibiotic decreases efficacy Ineffective in purulent lesions Rich in methionine, purines & thymidine from cells that have lysed, so synthesis of proteins, RNA and DNA can continue Sulfonamides Introduced the Problem of Drug Resistance Development of sulfonamide resistance was rapid Sulfonamides were introduced to treat bacillary dysentery during World War II 4 years later, most isolates were resistant About 10% were resistant to 3 biochemically unrelated antibiotics This pattern has been repeated with each new drug Resistance to multiple drugs is more common than to a single drug R factors, transposons, and integrons Dynamics of Drug Resistance People who receive an antibiotic are more likely to harbor bacteria resistant to that antibiotic and biochemically unrelated antibiotics People who frequent environments in which antibiotics are used are more likely to harbor drug-resistant bacteria, even if they have not received antibiotics. This applies to patients as well as to staff. The probability of harboring drug-resistant bacteria returns to normal within a few weeks after antibiotic therapy is discontinued or after absence from the antibiotic-rich environments The prevalence of drug-resistant bacteria in the community is increasing due to increasing use of antibiotics in the environment Antibiotics, use them and lose them Resistance to Sulfonamides Reduced uptake (Antiporter) Transposons Altered dihydropteroic acid synthase Reduced sensitivity to sulfonamides Transposons & plasmids Increased & plasmids levels of synthase or synthase activity Mutation or plasmid Increased synthesis of PABA (rare) Mutation Loss of end-product inhibition Promoter up mutation Impact of Sulfonamide Discovery Shattered vitalist dogma on treatment of infection Proved in vitro effects are relevant Initiated successful searches for antibiotics Penicillin and streptomycin Launched huge search for metabolic analogs Produced thousands of rat poisons A few anticancer agents An immunsuppressant One antibacterial drug (Trimethoprim) Trimethoprim Competitive inhibitor of dihydrofolic acid reductase The competitive substrate is dihydrofolic acid Trimethoprim blocks a step in the biosynthesis of tetrahydrofolic acid Site of Action of Trimethoprim Dihydropteridine + PABA Sulfonamides Inhibit Dihydropteroic acid + glutamic acid dTMP Dihydrofolic acid Trimethoprim Inhibits NADPH NADP Tetrahydrofolic acid (THF) dUMP 5,10-methylene THF 5-methyl THF methionine & purines Site of Action of Trimethoprim Dihydropteridine + PABA Sulfonamides Inhibit Dihydropteroic acid + glutamic acid Dihydrofolic acid dTMP Trimethoprim NADPH Inhibits NADP Trimethoprim acts rapidly, sulonamides act slowly With trimethoprin, dUMP ⇒ dTMP rapidly depletes THF by conversion to DHF, and there is no DHF ⇒ THF With sulfonamides, there is no net synthesis of THF, but DHF ⇒ THF proceeds Tetrahydrofolic acid (THF) dUMP 5,10-methylene THF 5-methyl THF Depletion of THF pool takes 3-4 generations Synthesis of pyrimidines & purines does not deplete THF methionine & purines Site of Action of Trimethoprim Dihydropteridine + PABA Sulfonamides Inhibit Bactericidal in blood Ineffective in purulent lesions Dihydropteroic acid + glutamic acid Dihydrofolic acid dTMP Trimethoprim NADPH Inhibits NADP But trimethoprim is not antagonized by PABA Trimethoprim and sulfonamides are synergistic Inhibitors of sequential steps are often synergistic 5,10-methylene H4F Sulfonamides reduce DHF 5-methyl H4F which competes with methionine & purines trimethoprim Tetrahydrofolic acid (H4F) dUMP Trimethoprim is like sulfonamides Trimethoprim and Sulfonamides are Synergistic Sulfamethoxazole inhibits an early step in the pathway and lowers the concentration of dihydrofolic acid Dihydrofolic acid and trimethoprim compete for binding to dihydrofolic acid dehydrogenase Less trimethoprim is required for inhibition of dihydrofolic acid reductase in the presence of sulfamethoxazole Trimethoprim and Sulfonamides are Synergistic The synergism permits use of smaller doses than if either drug were used alone The use of two drugs together reduces the frequency of resistance The two drugs are marketed as a combination in the fixed ratio of 5 parts sulfamethoxazole to 1 part trimethoprim There are only a few indications for the use of either drug alone Selectivity of Trimethoprim Both bacteria and humans have dihydrofolate reductase The human enzyme is 60,000-fold less sensitive to trimethoprim There is no toxicity due to the antibacterial action of trimethoprim Folic acid deficiency can occur in patients with inadequate dietary consumption Normal bacterial flora can no longer make folic acid to compensate Resistance to Trimethoprim Dihydrofolate reductases with decreased sensitivity to trimethoprim Reduced affinity for trimethoprim Located in the intervening sequences of transposons On a plasmid, but may transpose to the chromosome It is not a mutant form of the bacterial enzyme, but a new gene Mutation of bacterial dihydrofolate reductase is only important in the lab Resistance to TMP/Sulfa Resistance to TMP makes the combination ineffective Resistance to Sulfonamide maintains considerable potency Drugs to Remember TMP/Sulfonamide Combination Trade name Bactrim Drugs that Affect Nucleic Acid Synthesis Quinolones Quinolones Nalidixic was the first quinolone Too toxic for systemic use (newer quinolones can be used systemically) Rapidly excreted in the urine Effectively used to treat urinary tract infections Inhibits the A subunit of DNA gyrase Human analog (topoisomerase II) is several hundred fold less sensitive Rapidly inhibits DNA synthesis Bactericidal unless growth is prevented Quinolones O O F COOH H3C N N N C2H5 Nalidixic Acid COOH N R2N R1 6-FluoroQuinolones Ciprofloxacin R1 = , R2 = H: Norfloxacin R1 = —C2H5 , R2 = H: Ofloxacin R1 = —C 2H5 , R2 = CH3: Resistance to Quinolones Missense mutations in gyrA Missense mutations in a gene for a membrane protein, which reduces the uptake of fluoroquinolones Development of resistance to ciprofloxacin among nosocomial pathogens Between 1989 and 1992, resistance among S. aureus increased 123% By the end of 1992, More than ¼ of all S. aureus strains were resistant to ciprofloxacin Ciprofloxacin resistance was 80% among methicillin resistant S. aureus Resistance to Quinolones Most frequent among important nosocomial pathogens such as S. aureus and P. aeruginosa These species were not highly susceptible to the first fluoroquinolones Resistance developed rapidly because the drugs were used at levels close to the MIC Ciprofloxacin resistant organisms are cross resistant to other fluoroquinolones Plasmid encoded resistance is not a problem A single copy of the sensitive gyrA gene makes the bacteria susceptible Errors in DNA synthesis and repair are lethal Drugs to Remember Ciprofloxacin (Cipro) Levofloxacin Drugs that Inhibit Cell Wall Synthesis Penicillins Cephalosporins Vancomycin Penicillins Penicillin G was the first penicillin in 1942 Advantages compared to sulfonamides Much greater potency Much less toxicity Effective against organisms that were resistant to sulfonamides Effective in wounds and purulent lesions 6-Aminopenicillanic Acid H H S H2N C N O CH3 CH3 H COOH β-lactam ring Thiazolidine ring Peptidoglycan Cross Linking L Ala D Glu m Dap D Ala D Ala D Ala Site of action of penicillins TRANSPEPTIDASE L Ala D Glu m Dap D Ala TRANSPEPTIDASE TRANSPEPTIDASE D Ala D Ala m Dap L Glu L Ala glycan ( N acetyl glucosamine-N acetyl muramic acid)n Peptidoglycan Cross Linking CH3 O CH3 O Ala – Glu –DAP - N H HO O CH3 C C H N H O CH3 C C H C H N H C N H2 NH N C H C - DAP – Glu - Ala glycan ( N acetyl glucosamine-N acetyl muramic acid)n free amino group of DAP (m-diaminopimelic acid) cross link NH2 OH Substrate-Enzyme Intermediate in the Cross Linking Reaction CH 3 O Ala – Glu –DAP - N H C H C CH3 O O N H2 C H C Transpeptidase O = Serine hydroxyl group in active center of transpeptidase OH β-lactam Inactivation of Transpeptidases H H S C C CH3 H2N C C CH3 N C O H COOH + Transpeptidase H H S C C CH3 H2N C C HN C O Serine OH of Transpeptidases O H Transpeptidase CH3 COOH Inactivation of Transpeptidases by β-lactams H H S C C CH3 H2N C C C O O CH3 HN H COOH Transpeptidase Serine OH of Transpeptidases Transpeptidases (Penicillin Binding Proteins) MW PBP Activity Function 91,000 87,000 1a Transpeptidases 1b 66,000 2 Transpeptidase? Maintenance of rod shape 60,000 3 Transpeptidase Peptidoglycan synthesis Septum formation 49,000 4 42,000 40,000 5 6 Peptidoglycan synthesis Cell wall elongation D-alanine Control extent of x links carboxypeptidases Selectivity & Side Effects of β-lactams Selective toxicity targets of β-lactams are uniquely bacterial The corresponding structures do not occur in humans The Side effects The earliest penicillins are exceptionally benign Some of the later derivatives have side effects related to their side chains A nonspecific side effect is superinfection, such as overgrowth of the large intestine with Clostridium difficile (pseudomembranous colitis) Hypersensitivity is a common and serious problem Haptene Formation: Reaction of β-lactams with Serum Proteins O R C H N H C H C S CH 3 C HN O ε amino group of a Lys residue NH CH 3 CO O H Serum protein Resistance to β-lactams Resistance of Staphylococci to penicillin G became a major problem within 10 years Resistance has since appeared in several additional bacterial species Most group A (β hemolytic) Streptococci are still highly sensitive Resistance is due to β-lactamase Resistance to β-lactams Destruction by β-lactamase H H S C C CH3 H2N C C HN C O Serine OH O H β-lactamase + H2O CH3 COOH Penicilloic acid + Free β-lactamase β-lactamases of Staphylococci Primarily penicillinases Inducible & extracellular Inoculum size has large effect on MIC MIC for β-lactamase negative is < 0.5 μg/ml for 10 – 106 cells MIC for β-lactamase positive is < 0.5 μg/ml for 10 – 103 cells MIC for β-lactamase positive Staph is 1250 μg/ml for 106 cells Large initial dose is important (kill before induction) Destruction of penicillin by a few bacteria can protect a sensitive pathogen (secretion of β-lactamase) One of the major limitations of the early penicillins Limitations of Early Penicillins Hypersensitivity by a significant proportion of the population Need to use parenteral routes of administration (no oral administration) Development of resistance among important groups of pathogens Narrow antibacterial spectrum Oral Penicillin Penicillin G is hydrolyzed by acid in the stomach Penicillin V is acid-stable Made by adding phenoxyacetic acid to the medium of the mold producing penicillin Penicillin G is now so inexpensive that it can be used orally by giving a larger dose Natural Penicillins O C C N H2 H CH3 Acid labile CH3 O COOH PENCILLIN G (benzylpenicillin) O O C C N H2 H CH3 Acid stable CH3 O COOH PENICILLIN V (phenoxymethyl penicillin) β-Lactamase Refractory Penicillin Penicillin G is hydrolyzed by β-lactamase Methicillin is refractory to β-lactamase hydrolysis Steric hindrance of the side chain prevents the hydrolysis Penicillin G forces the β-lactamase into its active conformation, so use with methicillin will decrease the effectiveness of methicillin These drugs are made semi-synthetically Preparation of Semisynthetic Penicilins S H2N CH3 N CH3 COOH 6-AMINOPENICILLANIC ACID O + Acid anhydrides or Acid chlorides OCH3 O C O S N N OCH3 O METHICILLIN CH3 CH3 COOH C S N N OC2H5 O NAFCILLIN CH3 CH3 COOH Broad Spectrum Penicillin Penicillin G cannot pass through the outer membrane of gram negative bacteria Ampicillin has a charged amino group that allows it to pass through the outer membrane Ampicillin is also acid-stable These drugs are semi-synthetic Penicillin G and Ampicillin O C H2 C S N H N CH3 Narrow Spectrum CH3 O COOH PENICILLIN G (Benzyl penicillin) O H C C S N H NH2 N O AMPICILLIN CH3 Broad Spectrum CH3 COOH Broad Spectrum β-Lactamase Refractory Penicillin? There are none The large side chains that make methicillin refractory to β-lactamase prevent it from crossing the outer membrane A partial solution is to combine a broad spectrum penicillin with a β-lactamase inhibitor Active Site Directed Inhibitors of β-Lactamases O O N O O S C C H 2O H H COOH C lavulanic A cid N O CH3 CH3 COOH Sulbactam Inhibition of β-Lactamases by Clavulanic Acid O CHCH2OH N O I O O II COOH β-lactamase + β-lactamase CHCH2OH HN O COOH CH2CH2OH HN O COOH β-lactamase Effect of Clavulanic Acid on Ampicillin Resistance Antibiotic MIC (μg per ml) E. coli β-lactamase - E. coli β-lactamase + Ampicillin alone 2 > 2,000 Ampicillin + Clavulanic Acid 2 4 Intrinsic Resistance to β-Lactams Methicillin resistant Staph. aureus (MRSA) Still cannot hydrolyze methicillin Resistant by an intrinsic mechanism Resistance developed rapidly (in 10 years of methicillin use) Resistance is carried on a transposon, frequently with other resistance genes Resistance is easily transmitted to other bacteria Pencillin Binding Proteins (PBP) of Methicillin Susceptible & Resistant S. aureus Susceptible PBP 1 2 3 4 Resistant 2A Genetics of Methicillin Resistance mecA encodes PBP 2A mecA is a fusion gene mecA is on a transposon Transmitted by a plasmid, but stability requires transposition to the chromosome Production of PBP 2A by mecA is essential but not sufficient for methicillin resistance Host (S. aureus) functions are also required Depending on host functions, resistance is often heterogeneous, leading to incorrect sensitivity reports The mecA transposon is an attractant for other resistance genes Drugs to Remember Penicillin Ampicillin Nafcillin Amoxicillin/Clavulanate Combination Augmentin Other β Lactam Antibiotics Cephalosporins Carbapenems Monobactams Cephalosporins About 20 currently in use Tend to be substrates for β-lactamases less frequently than penicillins 1st generation (Cefazolin) Antibacterial spectra & potency 2nd generation (Cefoxitin) like penicillins More potent & better against gram 3rd generation (Cefotaxime) negatives Even more potent & highly effective against gram negatives but at the expense of reduced potency for gram positives 4th generation (Ceftazidime) Enhanced activity against gram negatives without loss of potency for gram positives Core Structures of Penicillins & Cephalosporins H H S H 2N CH3 C N O H CH3 H CO O H 6-Aminopenicillanic Acid H S H 2N R N O COOH 7-Aminocephalosporanic Acid O R = CH2 O HC CH3 Cross Hypersensitivity of Cephalosporins with Penicillins About 2% of population are hypersensitive to cephalosporins About 8% of people who are hypersensitive to penicillins are also hypersensitive to cephalosporins Penicillins versus Cephalosporins Haptene Formation Penicillins + Serum protein Cephalosporins + Serum protein Rare Frequent O R C H N H C O H C S CH3 C HN O NH CH3 COOH Serum protein Penicilloyl protein R C N H if at all H H C C S C HN O R1 NH COOH Serum protein Cephasporyl protein Resistance to Cephalosporins β-lactamases Penicillins only Cephalosporins only Penicillins & Cephalosporins Specificities of β-lactamases are not predictable Some bacteria may have more than one β-lactamase Assumptions about sensitivity can lead to unpleasant surprises Carbapenems versus Penicillin Carbapenems H Penicillins H R1 H CH S R2 N O COOH H atoms are trans C replaces R1 attached directly R1 N H O H S C N CH3 CH3 COOH H atoms are cis S in fused ring R1 attached via amino group Monobactams H R H NH CH 3 N O SO3 _ Drugs to Remember Cephalosporins Cefazolin Cefotaxime Ceftazidime Carbapenems Imipenem Vancomycin Inhibits peptidoglycan synthesis The mechanism is different from that used by penicillin Binds to the D Ala – D Ala substrate Narrow spectrum of action Complex glycopeptide Cannot cross the outer membrane Resistant to β-lactamases Antibiotic of last resort Vancomycin Target (D Ala – D Ala) CH3 O CH3 O Ala – Glu –DAP - N H HO O CH3 C C H N H O CH3 C C H C H N H C N H2 NH N C H C - DAP – Glu - Ala glycan ( N acetyl glucosamine-N acetyl muramic acid)n free amino group of DAP (m-diaminopimelic acid) cross link NH2 OH Vancomycin Resistance A Depsipentapeptide instead of the normal Pentapeptide Pentapeptide L Alanyl - D Glutamyl - m DAP - D Alanyl - D Alanine VanSens Depsipentapeptide L Alanyl - D Glutamyl - m DAP - D Alanyl - D Lactate VanRes VanSens Vancomycin can bind to D Alanyl - D Alanine VanRes Vancomycin cannot bind to D Alanyl - D Lactate Vancomycin Resistance I Synthesis of Pyruvate + NADH D Alanine + D Lactate the Depsipentapeptide vanH vanA D Lactate + NAD D Alanyl - D Lactate L Alanyl - D Glutamyl - m DAP + D Alanyl - D Lactate van? L Alanyl - D Glutamyl - m DAP - D Alanyl - D Lactate (Depsipentapeptide) Vancomycin Resistance II Destruction of Existing Vancomycin Binding Sites D Alanyl - D Alanine vanX D Alanine + D Alanine L Alanyl - D Glutamyl – m DAP - D Alanyl - D Alanine vanY L Alanyl - D Glutamyl – m DAP - D Alanine + D Alanine Drugs to Remember Vancomycin Drugs that Act on Ribosomes Aminoglycosides Chloramphenicol Macrolides Clindamycin Tetracycline Mechanisms of Action Act on subunits of the bacterial ribosome to disrupt translation Aminoglycosides affect the 30 S subunit and are bactericidal The others are bacteriostatic Tetracycline affects the 30 S subunit Chlorampenicol, Macrolides and Clindamycin affect the 50 S subunit Gentamicin (Aminoglycoside) Aminosugar Aminocyclitol R1 CH NH NHR2 O 2 NH2 OH O NH 2 O OH Gentamicin C1 Gentamicin C2 Gentamicin C1a R1 = CH3 R2 = CH3 R1 = CH3 R2 = H R1 = H R2 = H NHCH CH 3 3 OH Aminosugar O Selective Toxicity Inhibits 30 S ribosomal subunit Difference between inhibition of eukaryotic and bacterial ribosomes is not very large Inhibits mitochondrial ribosomes Mammalian cell and mitochondrial membranes are barriers Mechanisms of Resistance Proteins modify and inactivate the compounds Resistance is additive Proteins are encoded on plasmids Resistant This ribosomal proteins occurs very rarely Resistance is very high Kanamycin Sites of Inactivation ACII ACIII ACI AC Types of Inactivation CH2-NH2 NH2 O HO NH2 OH OH O PI CH2OH OH O PII (AC) HO O NH2 OH AD AC (AC) AD P N-Acetyl transferases O-Acetyl transferases O-Adenyl transferases O-Phosphatases Blocked reaction Chloramphenicol I II III O H C H 2O H O O 2N C H C H N H C C H C l2 Chloramphenicol Binds to the 50 S ribosomal subunit Does not inhibit mammalian 80 S subunit Does inhibit mitochondrial 70 S subunit Aplastic anemia is possible 1 in 25,000 to 40,000 administrations Life-threatening Never a drug of first choice Resistance as for aminoglycosides Erythromycin CH3 H3C N HO CH3 O O H3 C CH3 CH3 O HO HO OH H3 C CH3 CH2 H3CO CH3 OH O O CH3 H3C O O CH3 Erythromycin Macrolide antibiotic Does not inhibit mammalian 80 S subunit Does inhibit mitochondrial 70 S subunit Does not cross the mitochondrial membrane Resistance by rRNA methylation Often an alternative for penicillin to treat allergic patients Clindamycin CH3 CH3 N H 3C C C H2 H2 O Cl CH N H CH O HO OH SC H 3 OH Clindamycin Similar spectrum as erythromycin Binds to the 50 S subunit Frequent association with bowel superinfection Pseudomembranous colitis Clostridium difficile infections Used to treat anaerobic infections Tetracylcines OH O OH O OH O NH2 7 6 5 OH N(CH3)2 Bacteriostatic inhibitors with broad spectrum Block the binding of aminoacyl-tRNAs to the A site of the ribosome 30 S subunit Resistance due to efflux and insensitive ribosomes Tetracylcines OH O OH O OH O NH2 7 6 5 OH N(CH3)2 Drug Position Chlortetracycline 6 CH3 ; OH Tetracycline CH3 ; OH Doxycycline Minocycline 5 OH 7 Cl CH3 N(CH3)2 Drugs to Remember Gentamicin Erythromycin Clindamycin Tetracycline Drugs that Disrupt Cell Walls Nystatin Polymyxin L-Leu L-Phe (α) L-Dab (α) L-Dab (α) L-Dab L-Thr (γ) L-Dab (α) L-Thr (α) L-Dab 6-Methyloctanoic POLYMYXIN B1 L-Dab = L-α, γ-Diaminobutyric acid (α) and (γ) indicate NH2 groups L-Dab involved in peptide linkages Polymyxins Too toxic for systemic use Effective against gram negative but not gram positive bacteria Bactericidal, disrupting the outer membrane Used in topical creams and ointments Newer Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic Streptogramins Oxazolidinones Lipopeptides Glycylcylines Ketolides Newer Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptogramins Quinupristin/dalfopristin (Synercid) was approved by the FDA in 1999 Effective against Vancomycin Resistant Staph. aureus (VRSA) and Enterococci (VRE) Oxazolidinones Lipopeptides Glycylcylines Ketolides Streptogramins N O N HN O N O O O O N OH O H N N N O NH O O O O OH N O N O P ristinom ycin Ia Pristinom ycin IIa Quinupristin/Dalfopristin Act synergistically on the bacterial ribosome to disrupt protein synthesis Active against S. aureus and E. faecium but not against E. faecalis Must be administered intravenously High incidence of adverse effects and drug interactions Wholesale cost for 10 day treatment is about $3,000 plus hospitalization No longer used very often New Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptogramins Oxazolidinones Linezolid (Zyvox) was approved by the FDA in 2000 Effective against Vancomycin Resistant Staph. aureus (VRSA) and Enterococci (VRE) Lipopeptides Glycylcylines Ketolides Oxazolidinones O R1 N O R2 Linezolid Inhibits protein synthesis at the bacterial ribosome Bacteriostatic against staphylococci and enterococci Active against S. aureus, E. faecium and E. faecalis Administered intravenously or orally Generally well-tolerated Wholesale cost for 10 day treatment is about $1,000 New Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptogramins Oxazolidinones Lipopeptides Daptomycin (Cubicin) was approved by the FDA in 2003 Effective against Vancomycin Resistant Enterococci (VRE) Glycylcylines Ketolides Daptomycin (Cubicin) Daptomycin (Cubicin) Binds to the cell membrane of grampositive bacteria and causes membrane depolarization Effective against Vancomycin Resistant Staph. aureus (VRSA) and Enterococci (VRE), including E. faecium and E. faecalis Administered intravenously Approved for treatment of complicated skin and skin structure infections New Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptogramins Oxazolidinones Lipopeptides Glycylcylines 9-Aminotetracyclines acylated with N-dimethylglycine Tigecycline was approved by the FDA in 2005 Ketolides Glycylcyclines O H3C OH H N OH O OH O 9 N H3C O NH2 8 7 6 5 OH N(CH3)2 Glycylcyclines are not substrates for the efflux process and they block insensitive ribosomes Tigecycline H 3C H 3C H 3C N H OH H N O O OH OH O O 9 NH2 8 7 6 5 OH N (C H 3 ) 2 Tigecylcine (Tygacil) Active against methicillin-resistant S. aureus and probably VRE (in vitro) Broad spectrum Approved for complicated intra-abdominal and skin and skin structure infections Not a substrate for tetracycline antiporters or ribosome protection proteins Intravenous administration Bacteriostatic New Antibiotics for Use Against Antibiotic Resistant Bacteria Semisynthetic streptogramins Oxazolidinones Lipopeptides Glycylcylines Ketolides Telithromycin (Ketek) was approved by the FDA in 2004 Effective against multi-drug resistant Streptococcus pneumoniae Telithromycin (Ketek) Telithromycin (Ketek) Structurally related to the macrolides, which include Erythromycin Blocks protein synthesis by binding to 23S rRNA of the 50S ribosomal subunit Effective against gram-positive S. aureus (MRSA, not VRA) and S. pneumoniae (increasingly resistant to penicillin and macrolides) gram negative Haemophilus influenzae Mycoplasma pneumoniae and Chlamydia Telithromycin (Ketek) Approved for treatment of bronchitis, sinusitis and pneumonia Alternative to a fluoroquinolone for macrolide-resistant pneumococci Cost is $114 for 10 day course Comparable cost to fluoroquinolones and newer macrolides such as Clarithromycin Erythromycin costs about $6 Use with caution because of reports of serious hepatotoxicity Drugs to Remember Linezolid Daptomycin Tigecycline Antibiotic Resistance Current Status of Resistance Introduction of new antibiotics had been keeping up with resistance Declining investment in antibiotic discovery during the 1980s altered the balance Accelerated investment in the 1990s is beginning to yield new drugs Avoidance of resistance to new drugs has been a consistent but never achieved design objective The Problems in Avoiding Resistance Mobile genetic elements Multiple resistance and association with virulence markers Increasing use of drugs is associated with increasing frequency of resistance Worst case scenarios are already here for some nosocomial infections (Staphylococci and Enterococci) Antibiotic Resistance in the US Sept. 2002 – ASM Meeting Methicillin-Resistant Staph. aureus >50% of nosocomial bloodstream infections 31% of Staph infections outside the hospital 71% of Staph infections in nursing homes First case in US of vancomycin resistant Staph. aureus (from Enterococcus) Campylobacter jejuni and coli Most common cause of diarrhea 50% are resistant to Ciprofloxacin (Cipro) Retarding Emergence of Resistance Maintenance of therapeutic levels Ensure patient compliance Avoid the use of drugs when the MIC is at or only slightly below the attainable level Prevent biofilms and treat them aggressively Use combinations of antibiotics when indicated (but not otherwise) Avoid over and ill-advised use of antibiotics Prescriptions for infections that won’t respond Tendency to use hot new drugs Self medication Antibiotic Resistance of Bacteria from Sewers Serving Isolated Locations Sewer Serving General Hospital Mental Hospital Residential Area Percent of Bacteria Resistant to Streptomycin Chloramphenicol Tetracycline 34.7 0.7 32.0 6.5 0.3 0.4 0.7 0.007 0.1 Gentamicin Resistant P. aeruginosa in Burn Patients 1965 - 90 % susceptible 1968 - 636 kg (0.7 tons) of topical gentamicin used 1969 - 9 % susceptible late 1969 - gentamicin discontinued 1970 - 95 % susceptible Antibiotic Treatment of Adults with Sore Throat 1989-1999 (JAMA 2001, vol. 286:1181) 6.7 million annual visits in the US Antibiotics were prescribed in 73% of cases Decreasing use of penicillin and erythromycin Increasing use of non-recommended, extended-spectrum macrolides and fluoroquinolones Antibiotic Treatment of Adults with Sore Throat Most sore throats are due to viral upper respiratory tract infections Group A β-hemolytic Streptococci is the only common cause warranting antibiotics Streptococci cultured in 5-17% of cases Penicillin and erythromycin are still recommended in most cases Other drugs increase likelihood of resistance to those drugs and greatly increase the cost (> 20-fold for quinolones versus penicillin) Societal Contributors Antibiotic additives in stock feed Chlorine treatment of water Reduces number of bacteria by > 100 Survivors are resistant to antibiotics Mercury and other contaminants in water Bacteria resistant to mercury are also resistant to antibiotics Antibacterial soaps Any inhibitor selects for resistance to other inhibitors, including antibacterial drugs Criticized by the AMA and CDC, which agree that regular soap and water is equally effective Current Status of Antibiotic Discovery Empiricism At first highly successful Now marginal Rational approach Molecular modeling is being used extensively Low yield so far, but promising Novel agents from non-microbial biological systems New or Improved Antibiotics in Development Synthetic Vancomycins For resistance to Fluoroquinolones New Antibiotics in Development Synthetic A For Vancomycins promising but unproven prospect resistance to Fluoroquinolones Synthetic Vancomycins The sugar groups on the peptide backbone were modified (Science 1999, vol. 284:508) Completely synthetic drug The modified drug was more efficient at killing both vancomycin-sensitive and vancomycinresistant organisms Mechanism of action is different, blocking transglycosylation rather than transpeptidation Additional modifications are being tried New Antibiotics in Development Synthetic Vancomycins For resistance to Fluoroquinolones 2-Pyridones O O F COOH F COOH N N N CH3 N HN NH2 2-Pyridone Ciprofloxacin Inhibits DNA gyrase A, like quinolones May be more effective against gyrA mutants Approaches to Identify New Antibacterial Drugs Peptides from higher organisms Magainin from frogs, reached phase III trials but never proceeded further Steroids from higher organisms Squalamine from sharks Inhibitors of additional pathways Block lipid A synthesis, which is an essential component of the outer membrane of gram negative bacteria Functional Genomics The genomes of more than 20 microbial organisms have been sequenced Sequence data are used to identify essential targets by comparative genomics The targets are experimentally tested Drugs are developed to block those targets, based on structural predictions The Future of Antibiotics The best long-term solution is to minimize the development of resistance Doctors have a critical role in accomplishing this goal