Staphylococcus aureus…………quickly posed a problem Produce a B-lactamase (Penicillinase) Plasmid mediated (easily spread) Secreted into the surrounding medium Destroyed the Penicillin before it could reach the PBP’s S O S O S O

CH3 O O N R2 C NH S CH3 CH3 R1 N O COOH R1 R2 Oxacillin H H Cloxacillin Cl H Dicloxacillin Cl Cl Isoxazoyl Penicillins

If chemical modification can overcome B-lactamase activity Maybe chemical modification can also increase spectrum of activity ?

NH2 O C NH S CH3 CH3 N O COOH Ampicillin

Aminopenicillins Modified to broaden their spectrum of activity better binding affinity to the PBP’s of Enterococci Listeria Some Gram –ve bacteria But a cost of slight decreased activity against Streptococci S. pneumonia / Group A strept Not stable to the B-lactamase produced by S. aureus Inactivated by many of the B-lactamases produced by Gram –ves (limited gram negative coverage)

Amino-Penicillin Amoxicillin Ampicillin Bacampicillin First penicillins discovered to be active against gram negative rods like E. coli and H. Influenzae.

Amino-Penicillin Same coverage like Pen VK plus: - Listeria monocytogenes - Enterococcus - Proteus mirabilus - E coli - H. flu

Amino-Penicillin Amoxicillin is more completely absorbed than Ampicillin. Serum levels are twice as high. Small amount is left in GI tract, so less diarrhea. However, more complete absorption makes it less effective for Shigella enteritis. Both have same antibacterial spectrum. Bacampicillin is more expensive and no advantage.

Amino-Penicillin Primarily used for: Otitis media Sinusitis Bronchitis Urinary tract infection esp. Enterococcus Bacterial diarrhea Salmonella infection Shigella infection. H. Influenzae and E. Coli are increasingly becoming resistant.

Maybe if we change its structure a lot….. …we will get even more gram negative coverage !

Carboxypenicillins Carbenicillin 1st penicillin developed with expanded gram –ve activity that included anti-pseudomonal activity But decreased activity against Streptococci and Enterococci Required large doses to be effective clinically too toxic to be used clinically Replaced by the next drug developed in this class…Ticarcillin

similar to Ampicillin wrt Streptococcal activity Enterococcal activity Ureidopenicillins similar to Ampicillin wrt Streptococcal activity Enterococcal activity Better anti-pseudomonal activity then Ticarcillin Better Klebsiella activity then Ticarcillin (75% vs 5%) Not orally absorbed thus available only IV 3 preparations Azlocillin (no longer available) Mezlocillin (USA) Piperacillin (Canada / USA) Piperacillin is also available as a combination with a B-lactamase inhibitor to increase its spectrum of activity (Piperacillin + Tazobactam = Pip/Tazo)

O C NH S CH3 CH3 N O COOH Penicillin G

O CH3 C N NH O N C NH S CH3 O O CH3 N O COOH Piperacillin

5 Classes of Penicillins Natural Penicillins Penicillin G / Penicillin V Penicillinase Resistant Penicillins Methicillin Nafcillin Isoxazoly Penicillins (Cloxacillin / Dicloxacillin / Oxacillin / Flucloxacillin) Aminopenicillins Ampicillin / Amoxicillin Carboxypenicillins Ticarcillin Ureidopenicillins Piperacillin / Mezlocillin / Azlocillin

Inducible Constituitive B-lactamases Chromosomal Plasmid Inducible Constituitive High [ ] High [ ] Low [ ] TEM 1-27 SHV 1-8 OXA 1-3 PSE 1-5 SPACE Serratia Pseudomonas Acintobacter Citrobacter Enterobacter Can NOT be overcome by Can be overcome by B-lactam – B-lactamase Inhibitor B-lactam – B-lactamase Inhibitor Combinations Combinations

Antibiotics Biosynthetic Chemical Penicillin (1945)

Antibiotics Biosynthetic Chemical Penicillin (1945) Search for other Compounds Chemical Modification

We like to think we are smart Chemical Manipulation

Bacteria have been fighting each other since before we evolved Nature has been doing chemical modifications for millions of years There have been many chemical modifications

R S CH3 CH3 N O COOH B-lactam

R C CH3 R S CH3 CH3 CH3 N N O COOH O COOH Carbepenem B-lactam

Carbapenems 1st isolated from Streptomyces cattleya in 1976 Base compound was thienamycin - methylene replacement for the Sulphur in the 5 membered ring R C CH3 R S CH3 CH3 CH3 N N O COOH O COOH Carbepenem B-lactam

Carbapenems stable to B-lactamases due to the side chain being in a trans rather then the typical cis configuration seen in B-lactams R S R N N O O B-lactam Carbapenem

2 Compounds in clinical use : Imipenem Meropenem Imipenem Meropenem

Imipenem Spectrum of Activity Gram +ve cocci - Staphylococcus aureus - Streptococci sp - S. pneumoniae - Enterococci Gram –ve rods - except Pseudomonas cepacia and Xanthomonas maltophilia Anaerobes - except Clostrium difficile and a % of Bacteroides sp

Mechanism of Action - binds to the PBP’s leading to cell lysis Considered Bacterocidal (except for Enterococcus – bacteriostatic) Resistance - not due to the typical B-lactamases 1. Enzyme that will hydrolyze the imipenem 2. Loss of an outer membrane protein that would otherwise facilitate entry

R S CH3 R S CH3 CH3 CH3 N N O COOH O COOH Side Chain Modifications Penicillin R C CH3 R CH3 S N R O COOH N O Carbapenem Cephalosporin COOH

Cephalosporins 1940’s Cephalosporium acremonium was isolated from the seawater At the harbour sewage outlet of Cagliari, Sardinia Produced a substance that inhibited the growth of both gram +ve and gram –ve bacteria 1948 a culture of the fungus C. acremonium was sent to Oxford 3 antimicrobial substances produced by this fungus were identified Cephalosporin P gram +ve activity only Cephalosporin N re-identified as a penicillin Cephalosporin C foundation for current Cephalosporin family

Cephalosporin C - B-lactam ring fused to a 6 membered dihyrdothiazine ring

Binds to PBP’s and interferes with synthesis of the peptidoglycan Mechanism of Action Binds to PBP’s and interferes with synthesis of the peptidoglycan cell wall Bactericidal Stable to many B-lactamases produced by gram +ve and gram –ve’s Better ability to penetrate the outer LPS membrane of gram –ve bacteria LPS Peptidoglycan Cell membrane Gram –ve Gram +ve

Lipopolysaccharide (LPS) Mechanisms of Resistance Alteration of the PBP target Production of B-lactamases that inactivate cephalosporins (Cephalinosporinases) Decreased ability of the Antibiotic to reach its target (LPS of gram –ve bacteria) PBP’s B-Lactamases Cell Memebrane Cell Wall Lipopolysaccharide (LPS)

Decreased affinity of PBP’s to bind cephalosporins are seen in Streptococcus pneumoniae Haemophilus influenzae Neisseria gonorrhea MRSA Major mechanism of resistance in gram +ve bacteria

Cephalosporinases (serine proteases) hydrolyze the amide bond of the B-lactam ring thus inactivating the antibiotic Major mechanism of resistance in gram –ve bacteria Can be encoded chromosomally or on plasmids Can be constitutive or inducible Effectiveness depends on Affinity of the Cephalosporinase for the Cephalosporin Concentration of the Cephalosporinase produced Concentration of the Cephalosporin present

Plasmid mediated B-lactamases are often produced in large numbers Number of different classes (TEM, SHV, etc) Mutations lead to B-lactamases with increased spectrum of activity ESBL’s (Extended Spectrum B-lactamases) resistance to the 3rd generation cephalosporins also will destroy the monobactams (Aztreonam) however the Carbapenems are not affected (Imipenem / Meropenem)

Classification – based on the spectrum of activity 1st generation : gram +ve cocci 2nd generation : variable g +ve but increased gram –ve rod activity 3rd generation : variable g +ve but greatly increased g -ve activity 4th generation : g +ve and increased g -ve activity

1st Generation Half Life Peak Protein CSF [ ] Bound Penetration Cephalothin (Ceporacin) IV 1hr 30 70 - Cefazolin (Ancef) IV 1hr 80 80 - Cephalexin (Keflex) po 1hr 18 10 - Cefadroxil (Duricef) po 1hr 16 20 - 2nd Generation Cefoxitin (Mefoxin) IV 0.8 hr 150 70 - Cefotetan (Cefotan) IV 3.5 hrs 230 90 - Cefuroxime (Kefurox/Ceftin) IV / po 1.3 hrs 100 35 1-2/- Cefaclor (Ceclor) po 0.8 hrs 13 25 - Cefprozil (Cefzil) po 1.2 hrs 10 42 - 3rd Generation Cefotaxime (Claforan) IV 1.0 hrs 130 35 5.6-44 Ceftriaxone (Rocephin) IV 8 hrs 250 90 1.2-39 Ceftizoxime (Cefizox) IV 1.7 hrs 1330 30 0.5-29 Ceftazidime (Fortaz) IV 1.8 hrs 160 17 0.5-30 4th Generation Cefepime (Maxipime) IV 2.1 hrs 130 20 3.3-5.7

1st Generation Half Life Peak [ ] Protein Bound Cephalothin (Ceporacin) IV 1hr 30 70 % Cefazolin (Ancef) IV 1hr 80 80 % Cephalexin (Keflex) po 1hr 18 10 % Cefadroxil (Duricef) po 1hr 16 20 % What does this mean ? ….. Best 1st Generation Oral Drug is Cephalexin (Keflex) - higher peak concentration - less protein binding -- means more free drug to act on bacteria Best 1st Generation IV drug is Cefazolin (Ancef) - even though more is protein bound (80vs70%), the higher peak means.. -- more free drug to act on bacteria NO CSF Penetration thus do not use in Meningitis

2nd Generation Half Life Peak Protein CSF [ ] Bound Penetration Cefoxitin (Mefoxin) IV 0.8 hr 150 70 - Cefotetan (Cefotan) IV 3.5 hrs 230 90 - Cefuroxime (Kefurox/Ceftin) IV / po 1.3 hrs 100/9 35 1-2/- Cefaclor (Ceclor) po 0.8 hrs 13 25 - Cefprozil (Cefzil) po 1.2 hrs 10 42 - Cefoxitin Cefuroxime Cefaclor Cefprozil

3rd Generation Half Life Peak Protein CSF [ ] Bound Penetration Cefotaxime (Claforan) IV 1.0 hrs 130 35 5.6-44 Ceftriaxone (Rocephin) IV 8 hrs 250 90 1.2-39 Ceftizoxime (Cefizox) IV 1.7 hrs 1330 30 0.5-29 Ceftazidime (Fortaz) IV 1.8 hrs 160 17 0.5-30 Cefixime (Suprax) po 3.5 hrs Cefotaxime Ceftriaxone

R S CH3 R S CH3 CH3 CH3 N N O COOH O COOH Side Chain Modifications Penicillin R C CH3 R CH3 S N R O COOH N O Carbapenem Cephalosporin COOH

R S CH3 R S CH3 CH3 CH3 N N O COOH O COOH Side Chain Modifications Penicillin R C CH3 R CH3 S Monobactam N R O COOH N O Carbapenem Cephalosporin COOH

Monobactams Isolated from Chromobacterium violaceum in 1981 Monocyclic B-lactam antibiotic No activity against gram +ve bacteria or anaerobes Binds to PBP’s of gram –ve bacteria only Hydrolyzed by cephalosporinases of Pseudomonas cepacia Xanthomonas maltophilia Acinitobacter Klebsiella oxytoca Haemophilus

Pharmacology no oral absorption thus available as IV formulation only Renal clearance Complications mild rash Mild transaminitis NO MAJOR SIDE EFFECTS Lack of cross reaction with other B-lactam antibiotics thus can be used in Pen/Ceph allergic patients

Aztrenoam monobactam in clinical use Limited to Gram –ve infections only

Penicillins Carbepenems Monobactams Cephalosporins B-lactamase Inhibitors Structural Overview

Antibiotics Biosynthetic Chemical Penicillin (1945) Search for other Compounds Chemical Modification

Antibiotics Biosynthetic Chemical Penicillin (1945) Search for other Compounds Chemical Modification Carbapenems Monobactams Cephalosporins Different Mechanisms B-lactam Antibiotics

Glycopeptides

1st compound was isolated from Streptomyces orientalis in 1956 Glycopeptides 1st compound was isolated from Streptomyces orientalis in 1956 (bacteria) Complex structure amino acids + sugars Vancomycin

Mechanism of Action : Inhibit cell wall synthesis - binds to peptidoglycan precursors - blocks additional polymer extension

N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys Cell wall Cell membrane Transpeptidases (PBP’s)

N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys

Vancomycin blocks cell wall synthesis N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys Mechanism of action of Vancomycin Vancomycin blocks cell wall synthesis By binding to the d-alanyl-d-alanine site on the growing peptidoglycan chain N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys

Useful drug to treat B-lactam resistant staphylococcus aureus Vancomycin Useful drug to treat B-lactam resistant staphylococcus aureus Staphylococcus aureus No b-lactamase B-lactamase Alter PBP’s (Penicillinase) (MRSA) Penicillin Cloxacillin Vancomycin

Vancomycin Useful drug to treat B-lactam resistant enterococcus Enterococcus species No b-lactamase B-lactamase Alter PBP’s Ampicillin Vancomycin

Resistance to Vancomycin Change in d-alanyl-d-alanine precursors - Van A, B, C mutations of VRE Excess cell wall production - VISA Biofilm production seen on prosthetic devices /foreign bodies Biofilms produced by bacteria impair the penetration of the glycopeptide to the bacterial cells Gram negatives constitutively resistant (LPS)

N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys Cell wall d-lactate Cell membrane Transpeptidases (PBP’s)

Mechanism of resistance N-acetylglucosamine N-acetylmuramic acid d-ala L-glu Lys d-lactate Mechanism of resistance to Vancomycin

VRE Gene Complex Van H gene Van A gene Van X gene Van R & S genes - synthesizes d-lactate Van A gene - binds d-lactate to d-alanine Van X gene - hydrolyzes d-ala-d-ala Van R & S genes - regulatory gene regions Van Y& Z genes - accessory proteins

Antibiotics Biosynthetic Chemical Penicillin (1945) Search for similar compounds Chemical modification

Biosynthetic Chemical Antibiotics Biosynthetic Chemical B-lactam based antibiotics Penicillins Carbapenems Monobactams Cephalosporins

Biosynthetic Chemical Antibiotics Biosynthetic Chemical B-lactam based antibiotics Penicillins Carbapenems Monobactams Cephalosporins Vancomycin (1956) Search for similar compounds Chemical modification

Naturally occuring glycopeptides Vancomycin Streptomyces orientalis Teicoplanin (Targocid) Actinoplanes teichomyceticus Daptomycin (Cubicin) Streptomyces roseosporus Ramoplanin Actinoplanes ATCC 33076 Semisythetic glycopeptides (2nd Generation) Oritavancin Dalbavancin Telavancin

Glycopeptide Antibiotics Glycopepetides Lipopeptides Lipoglycopeptides Vancomycin Teicoplanin Daptomycin (Cubicin) (LY146032) Ramoplanin Oritavancin (LY33328) Dalbavancin Telavancin Semisynthetic derivatives Inhibition of cell wall synthesis Inhibition of cell wall synthesis + Increase cell membrane permeability Cidal

Vancomycin Oritavancin Teicoplanin Dalbavancin

Role of the newer glycopeptide antibiotics…. Combat emerging resistance of MRSA VRE

Biosynthetic Antibiotics that inhibit protein synthesis Lincosamides 1962 Aminoglycosides 1943 Stretpogramins 1953 Macrolides 1952 Tetracyclines 1953