Biosynthesis of Peptidoglycan 30 enzymes M G M G M G. Three stages: 1) Precursor formation: Cytoplasm M G M G M G

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1 Online edical Tourism Agency Health Options Worldwide (HOW) Discusses India's Resistant edical Travel "Super Bug Dolly ehta ND-1 (New Delhi metallo-beta-lactamase bacteria) was found in British patients returning from Bangladesh, India, and Pakistan after medical treatment. The ND-1,, is also being carried over by patients in the United States and South Asia, who also have received surgical care in India. "Scientists say it is highly resistant to antibiotics and is nearly impossible to treat," said David Goldstein, President of Health Options Worldwide Gram positive bacterial cell wall Penicillins (PCN or PEN) Cephalosporins Clavulanate Carbapenems Bacterial cell wall Proteoglycan (PG) (15-50 nm thick) Plasma membrane Gram negative bacterial cell wall Vancomycin Lipopolysacharides (LPS) Plasma membrane periplasm PG (2 nm thick) LPS: lipid A + core polysaccharides, O antigen Gram negative bacterial cell wall Peptidoglycan N acetylmuramic acid (NAA) N acetylglucosamine (NAG) G Penta peptide Glycine 1

2 G G G G G G Biosynthesis of Peptidoglycan 30 enzymes Three stages: 1) Precursor formation: Cytoplasm 2) Binding with phospho-c55 lipid carrier to form long polymer: Cell membrane G G G 3) Cross-linking in cell wall L-alanine racemase UDP CYTO Penicillin: -Lactam antibiotics D-alanine synthetase P-C55 lipid UDP UP G UDP UDP P-P-C55 G P-P-C55 G P-P-C55 Drug of choice for a large number of diseases Discovered by Alexander Flemming Produced by penicillium EB WALL P P-P-C55 lipid G transglycolase G G G G transpeptidase -lactum ring Thiazolidine ring O S CH3 R C NH CH CH C CH3 amidase 6APA O = C N CH COOH Penicillinase/ -lactamase 6-aminopenicillanic acid i.e. penicillin nucleus (required for biological activities) R decides: = Penicillin subtype Antibacterial activity resistance to -lactamase Classification Natural Penicillins -lacatamse resistant Penicillin Aminopenicillins/ modern spectrum Carboxypenicillin Ureidopenicillins/ extended penicillin Spectrum Gram (+) cocci, hydrolyzed by penicillinase so ineffective against most strains of S. aureus Less active agnst bacteria sensitive to Penicillin G First choice for S aureus and S epidermidis Gram (-) e.g Hemophillus influenzae, E.Coli, Neissaria sp. Administered with -lactamse inhibitor such as clavanate to prevent hydrolysis Gram (-) e.g. pseudomonas sp, enterobacter sp. Inferior to ampicillin against Gram + cocci Pseudomonas sp, 10 times more effective than carboxypenicillin R1 CH 2 - OCH 2 - OCH3 OCH3 CH 2 - NH2 CH- COOR stability for stomach acids 2

3 echanism of Inhibition All -Lactam antibiotics binds penicillin binding proteins (PBP) belong to acyl serine transferases N TG PBP domain structure Ser TP C PBPs: required for final stages of cell wall synthesis i.e. peptidoglycan (bind covalently) -lactum antibodies Acylation of PBPs Inhibition of PBPs Structural irregularities Transglycolase (TG) (insensitive to penicilin) formation of linear glycan strands Transpeptidase (TP) (sensitive to penicillin) cross-link the peptide subunits) serine residue (required for covalent bond formation) at the active site is conserved in all members of the PBP family. Cell lysis high-molecular-weight (HW) PBPs include transpeptidase G G G G G G low-molecular-weight (LW) PBPs may maintain shape of bacteria G G G PBP s (40kD-91kD): Number of PBPs varies within bacterial strain. i.e. S aureus has 4 PBPs whereas E coli has 7 Affinity of PBPs to antibiotics is variable Penicillin Protein Apparent molecular weight Binding of penicillin( % total ) olecules/cell Lytic PBP1 (leads to death of bacterial cell) Non-lytic (PBP2/3) (affect holin-like proteins in bacterial cell memb which alter membrane potential) 3

4 echanisms of Penicilin Resistance A. Elaboration of normal PBPs B. Inability of agent to penetrate to site of action C. Increased expression of efflux pumps i.e E. coli D. Production of -lactamase A. Elaboration of PBPs a) decreased affinity for -lactams a1. formed by homologous recombination between PBPs of different bacterial species a2. by transposans from unknown org b) structural differences in PBPs B. Inability of agent to penetrate to site of action b1. Gram (-) bact outer layer of LPS Small hydrophilic antibiotics can pass through channels porins i.e. amoxicillin, ampicillin>penicillin G P aeruginosa resistant to most antibiotics lacks porins C. Increased expression of efflux pumps i.e E. coli ajor facilitator superfamily (FS) Adenosine triphosphate binding cassette (ABC) Small multidrug resistance (SR) Resistance nodulation cell division (RND) Gram (-) ultidrug and toxic compound extrusion (ATE) D. Production of -lactamase d1. -lactamases class A-D: Hydrolyse lactam ring of penicillin's Class A, C and D works by hydrolyzing serine ester Class B (Zn-dependent) Class A extended spectrum -lactamase; degrade penicillin, some cephalosporin's and carbapenems Class B destroy all -lactums except aztreonam Class C cephalosporin's Class D cloxacillin 4

5 d2. Site of liberation Gram (+), lactamase is secreted extracellularly in large amounts Gram (-), lactamase is located in the periplasmic space, small amounts. Primary mechanism of acquired resistance! d3. Other factors: surviving bacterial cell, biofilms produce bacteria in prosthetics General features of the Penicillins Distribution widely distributed; concentration varies in diff tissues. therapeutic concentrations is achieved readily in joint fluid, pleural fluid, pericardial fluid, and bile Do not penetrate phagocytic cells, very low conc in prostatic fluids, brain tissue, and intracular fluid <1% in CSF when meninges are normal; ~5% when inflamed meningis Active transport process pumps penicillin's from CSF to the bloodstream. This mechanism is blocked by Probenecid Excretion Eliminated by glomerular filtration. Higher urine concentrations. O S CH3 R C NH CH CH C B A CH3 amidase O =C N CH COOH Penicillanase O CH CH R CH + NH2 CH O =C N 6-APA OH H Penicilloic acid = = Natural Penicillin Penicillin G Penicillin V Pen V is superior to Pen G because of acid stability and absorption Repository Forms of Penicillin G: Penicillin G procaine (Wycillin) (benzyl penicillin with local anasethetic agent procaine) slowly absorbed after I injection; Wycillin will maintain adequate plasma levels for 24 hours. Syphillis, RTI, anthrax Penicillin G benzathine (Bicillin L-A, Permapen); slowest rate of absorption after I absorbtion. Can maintain adequate plasma levels for 10 days. Distribution bound with albumin significant amount appear in liver, bile, kidney, semen, lymph, intestine Excretion rapidly eliminated from the body by kidney 10% by glomerular l filtration; ti 90% by tubular secretion 60-90% urine within Ist hr after injection rest metabolized to penicilloic acid Renal clearance ~ total renal plasma flow ( 3 million u (1.8 g)/hr) lower in neonates and infants (3hrs in1wk old baby) 5

6 Renal dysfunction: i.e Anuria increases the half life of Pencillin G from 0.5 hr 10hr impairment of renal function 7-10% antibiotic may be inactivated by liver/hr Therapeutic uses Penicillin G: Penicillin V: cellulitis, bacterial endocarditis,gonorrhea Pneumonia, Steptococcal infections, syphilis, meningococcal infections tonsilitis, pharyngitis, skin infection, odontogenic infection Prophylactic uses: Affords protection agnst Steptococcal infections Rheumatic fever -lactamase resistant Penicillin narrow spectrum also called as anti-staphylococcal aureus penicillin) Isoxazoyl penicillin (oxacillin, cloxacillin, dicloxacillin) (dicloxacillin most active) Relatively stable in an acid medium Absorbed rapidly but incompletely (30-80%) increases after empty stomach Nafcillin Very effective agnst S aureus Inactivated in the acidic medium Temocillin anti-pseudomonas aeruginosa or acinetobacter spp Eliminated rapidly by kidney. Also hepatic Aminopenicillins (oderate spectrum) Ampicillin (Principen) Amoxicillin Amocxicillin superior than ampicillin acid stability, absroption, half life Excretion Ampicillin (Principen) Appears in bile, undergo enterohepatic circulation and is excreated in feces Amoxicillin Eliminated in urine; probenecid delays excreation of drug; Use Upper respiratory infections, UTI, eningitis, salmonella infections Amoxicillin less effective than ampicillin for shigellosis 6

7 Antipseudomonal penicillins: (extended spectrum) Carboxypenicillin and Ureidopenicillin -lactamase sensitive Carbenicillin Indanyl sodium (Geocillin) only used for managing UTI caused by Proteus Ticarcillin 2-4 times effective for P aeruginosa than Carbenicillin, which is toxic Piperacillin (Pipracil) extends the spectrum of ampicillin to include most strains of P aeruginosa VIII. Untoward Effects: Hypersensitivity: OST common side effect (0.7%-4%) Allergy to one penicillin greater risk to other penicillins O S CH3 R C NH CH CH C CH3 B A amidase O =C N CH COOH Penicillanase R O = = CH + NH2 6-APA CH CH O =C OH CH N H Haptens Penicilloic acid IgE Abs Hapten Immunologic Classification of Hypersensitivity Reactions (Gell and Coombs) non-immunogenic compound of low molecular weight + binding to protein or cell Immunogenic Immune Response (IgE, IgG,Ig, Lymphocyte) Gell-Coombs Classification Time of Onset ediator(s) Clinical Signs Type I <1 h IgE Type II >72 h Type III Type IV >72 h >72 h Anaphylaxis: urticaria, angioedema, wheezing, laryngeal edema, hypotension Skin Testing Indicated Yes IgG/Ig g +comple Immune cytopenia, some organ No ment inflammation Serum sickness, IgG or Ig drug fever, immune complexes vasculitis, tissue injury T cells and cytokines Contact dermatitis, some organ inflammation No No Ig = Immunoglobulin. Beta-lactam antibiotics can cause all 4 types of hypersensitivity reactions. Ann Yates, am J ed: 2008 Beta-Lactam Reactions Based on Time of Occurrence and Relation to IgE Immediate (<1 h) and accelerated (1-72 h), IgE mediated Urticaria Angioedema Laryngeal edema Bronchospasm Hypotension Late reactions (>72 h), possibly IgE mediated orbilliform rash Urticaria Severe late reactions (>72 h), not IgE mediated Hemolytic anemia Neutropenia Thrombocytopenia Serum sickness Interstitial nephritis Hepatitis Pulmonary infiltration Eosinophilia Vasculitis, Stevens-Johnson syndrome, Drug fever Decreased platelet aggregation (carbenicillin and ticarcillin) Neutropenia (especially the -lactamase -resistant penicillins) Hypernatremia and hypokalemia (carbenicillin) Pseudomembranous colitis: due to effect on microflora anagement of patient potentially allergic to penicillin: history skin tests (not confirmatory) desensitization achieved by administering gradually increasing dose of penicillin 42 7

8 Drug-drug Interactions Chemically antagonize aminoglycosides. UST NOT be administered simultaneously through the same I.V. line; should be staggered by about 1 to 2 hours. Carboxy- or Ureidopenicillins and aminoglycosides are synergistic in their anti-pseudomonas activity. R1 C = O Cephalosporins NH CH CH 7ACP O =C N R1 decides: antibacterial activity resistance to -lactamase stability for stomach acids S CH2 C R2 (effects C metabolism and C = O pharmacokinetic) OH 7ACP: 7-aminocephalosporanic acid Cephalosporins Acylation of PBPs Inhibition of PBPs Classification: Best indicated by generation based on antimicrobial activity G G G G G G Inhibits cross linking of peptidoglycan G G G Structural irregularities Cell lysis Ist generation Cefazolin (ANCEF, ZOLICEF, others) Cefadroxyl (DURACEF) Cefalexin monohydrate (KEFTAB) Cefradine (VELOSEF) II nd generation Cefuroxime (ZINACEF) Cefuroxime axetil (CEFTIN) Cefprozil (CEFZIL) Cefmatazole (ZEFAZONE) Loracarbef (LORABID) Useful spectrum good against Gram (+); modest against Gram (-) Streptococci (except penncillinresistant strains); Staphylococcus aureus (except ethicillin-resistant strain) Increased activity it against Gram (-) but much less active than IIIrd generation Gram (-) e.g., Enterobacter sp, Klebsiella sp., haemophilus influenza; Not active against gram + as Ist generation 47 IIIrd generation Cefotaxime (CLAFORAN) Ceftriaxone (ROCEPHIN) Cefdinir (ONICEF) Cefditoren pivoxil (SPECTRACEF) Ceftizoxime (CEFIZOX) Ceftibuten (CEDAX) Cefpodoxime proxetil (VANTIN) Cefoperazone (CEFOBID) Ceftazidime (FORTAZ, others) IV generation Cefepime (AXIPINE) Useful spectrum Less active than Ist generation against Gram (+) but more active against Enterobactericeae including -lactamase producing bacteria Active agnst Pseudomonas Extended spectrum of activity than IIIrd generation and have increased stability against hydrolysis by - lactamase 48 8

9 Ceftobiprole (Zeftera/Zevtera) 5th generation active against RSA (methicillin-resistant Staphylococcal aureus, penicillin-resistant Streptococcus pneumoniae, Pseudomonas aeruginosa It has been shown to be statistically non inferior to the combination of vancomycin and ceftazidime for the treatment of skin and soft tissue infections. Ceftobiprole inhibits the PBP. Ceftobiprole is resistant to staphylococcal lacatmase. ] echanism of Resistance: Same as penicillin's. i.e. Altered PBPs or lactamase function First generation cefazolin is more susceptible to - lactamase from S aureaus than is Cephalothin Third generation: susceptible to hydrolysis by inducible chromosomally encoded (Class 1 -lactamase) Fourth generation: less susceptible 50 General features of the Cephalosporins Distribution absorbed readily after oral administration Several cephalosporins can penetrate into CSF meningitis Can also cross placenta High concentrations also seen in synovial, bile and pericardial fluids Penetration in aqueous humor of eye is high Excretion Primarily excreted by kidney dosage should be adjusted in patients with renal insufficiency Cefoperazone (excreted in bile) cefotaxime is deacelated in vivo; the metabolite less active Specific Agents: Ist generation: Cefazolin Well-tolerated after either I or IV Conc in plasma after 1g I administration reach to 64 ug/ml Excreted by glomerular l filtration ti and is bound to plasma proteins (85%) II nd generation: Cefoxitin Resistant to -lactamse produced by Gram (-) rods For Gram (+) < active than I st generation cephalosporins ore active than I st or II nd generation agents agnst -fragalis Conc in plasma after 1g I administration reach to 22 ug/ml; half life 40 min Preferred among Ist generation as can be administered less frequently due to longer half-life

10 Cefotetan ore active than Cefoxitin agnst Gram (-) Conc in plasma after 1-g I administration reach to 70 ug/ml; half life 3.3 hrs III rd generation: Ceftazidime Active agnst Gram (+) excellent for Pseudomonas and Other Gram (-) bacteria half life 1.5 hrs; not metabolized IIIrd generation: Cefotaxime Resistant to many -lactamase and has a good activity agnst most Gram (+) and (-) bacteria except B. fragilis Half life in plasma 1 hr etabolized desacetylcefotaxime 55 IV th generation: Cefepime Active agnst many enterobact which are resistant to other Cephalo Excellent penetration in CSF; Conc in plasma after 2-g IV administration reach to ug/ml; half life 2 hrs 56 Therapeutic Uses: First generation: skin and soft tissue infections, surgical prophylaxis of wound infection. Third generation: infections caused by Klebsiella, Enterobacter, Proteus etc, ceftriaxone: all forms of gonorrhea, severe lyme diseases cefotaxime or ceftriaxone: used to treat meningitis due to pneumococci, meningococci, and Haemophillus influenza Untoward Reaction: Hypersensitivity: The frequency of cross-reactivity with penicillin-sensitive individuals is 5 to 15%. CONTRAINDICATED in patients with a history of anaphylaxis to a penicillin. Nephrotoxic Renal tubular necrosis i.e. cephaloridine (4g/day) Fourth generation noscomal infections where resistance to -lactum antibiotics is expected. 57 Hyperprothrombinemia, Platelet dysfunction Thrombocytopenia Disulfiram-like Effect: cefamandole, cefotetan, moxalactam, cefoperazone. 58 Drug-drug Interactions: OTHER -LACTA Antibiotics (Not penicillin or cephalosporins) Concurrent administration of Cephalosporins or gentamicin cause nephrotoxicity (in >60 yr old patients) Carbapenems (fused -lactum ring and a 5-membered ring sys) Imipenem, eropenem, Ertapenem, Aztreonam

11 i. echanism of action: Binds to PBPs, disrupting cell wall synthesis and is bactericidal. ii. Spectrum: Broad-spectrum covers Gram (+) & Gram (-) e.g. Streptococci, Enterococci. Resistant to most forms of -lactamase, including that produced by staphylococcus. 61 iii. etabolism: not absorbed orally Imepenem hydrolyzed by dipeptidase, so always administered with cilastatin, an inhibitor of dipeptidase eropenem, Ertapenem (long half life) are resistant to dipeptidase most of it is recovered in urine as the active drug; renal insufficiency iv. Side effects: patients allergic to the penicillins may demonstrate cross-reactivity with imipenem. nausea and vomiting. Seizures have been reported with high doses. 62 iv. Therapeutic Use: urinary tract and lower respiratory infections intra-abdominal and gynecological infections effective against cephallosporin resistant bacteria prudent to use imipenem for empirical treatment of serious infections in hospitalized patients who have recvd other -lactums should NOT be used as monotherapy against pseudomonas due to risk of resistance during therapy 63 Aztreonam (AZACTA) A monocyclic -lactam (a monobactam). i. echanism of action: Interacts with PBPs and induces the formation of long filamentous bacteria ii. Spectrum: It more closely resembles the spectrum of the aminoglycosides. No activity against Gram (+) and anaerobic bacteria are resistant. Aztreonam is resistant to the -lactamase produced by Gram (-) organisms. iii. Side effects: well tolerated. Penicillin allergic patients do not exhibit cross-reactions with aztreonam. 64 -Lactamase Inhibitors: echanism of action: i. Inhibits -lactamase prevent the destruction of - lactun sensitive antibodies. ii. Very efficient against -lactamase that degrade ceftazidine/cefotaxime. However, inactive against -lactamase produced by treatment with IInd and IIIrd generation cephalosopirns. iii. Poor antimicrobial activity, but binds irreversibly with - lactamase from both gram (+) or gram (-) bact so known as SUICIDE" inhibitor of -lactamase 65 iii. well absorbed; included in combination with amoxacillin (Augmentum) or with ticaricillin (TIENTIN) 66 11

12 Vancomycin Complex tricyclic glycopeptide antibiotic echanism: Inhibits cell wall polymerization by binding to terminal D-Ala-D-Ala terminus of incoming complex attached to carrier Antibacterial activity: Gram (+) Gram( ) are resistant because D-ala-D-ala (target) is substituted with D-ala-D-ser or D-ala-D-lactate ( G )n + G P-P-C55 G ( G )n Vancomycin P-P-C Absorption, Distribution and excretion: Oral absorption poor; slow IV is preferred, NEVER I (dose should be adjusted to maintainmdesirable trough levels) appears in body fluids and CSF 90% excreted by glomerular filtration; accumulates if renal function is impaired (can be cleared by hemodialysis) Untoward Effects: Hypersensitive Reacns (macular skin rashes, anaphylaxis, Chills) Rapid administration flushing, tachycardia, hypotension, erythematous or urticarial reacn flushing red-neck or red-man syndrome by directly inducing i toxicity it in mast cells auditory impairment (ototoxicity) and nephrotoxicity; caution with the use of aminoglycosides