Mechanisms of Antimicrobial Action and Resistance Alan L. Goldin, M.D./Ph.D.
Sections in Medical Microbiology & Immunology Chapter 10 Mechanisms of action Pages 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 growth of micro-organisms Antibacterial Inhibits 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
Tube Dilution Method for Determination of MIC MIC 128 64 32 16 8 4 2 1 0.5 μg Antibiotic per ml
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 Amikacin Tetracycline 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 1 0.5 μg Antibiotic per ml
Tube Dilution Method for Determination of MBC MIC 128 64 32 16 8 4 2 1 0.5 μg Antibiotic per ml
Tube Dilution Method for Determination of MBC MIC 128 64 32 16 8 4 2 1 0.5 μg Antibiotic per ml
Tube Dilution Method for Determination of MBC MBC MIC 128 64 32 16 8 4 2 1 0.5 μg Antibiotic per ml
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 sub- MIC 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 NH 2 H 2 N N N S NH 2 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 H 2 N S NH 2 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 H 2 N S NHR H 2 N C 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 H 2 N S NHR H 2 N C 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) SULFNAMIDES 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 & plasmids Altered dihydropteroic acid synthase Reduced sensitivity to sulfonamides Transposons & plasmids Increased 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 ne 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 Trimethoprim Inhibits Dihydrofolic acid NADPH NADP Tetrahydrofolic acid (THF) dump 5,10-methylene 5-methyl THF THF methionine & purines
Site of Action of Trimethoprim dtmp dump Dihydropteridine Sulfonamides Inhibit Dihydrofolic acid Trimethoprim Inhibits Dihydropteroic acid + PABA + glutamic acid NADPH NADP Tetrahydrofolic acid (THF) 5,10-methylene THF 5-methyl THF 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 Depletion of THF pool takes 3-4 generations Synthesis of pyrimidines & purines does not deplete THF methionine & purines
dtmp dump Site of Action of Trimethoprim Dihydropteridine Sulfonamides Inhibit Dihydrofolic acid Trimethoprim Inhibits Dihydropteroic acid + PABA + glutamic acid NADPH NADP Tetrahydrofolic acid (H 4 F) 5,10-methylene H 4 F 5-methyl H 4 F methionine & purines Trimethoprim is like sulfonamides Bactericidal in blood Ineffective in purulent lesions But trimethoprim is not antagonized by PABA Trimethoprim and sulfonamides are synergistic Inhibitors of sequential steps are often synergistic Sulfonamides reduce DHF which competes with trimethoprim
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 n 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 CH F CH H 3 C N N N N C 2 H 5 R 2 N R 1 Nalidixic Acid 6-FluoroQuinolones Ciprofloxacin Norfloxacin floxacin R 1 =, R 2 = H: R 1 = C 2 H 5, R 2 = H: R 1 = C 2 H 5, R 2 = CH 3 :
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 2 N H H N H S CH 3 C CH 3 CH β-lactam ring Thiazolidine ring
Peptidoglycan Cross Linking L Ala D Glu m Dap D Ala D Ala D Ala TRANSPEPTIDASE Site of action of penicillins 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 Ala Glu DAP - CH 3 N C C H H CH 3 N H 2 C H C H CH 3 CH 3 NH N H C C H N H C C H N H -DAP Glu -Ala glycan ( N acetyl glucosamine-n acetyl muramic acid) n free amino group of DAP (m-diaminopimelic acid) NH 2 cross link
Substrate-Enzyme Intermediate in the Cross Linking Reaction CH 3 CH 3 Ala Glu DAP - N C C H H N H 2 C H C H Transpeptidase = Serine hydroxyl group in active center of transpeptidase
β-lactam Inactivation of Transpeptidases H 2 N H C C H C N H S C CH 3 C CH 3 CH + Transpeptidase Serine H of Transpeptidases H 2 N H H C C C HN S C H Transpeptidase CH 3 C CH 3 CH
Inactivation of Transpeptidases by β-lactams H 2 N H C H C S C CH 3 C HN H C CH 3 CH Transpeptidase Serine H of Transpeptidases
Transpeptidases (Penicillin Binding Proteins) MW PBP Activity Function 91,000 87,000 1a 1b Transpeptidases Peptidoglycan synthesis Cell wall elongation 66,000 2 Transpeptidase? Maintenance of rod shape 60,000 3 Transpeptidase Peptidoglycan synthesis Septum formation 49,000 42,000 40,000 4 5 6 D-alanine carboxypeptidases Control extent of x links
Selectivity & Side Effects of β-lactams Selective toxicity The targets of β-lactams are uniquely bacterial The corresponding structures do not occur in humans 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 H H R C ε amino group of a Lys residue N H C C C HN NH Serum protein S CH 3 CH 3 CH
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 H 2 N Destruction by β-lactamase Serine H H C C H C HN H β-lactamase S C C CH 3 CH 3 CH + H 2 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 10 6 cells MIC for β-lactamase positive is < 0.5 μg/ml for 10 10 3 cells MIC for β-lactamase positive Staph is 1250 μg/ml for 10 6 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) ne 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
ral 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 CH C C N 3 H 2 H CH 3 CH PENCILLIN G (benzylpenicillin) Acid labile C H 2 C N H CH 3 CH 3 CH PENICILLIN V (phenoxymethyl penicillin) Acid stable
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 H 2 N S CH 3 N CH 3 CH 6-AMINPENICILLANIC ACID + Acid anhydrides or Acid chlorides CH 3 C N S CH 3 C N S CH 3 CH 3 N CH 3 CH C 2 H 5 N CH 3 CH METHICILLIN NAFCILLIN
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 C H 2 C N H PENICILLIN G (Benzyl penicillin) N S CH 3 CH 3 CH Narrow Spectrum H C NH 2 C N H AMPICILLIN N S CH 3 CH 3 CH Broad Spectrum
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 N C H CH CH 2 H N S CH 3 CH 3 CH Clavulanic Acid Sulbactam
Inhibition of β-lactamases by Clavulanic Acid CHCH 2 H N I + β-lactamase CH CHCH 2 H CH 2 CH 2 H HN CH II HN CH β-lactamase 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 PBP 1 Susceptible 2 3 Resistant 2A 4
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
ther β Lactam Antibiotics Cephalosporins Carbapenems Monobactams
Cephalosporins About 20 currently in use Tend to be substrates for β-lactamases less frequently than penicillins 1 st generation (Cefazolin) Antibacterial spectra & potency like penicillins 2 nd generation (Cefoxitin) More potent & better against gram negatives 3 rd generation (Cefotaxime) Even more potent & highly effective against gram negatives but at the expense of reduced potency for gram positives 4 th generation (Ceftazidime) Enhanced activity against gram negatives without loss of potency for gram positives
Core Structures of Penicillins & Cephalosporins H 2 N H H N H S CH 3 C CH 3 CH H 2 N H H N S CH R 6-Aminopenicillanic Acid 7-Aminocephalosporanic Acid R = CH 2 HC CH 3
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 Frequent Cephalosporins + Serum protein Rare if at all R C N H H H C C C HN S CH 3 CH 3 NH CH Serum protein R C N H H C C H C HN S NH CH Serum protein R 1 Penicilloyl 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 Penicillins R 1 H H N S R 2 CH R 1 CH H N H H N S CH 3 C CH 3 CH H atoms are trans C replaces R 1 attached directly H atoms are cis S in fused ring R 1 attached via amino group
Monobactams R H NH N H CH 3 S 3 _
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) Ala Glu DAP - CH 3 N C C H H CH 3 N H 2 C H C H CH 3 CH 3 NH N H C C H N H C C H N H -DAP Glu -Ala glycan ( N acetyl glucosamine-n acetyl muramic acid) n free amino group of DAP (m-diaminopimelic acid) NH 2 cross link
Vancomycin Resistance A Depsipentapeptide instead of the normal Pentapeptide Pentapeptide L Alanyl - D Glutamyl - m DAP - D Alanyl - D Alanine Van Sens Depsipentapeptide L Alanyl - D Glutamyl - m DAP - D Alanyl - D Lactate Van Res Van Sens Vancomycin can bind to D Alanyl - D Alanine Van Res Vancomycin cannot bind to D Alanyl - D Lactate
Vancomycin Resistance I Synthesis of the Depsipentapeptide Pyruvate + NADH D Lactate + NAD vanh D Alanine + D Lactate vana 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 D Alanine + D Alanine vanx 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
Aminoglycosides Chloramphenicol Macrolides Clindamycin Tetracycline Drugs that Act on Ribosomes
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 R 1 Aminocyclitol CH NHR 2 NH 2 NH 2 H NH 2 H Gentamicin C 1 R 1 = CH 3 R 2 = CH 3 Gentamicin C 2 R 1 = CH 3 R 2 = H Gentamicin C 1a R 1 = H R 2 = H CH 3 NHCH 3 H Aminosugar
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 ribosomal proteins This occurs very rarely Resistance is very high
Kanamycin Sites of Inactivation AC II AC III AC AC I Types of Inactivation CH 2 -NH 2 NH 2 AC N-Acetyl transferases NH 2 (AC) -Acetyl transferases H H H AD P -Adenyl transferases -Phosphatases P I H CH 2 H Blocked reaction P II (AC) H NH 2 H AD
Chloramphenicol I II III H CH 2 H 2 N C H C H N H C CHCl 2
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 H 3 C N CH 3 H CH 3 CH 3 H H 3 C CH 3 H H H 3 C H 3 C CH 2 CH 3 CH 3 H 3 C CH 3 H CH 3
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 ften an alternative for penicillin to treat allergic patients
Clindamycin CH 3 CH 3 N Cl CH H 3 C C H 2 C H 2 N H H CH H SCH 3 H
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 H H H NH 2 7 6 5 N(CH 3 ) 2 H 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 H H H NH 2 7 6 5 N(CH 3 ) 2 H Drug Position 5 6 7 Chlortetracycline CH 3 ; H Cl Tetracycline CH 3 ; H Doxycycline H CH 3 Minocycline N(CH 3 ) 2
Drugs to Remember Gentamicin Erythromycin Clindamycin Tetracycline
Drugs that Disrupt Cell Walls Nystatin Polymyxin
L-Leu (α) L-Dab L-Phe (α) L-Dab (α) L-Dab L-Thr (γ) L-Dab (α) L-Dab = L-α, γ-diaminobutyric acid (α) and (γ) indicate NH2 groups L-Dab involved in peptide linkages L-Thr (α) L-Dab 6-Methyloctanoic PLYMYXIN B 1
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 xazolidinones 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) xazolidinones Lipopeptides Glycylcylines Ketolides
Streptogramins N N N N HN NH H H N N N N N H Pristinomycin Ia Pristinomycin 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 xazolidinones Linezolid (Zyvox) was approved by the FDA in 2000 Effective against Vancomycin Resistant Staph. aureus (VRSA) and Enterococci (VRE) Lipopeptides Glycylcylines Ketolides
xazolidinones R 1 N R 2
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 xazolidinones 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 xazolidinones Lipopeptides Glycylcylines 9-Aminotetracyclines acylated with N-dimethylglycine Tigecycline was approved by the FDA in 2005 Ketolides
Glycylcyclines H 3 C H 3 C N H N 8 9 H H H 7 6 5 NH 2 H N(CH 3 ) 2 Glycylcyclines are not substrates for the efflux process and they block insensitive ribosomes
Tigecycline H 3 C H 3 C H 3 C N H H N 8 9 H H H 7 6 5 NH 2 H N(CH 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 xazolidinones 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 ther 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 Vancomycins A promising but unproven prospect For 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 F N CH F CH N N N CH 3 HN NH 2 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