Antibiotics: Mode of Action and resistance

Antibiotic
    Chemical substance Natural, semi synthetic and wholly synthetic Effective at low concentration (μ/ml) Bacteria static or bactericidal
Dr Ellabib MS

 Criteria for antibiotic for bacteria  Selectively toxic
Bactericidal (killing) Bacteriostatic (growth inhibition)  No harm to patient  Destroy structures

Present in bacteria Not present in host
 Antibiotics work together with immune system

 Minimal inhibitory concentration

 Lowest level stopping growth e.g. zone of inhibition around a disk impregnated with antibiotic Antibiotics inhibit cell wall are bactericidal Without cell wall osmotic pressure cause bacteria to burst

Mode of Action
 Inhibitors of cell wall synthesis (Peptidoglycan)  Inhibit Peptidoglycan biosynthesis at various stages  Stage one: inhibit UDP –N-Acetylmuramic acid through inhibition of tranferase enzyme  Fosfomycin  Stage two: inhibit building pentapeptides side chain through inhibition of synthetase enzyme  Cycloserine  Stage three: inhibit reaction leading to formation of a linear Peptidoglycan polymer (pyrophosphtase enzyme)  Vancomycin & Bacitracin  Stage four: preventing cross-linking and formation of Peptidoglycan (transpeptidase enzyme)  Bound covalently to various proteins called penicillin binding proteins (PBPS)  B-lactam antibiotics

Chemical modification changes of Blactam and their biological activity
 Early B-lactam antibiotics  Penicillin G &V
 Inactive against gram negative  No penetration of outer membrane  Unstable to B-lactamases enzyme  Active against streptococcus pyogens

 Pencilliinase-resistant penicillin
 More stable  Staphylococcus species  E.g. Methicillin and Oxacillin

 Aminopencillin
 activity against gram positive  increased activity toward gram negative (E. coli)  Ampicillin, Amoxil

Antipseudomonase penicillin
Carboxypenicillin Increased activity against -ve and decreased activity against +ve Carbenicillin and ticarcillin Uredopenicillin Increased activity against –ve and preserved activity against +ve Piperacillin and mezalocillin B-lactamases inhibitory Binds strongly to beta-lactamase Inhibit activity Called suicidal agents Clavulanic acid & Tazobactam

Cephalosporin's

 First generation  Old and narrow spectrum cephalosporin's  Cephaloridine, Cephalexin  2nd generation  -ve and some anaerobic bacteria  Cefuroxime and cefoxitin  3rd generation  -ve such as pseudomonas  Ceftriaxone and Ceftazidime  4th generation  Broad spectrum  cefpime

Other B-lactam antibiotics
 Monobactams  -ve  Aztreonam  Carbapenems  -ve  Imipenem

Antifungal Antifungal agents
 Chitin synthetase  Polyxin and nikkomycin  Glucan synthesis
 cilofungin

Compounds inhibit cell membrane
 Concentration dependent  Effect integrity of CM  Leakage of K, proteins and nucleic acid  Disinfectants, antiseptics and polypeptides  Phenols  Release compounds absorbed at 200nm  Inhibits electron transport chains (metabolic activity)  Alcohols  Interact with ester fatty acid and thiol group of proteins  Used as 70% concentration

 Inhibit adenosine triphosphotase (ATPase)  Uptake of K+  Polymyxin B  Inhibit phospholipids of –ve but not +ve  Antifungal agents (inhibit Ergosterol)  Interact with phospholipids of CM directly  Pores and leakage  Amphotericin B and Nystatin  Indirectly inhibit ergosterol biosynthesis  Cytochrome P-450  Azoles antifungal (Miconazole, itraconazole, Clotrimazole )  Other non azoles such as terbifine morpholin and tolnaftate

Chlorohexidine

Inhibitor of protein synthesis

 Mostly Bacteriostatic  Selectivity due to difference in ribosome's  Some toxicity –eukaryotic 70S ribosome's  Classes of ribosome's subunit  80s ribosome's (eukaryotic)  Dissociated to 60s and 40s as Mg+ concentration  Protein to RNA (50:50)  70s (prokaryotic and eukaryotic)  dissociate to 30s and 50s  Protein to RNA (35:65)  50-55s ribosome's (mammalian mitochondria)

Antimicrobial that bind to the 30s Amino glycosides
 Such as Streptomycin, gentamicin, Amikacin  Gram positive and –Ve  Slight initial entry of the drug inside cell  Interact with chain elongation (PC) at 12s  Misreading of mRNA abnormal protein  formation of abnormal channels  Increase and irreversible entry through channels  Blockage initiation of ribosome's  Inhibit binding of aminoacyl-tRNA and peptide synthesis inhibit protein synthesis

Tetracycline's ( Rickettsias and mycoplasma)
Short acting Chlortetracycline Intermediate Demeclocycline Long acting Doxycycline Blocking binding of aminoacyl-tRNA acceptor site on the mRNA ribosome's complex Prevent the addition of new amino acids to the growing peptide chain

Antimicrobials bound to 50s subunit

Chloramphenicol
 Natural antibiotic  Meningitis, typhoid fever  Associated with bone marrow toxicity  Reversible effect  Bind to a region on 50s close to site bound aminoacyl-tRNA in peptidyltransferase center  Blocking addition of new amino acids  Prevent growing of protein chain  Inhibit peptide bound formation

Macrolides (erythromycin, claritromycin and spiramycin)  +ve bacteria, mycoplasma, legionella  Resistant common  Stimulate dissociation of peptidy-tRNA from ribosome's during translocation step  Interrupting completion of peptide chain formation

Lincosamides (Lincomycin & Clindomycin)
 Similar to chloramphenicol and erythromycin  +ve cocci  Resistant common  Anaerobic bacteria

Inhibitors of protein synthesis

Fusidic acid

Bind to 70s ribosome's Active against gram positive cocci Inhibits polypeptide chain elongation

Inhibitor of nucleic acid synthesis  To toxic  Some are used to treat tumor, viral and serious bacterial such as TB  DNA inhibition  result in cell division inhibition  Effect Extra chromosomal elements of DNA and plasmids  Effect bacterial response to environmental changes  RNA inhibition  Inhibit protein synthesis

Nucleic acid inhibitors (Two groups)

 Compound interfere with precursor of nucleic acid (purine, pyrimidine) Sulfonamides and trimethoprim Compounds interfere with nucleic acid synthesis at the polymerization stage

RNA polymerase inhibitors

 rifampin Mycobacterium tuberculosis Inhibit DNA-dependent RNA polymerase
RNA polymerase

Minimal or core enzyme

Sigma factor (RNA)

α, β, β1 and ω subunits

Rifampin continuous  Form a tight one to one complex with β subunit  Prevent protein synthesis (chain initiation)  DNA gryases or topoisomerase inhibitors  quinolones
 Nalidixic acid, ciprofloxacin  Called nick closing enzyme  Enzyme play important role in supercoil strand DNA  Supercoiling is completed the single strand DNA is abolished by an enzyme that seals the nicked DNA  The enzyme is known as DNA gryase or Topoisomerase 2

DNA gryase or Topoisomerase 2
Four subunits and two were identified as A and β  A subunit introduce the nick and seal the nick they produced initially Nalidixic acid  β subunit Responsible for supercoil Norofloxacin & ciprofloxacin May interfere with A and β subunit

 Sulfonamides  Trimethoprim

Inhibitors of Folic Acid Synthesis

Anti- Mycobacterial Antibiotics
Para-aminosalicylic Para-aminosalicylic acid (PSA)
 Bacteriostatic

 Dapsone
 Bacteriostatic

 treatment of leprosy (Mycobacterium leprae)

 Isoniazid (INH)
 bacteriostatic  inhibits synthesis of mycolic acids.

Furantoin  Gram positive and negative  Urinary tract infection  Damaging DNA Nitro-imidazole (metronidazole)  Anaerobic bacteria and protozoa  Reduced to Nitro radical compounds  Acts as nuclease and damaging DNA Griseofulvin  Antifungal agent  Dermatophytes infection only  Effect nuclear function  Interfere with microtubules during separation of chromosomes in cell division at the metaphase

Antibiotic Mechanisms of Action

Microbial resistance to antimicrobial agents
 Clinical resistance  By mutation or acquisition of a plasmid  Provides a selective advantage  Single or multiple steps

 Cross resistance Vs multiple resistance
 Cross resistance  single mutation  closely related antibiotics  Multiple resistance  multiples mechanisms  Unrelated antibiotics

Genetic basis of resistance

Mutation
 For the origin of some resistant variants

Acquisition
 Transfer of genetics material from R to S microorganism  Confined by genes on Chromosomal or plasmid

 Chromosomal mediated resistant
 Remain with the particular bacterial cell  And Offspring

 Plasmid mediated resistant
 Self replicating extra chromosomal DNA  Widely distributed in nature  Often transmissible  Often Carry resistant determinants to many drugs

 Other function of plasmid  Carry genes allow bacteria to attach to mucosal surface  Produce toxins  Invade and colonize host cells

Transformation of plasmid resistant
 Conjugation (direct cell to cell contact)  Transduction (bacteriophage vector)  Transformation (uptake DNA from environment)  Transposition (transformation via transposons

Transduction ( bacteriophages vector)

 Transposons  So called jumping genes  Movable DNA elements  Carrying resistant genes  Jump or hop from plasmid to plasmid  From plasmid to chromosomal  Found in many bacteria  Carrying resistant to many antibiotic  Main cause of hospital and community outbreaks resistant

viewpoint  Most important type of resistant  Transmissible  Usually highly stable  Convert resistant to different class of antibiotics  Often associated with other characteristics
 Requirements for antibiotic activity and mechanisms resistant

 Plasmid from Epidemiological

 Properties of antibiotic required for efficacy  Penetration to target site in sufficient amount  Evade inactivation enzymes by microorganism  Interaction with target molecules to initiate an effect

Steps of resistant mechanisms
 Cellular barrier to drug penetration  Reduce antibiotic activity  Altered target molecules  Interact less effectively with drug with retain function  May use additional pathway insensitive to the drug  Modifying or inactivating enzymes  Rendering antibiotic ineffective Two or more of these mechanisms give rise to high level of resistant than any one mechanisms alone

Resistant due to permeability
 Beta lactam  Poor permeability
 change in porin channels  Mutation  Such as Neisseria gonorrhoeae

 Outer membrane composition

 Lipoplysaccharides, lipids and proteins  Pseudomonas aeruginosa  Usually display resistant to other antibiotics E. coli deficient mutant in porin channels  Was sensitive to imipenem because  Due to its compact zwitterionic structure  Permit rapid penetration  PBP2 per cell 20  Require only few drug molecules for antimicrobial

Aminoglycosides
 loss or reduction in outer membrane proteins  Enterobacteriacae  Appearance of new outer membrane proteins  Or alteration in surface Lipoplysaccharides  Affect intracellular uptake of Aminoglycosides  Ps. aeruginosa  MLS Antimicrobials  Intrinsic low level resistant to erythromycin  Limited drug permeability  Enterobacteriacae  Reduced drug uptake  Staph. epidermidis  Chloramphenicol  Loss of outer membrane proteins  Diminish uptake of drug in Ps. aeruginosa (acquired)

 Tetracycline's
 Poor penetration  Change in outer membrane (proteins, porin)  Alteration in Lipoplysaccharides  Ps. Aeruginosa (intrinsic)  Poor penetration  plasmid-mediated changes in transport system  Acquired  Anaerobic bacteria

 Quinolones  Decrease level of major outer membrane proteins  Enterobacteriacae  Cross resistant with unrelated antibiotics

Folate pathway inhibitors  Permeability barriers  Intrinsically  Ps. aeruginosa  Trimethoprim  Acquired  Diminished level of putative proteins  Enterobacteriacae  Trimethoprim  Cross resistant to other unrelated drugs classes  Decrease intracellular penetration  Intrinsic or acquired  Gram negative bacteria  sulphonamides

Resistant due to altered target molecules  Beta-lactam antibiotics  Altered penicillin-binding proteins (PBPS)  Pneumococci  Staph. aureus
 Methicillin Resistant Staph. aureus (MRSA)  PBP2 alteration  Lower affinity to drug  Resistant gene located on transposable genetic element  Should be consider resistant to all beta-lactam drugs

 Aminoglycosides
 Mutation affecting binding to ribosomal target  Streptomycin and kanamycin  Alteration of 12s protein  Control binding of drugs to 30s ribosomal subunit  Staph. aureus, Ps. aeruginosa and mycobacterium

Macrolides-Lincosamides

 Barrier to penetration
 Intrinsic In Enterobacteriacae  Alteration in 50s ribosomal subunit
 Plasmid-mediated  Found on transposons  Staph. aureus., streptococci, Pneumococci, Bacteroides  Demethylation of adenine group of 23s rRNA  Decrease binding to 50s ribosomal subunit

Continuous
 Inducible or constitutive

 Inducible bacteria
 Show resistant to erythromycin and sensitive to Clindomycin  Sub inhibitory concentration of erythromycin  Increase methylase enzyme  Induced cells became resistant to all MLS

 Constitutive cell
 Resistant to MLS without prior antibiotic exposure

Quinolones  Mutation in DNA gryase A and B subunit  E. coli  Rifampin  Single step mutation in B-subunit RNA polymerase  E. coli  Folate pathway inhibitors  Low affinity to dihydropteroate synthetase  Hyper production of enzyme  Gram negative bacteria  Transposons  sulphonamide

Folate pathway inhibitors
 Low affinity to dihydrofolate reductase  Hyper production of enzyme  Trimethoprim

Resistant due to inactivation of antimicrobial
Beta-lactam antibiotics
 Most common and understand mechanisms  Dozens of enzymes  Differing in their substrate profile  Potential for inhibition by B-lactam inhibitors  Physical characteristics  Divided into Four groups based on  Preferred antibiotic substrate  Inhibition by Clavulanic acid  Based on Bush Scheme classification

Classification schemes of Bush
Enzyme class OR Groups characteristic One Example

cephalosporinase not inhibited chromosomal enzyme of Ps. by Clavulanic acid aeruginosa and Ent. Cloacae 2a-2c Pencilliinases and cephalosporinase plasmid mediated Temtype inhibited by Clavulanic acid Staph, Kleb, chromosm Group 3 Metalloenzymes Imipenem hydrolyzing enzyme of Ps. Maltophilia Group 4 Pencilliinases not inhibited chromosom enzyme of Clavulanic acid Ps. Cepacia

Gram positive bacteria  Presented by exoenzyme of Staph, Enterococcus
 Less active against cephalosporin and stable penicillin's  Inhibited by Clavulanic acid NB: Border line Oxacillin resistant (hyper producer of enzyme)  Gram-negative bacteria  Many has been described  Plasmid or chromosomal mediated  Common among nosocomial pathogens  Enterobacteriacae  Ps. aeruginosa and Acinetobacter spp

Plasmid-mediated Blactamase

Such as TEM (Hydrolyzing ampicillin) TEM3 (CTX) hydrolyzing cefotaxime TEM5 (CAZ) hydrolyzing Ceftazidime Inhibited by B-lactamase inhibitors Inducible chromosomally-mediated
 Produced at low concentration before antibiotic exposure  Hyper production after exposure  Resistant to many B-lactam antibiotics  Ps. aeruginosa, Enterobacter

Aminoglycosides
      Acetylation of amino groups Adenylation Phosphorylation of hydroxyl group Plasmid or chromosomal modifying enzymes Enzyme may be located on transposons Staph. aureus or Ps. Aeruginosa

 MLS  Esterase enzyme  Plasmid born  Klebseilla pneumoniae  E. coli  Erythromycin

Chloramphenicol
 Drug inactivation by enzyme  Major mechanisms of resistant  Gram positive bacteria  By acetyltransferase enzyme  Plasmid and inducible in Staph. aureus and Enterococci  Constitutive in gram negative and plasmid located on transposons  Anaerobic bacteria  Enzyme identified as nitroreductase  Bacteroides fragilis

Tetracycline's
 Transposons element mediated resistant  Promoting active drug efflux  Drug detoxification  Bacteroides fragilis Notes: active efflux I a mechanism involving certain protein by
which the cell pumping the drug out

Antimicrobial cross resistant
 Between drugs from same class  Beta-lactam (MRSA)  Aminoglycosides  Inactivating enzymes  Ps aeruginosa  MLS  Staph. Aureus  Different classes  Permeability mutant  Altered outer membrane protein

Different classes

 Gram negative bacteria  Quinolones, trimethoprim, chloramphenicol  Single or many plasmids  Resistant to multiple classes of antibiotics  Reduction of antimicrobial resistant and synergism  Synergism  Two drug combined together  0.25 of MIC of each drug  Exert higher activity than that achieved by each drug alone  Used to prevent resistant  Rifampin and streptomycin for TB

Continuous
 Rifampin plus Vancomycin for endocarditis  B-lactam plus aminoglycosides for –ve  Amphotericin B plus 5-flourocytosine for Cryptococcus neoformans  Empirical for unknown organisms or mixed infection  Metronidazole plus gentamicin

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