ANTIMICROBIAL DRUG

Dr. Muh. Nasrum Massi, Ph.D Bagian Mikrobiologi Fak. Kedokteran Unhas

Antimicrobial Agents

Microbiocidal kills, e.g. bacteriocidal Microbiostatic inhibits growth but does not kill, e.g. bacteriostatic Narrow spectrum drug specific for one genus, e.g. Mycobacterium, or one group of organisms, e.g. Gram negatives Broad spectrum good for all bacteria Selection of Drugs depends on: - microbial sensitivity - side effects - biotransformation will it remain active in the body? - distribution must reach correct site of infection

Antibiotic Resistance Mechanisms:

1. Antibiotic is inactivated by microbial enzymes, e.g. penicillinase will destroy penicillin 2. Antibiotic is prevented from reaching its target, e.g. efflux pumps in the bacterial membrane pump actively pump out drugs; e.g. antibiotic cannot penetrate across the cell wall or membranes 3. Target for the antibiotic has been altered by mutation Genes for several different drug resistance mechanisms are often found on R plasmids which can be readily transferred from bacterium to bacterium

Antibacterial Agents 1. Cell Wall Inhibiters

- excellent selective toxicity (means will only harm bacteria not us!) - targets peptidoglycan a) Penicillins and Cephalosporins Penicillins Pen G, Ampicillin, Amoxicillin, Methicillin Cephalosporins Cefoxitin, Cephalothin, Cephadrine o all have similar structures and inhibit the cross-linking of PG o only work on actively growing and dividing cells when PG is being made

o most differ due to particular side groups which are added to enhance activity, e.g. Pen G only works with Gram positives but once you add the amino group (NH2) to make Ampicillin the drug can get past the bulky LPS on Gram negative cell surfaces and work for them too o many penicillin resistant bacteria exist, make -lactamase (penicillinase) which cleaves the lactam ring structure of these antibiotics, cephalosporins are still left intact b) Vancomycin blocks linkage of NAG and NAM of the PG o used for MRSA (methicillin resistant Staphylococcus aureus) c) Bacitracin same function as vancomycin, very toxic so used externally as an ointment, e.g. Polysporin

2. Protein Synthesis Inhibiters

a. Aminoglycosides Streptomycin, Gentamycin, Neomycin o bind to ribosomes and block translation o good for aerobes, Gram negatives b. Tetracyclines Chlorotetracycline, Doxycycline, Methacycline o binds ribosome and prevents the addition of new amino acids to the growing peptide chain, blocks t-RNA o broad spectrum c. Erythromycin binds ribosome and prevents its movement along the mRNA o broad sprectrum d. Rifampcin (Rifampin) o blocks RNA Polymerase so inhibits transcription no mRNA made o no mRNA means no protein o can penetrate tissues so good for treating abcesses, spinal cords infections, brain infections o orange colour so when excreted can get orange urine, tears, sweat o broad spectrum

3. Membrane Inhibiters Polymyxin B o incorporates into the cell membrane and pokes holes causes cell lysis o very toxic targets our membranes too o used for antibiotic resistant bacteria , e.g. Pseudomonas aeruginosa 4. DNA Replication Inhibiters Quinilones Ciprofloxacin, Norfloxacin, Nalidixic Acid o binds DNA Gyrase and blocks unwinding of DNA o broad sprectrum 5. Sulfa Drugs (Sulfonamides) Para-amino benzoic acid (PABA) analogs Trimethoprim, Sulfamethoxazole PABA is the bacterial metabolic building block to make Folic Acid o Folic Acid is an essential metabolite o Drugs block folic acid production o We get folic acid from our diet and do not produce it like bacteria o Broad spectrum

6. Isoniazid (INH) o very narrow spectrum, only Mycobacterium species, e.g. Mycobacterium tuberculosis o blocks the synthesis of the unusual compounds found only in mycobacterial cell walls (mycolic acids)

Antifungal Agents
- some risk of toxicity as fungi are eucaryotes like us - most target fungal lipid production so tend to be quite specific for fungi 1. Polyenes o Amphoteracin B, Nystatin o Alter the permeability of fungal membranes by inserting into membranes and poking holes o Used topically for yeast infection, e.g. thrush, vaginitis 2. Imidazoles o Miconazole, Ketoconazole o Interfere with fungal sterol production o Side effects liver damage and liver is the site of our sterol production o Yeast infections 3. Griseofulvin o interferes with fungal cell division o taken orally but locates in the skin and nails o used for nail infections o must take for 6-8 months, or until infected nail grows out

Antiprotozoan Agents
- high toxicity problem - very few drugs, all highly specific for one type of protozoa 1. Quinine o Chloroquinine, Fluoroquinine o Antimalarial agent o Interferes with DNA replication of the malaria protozoa during a specific stage of its life cycle 2. Metronidazole (shelf name Flagyl) o inhibits metabolism of anaerobic organisms o used for certain protozoa (Giardia lamblia beaver fever), certain bacteria (Clostridium difficle), and yeast infections

Antiviral Agents
1. Nucleotide Analogs o block viral replication o many, e.g. Acyclovir Herpes infections; AZT targets Reverse Transcriptase of HIV 2. Interferons o just like our naturally produce compound o triggers cells to produce an enzyme that blocks viral replication o best for RNA viruses

INTRODUCTION

Antibiotic: chemical substance produced by mo (mainly fungi) low concentration: inhibit growth of other mo used to treat infectious disease Antimicrobials(AM): a substance that can prevent mo growth or can kill mo: Natural AM, synthetic or semisynthetic AM Chemotherapeuticals: chemicals used to prevent or treat disease. Antibiotic as chemotherapeutical can work as: Bacteriostatic and bactericidal

Desired antibiotic characteristic:

Prevent growth or disrupt pathogenic mo but cause no damage to host. Bactericidal > bacteriostatic. Does not result in bacterial resistance Effective to as much bacteria as possible Does not cause allergy or other side effects. Must be steadily active in plasma, exudate, and other body fluid. Soluble in water and stable.

Antibacterial spectra: Narrow spectra: sensitive to a small amount of bacteria. Broad spectra: sensitive to a large amount of bacteria.

Rational treatment: right target, right dose, right regiment and right method of drug administration For a rational antibiotic treatment, it is important to know: 1. Microbiology : the etiological agent of infection & still sensitive tests are needed 2. Clinical: infected tissue 3. Pharmacology: drug mechanism, toxic characteristic, side effects

ANTIBIOTIC CLASSIFICATION BASED ON ACTIVITY MECHANISM

A.
B. C.

D.

Antimicrobials that inhibit cell wall synthesis Antibiotics that inhibit cell membrane function Antimicrobials that inhibit DNA structure and function Antimicrobials that inhibit protein synthesis

A. Antimicrobial that inhibit cell wall synthesis

Bacterial cell wall contain mucopeptides=peptidoglycan Gram positive bacteria: >> peptidoglycan in comparison to Gram negative bacteria Peptidoglycan: polysaccharide that is cross bound with polypetide. the cross binding is the result of transpeptidase reaction. Inhibiting substance to this enzyme inhibit cross binding formation inhibit cell wall synthesis.

-lactam antibiotic
A.

Penicillin

Synthetic penicillin : methicillin, nafcillin, and isozazolyl penicillin (cloxacillin, dicloxacillin, oxacillin) Penicillin with broad spectrum: aminopenicillin ( ampicillin, amoxycillin), carboxy-penicillin ( : cerbenicillin, ticarcillin), and piperacillin. Penicillin with broad spectrum could not stand penicillinase used together with antibiotics which inhibit laktamase, exp. asam clavulanic, sulbactam.

B. Cephalosporin Produced by Cephalosporin. Structure and mechanism is similar to penicillin, structural basis of synthetic cephalosporin Spectrum: broad. Toxicity : more toxic than penicillin subtly nephrotoxic.

Circulating Cephalosporin:
First generation cephalosporin : oral and injection cefazolin, cephalexin, dan cephalothin. Second generation cephalosporin: ke>an injection: cefamandole, cefaclor, cefoxitin, dan cefuroxime. Third generation cephalosporin: ke>an injection cefotaxime, dan ceftriaxone. Fourth generation cephalosporin: Ke>an injection.

3. Other -laktam antibiotics

Carbapenem: imipenem: very broad spectrum Monobactam: Aztreonam : negative gram aerobic, member of Pseudomonas

B. Other cell wall synthesis inhibiting antibiotics

Cycloserine: autotoxic, should not be used Produced by: Streptomyces orchidceus Active to coliform, proteus & bsl tbc Vancomycin: inhibit peptidoglycan polymerization, narrow spectrum, bactericidal to Gram positive coccus > toxic than penicillin, renal toxicity target

Bacitracin: Produced by: Bacillus subtilis, Prevent peptidoglycan polymerization Bactericidal to Gram positive bacteria Nephrotoxic & autotoxic by parenteral administration > Used topically

C. Antibiotic that inhibit membrane function

Cell membrane functions as osmotic barrier to regulate diffusion of intracellular and extracellular fluid. Substance working on cell membrane is independent and starts soon after bacterial cell encounters the antibacterial agent This substance can hardly differentiate between microbial cell and host tissue cell toxic seldom used.

Antimicrobials that inhibit membrane function

Polymyxin: produced by Bacillus polymyxa (A, B, C, D, and E.) only polymyxin B and polymyxin-E (colisin) are used Mechanism: forms bond with outer part of cell membrane cell structure & osmotic features change. Bactericidal mainly for: P. aeruginosa Not active against fungi.

. Polyenes: Microlide antibiotics selectively inhibit growth of microorganism with sterol containing membrane. Important in this group: antifungal amphotericin B & nystatin yeast, fungi, other eukaryotic cells Does not inhibit procaryotic bacteria that lack sterol in their membranes.

D. Antimicrobials that interfere with DNA structure & function

Only few are clinically used because of its toxicity DNA have two main functions: duplication & transcription All substances interfere with DNA structure effects all levels of metabolism and cell growth

Antimikrob yang mengganggu DNA

Mitomycin: produced by Streptomyces spp. Bacteriostatic because interference in DNA replication Not recommended for treatment becase its toxic Quinolone (Nalidixic acid) & Fluoroquinolone (oflxacine, ciprofloxacin) All are synthetic, and have similar structure and unique mechanism

Obat golongan quinolone:

Nalidixic acid: first introduced from this group it was used for treatment without complication now replaced with new quinolones Norfloxacine & Ciprofloxacine Spectrum:belongs to enterococcus, streptococci, and Pseudomonas Ciprofloxacine is the most potential active for most part of gram negative and gram positive bacteria, used orally for respiratory tract infection and digestive tract infection

Metronindazole: for treatment of anaerobic infection and infection of several protozoans Not useful for facultative anaerobes and aerobes Synthetic as derivative of 5-nitromidazole Novobiocin: Produced by Streptomyces niveus, inhibit DNA multiplication Bactericide maily for Gram positive bacteria. Currently not used for treatment

E. Antimicrobials that inhibit protein synthesis

Protein synthesis is the end result of two processes: RNA synthesis on DNA template (Transcription) RNA dependent protein synthesis (Translation)

1. Antimicrobials that inhibit transcription

In transcription inhibition the following things occur: - change in DNA template

Inhibition in RNA polymerase.

Antibiotics that belong to this group are :

Actinomycin, produced by Streptomyces inhibit RNA polymerase along DNA template. Spectrum: positive Gram and negative Gram bacteriaGram not much used for its toxicity. Rifampicin: produced by Streptomyces mediterrane binds to RNA-polymerase enzyme so there is no RNA formation. Broad spectrum: mainly against Gram positive bacteria m & mycobacteria. Main drug to treat tuberculosis and leprae & for meningitis prophylaxis or meningococci.

2. Antimicrobials that inhibit translation

Inhibiton of translation occur if: a. There is disturbance to sub-unit 30S ribosome b. There is disturbace to sub-unit 50S ribosome.
Inhibition to sub-unit 30S ribosome Mechanism of activity of this group is: Prevent binding of m-RNA to template DNA Inhibit acceptance of aminocyl

Antimicrobials that belong to this group are:

1. Aminoglykosides: similar chemical, pharmacological, toxic, and antimicrobial features. Included: Streptomycin, Neomycin, Kanamycin, Gentamycin, Tobramycin, Amikacin, and Netilmicin. If administered together with antibiotic good synergy, because both are bactericidal. Toxic to the nervous system and kidney.

2. Tetracyclines

Natural antibiotic produced by Streptomyces , (tetracycline and derivatives: chlortetracycline, oxytetracyclin. Synthetic: methacyclin doxycyclin, and minocyclin Bakteriostatic with broad spectrum. Effective for intracellular bacterial infection & alternative drug to penicillin. Tetracycline is not useful for treatment because it is not secreted by kidney. Tetracycline toxicity: GI & candida superinfection & other fungi Not allowed for children & pregnant woman

2. Inhibition to sub-unit 50S ribosome

Inhibition to sub-unit 50S ribosome occur if there is disturbance in: binding of peptidyl-t-RNA peptidyl bond formation translocation

Antimicrobials that belong to this group are:

Chloramphenicol: first antimicrobia to be synthesized in laboratory. Broad spectrum, anaerobic bacteria included. First choice for treatment of tiphoid fever, and meningitis by N. meningitidis. Toxicity : can cause pansitopenia because of obstruction to haematopoetik system

The macrolides a (erythromycin, azitromycin): A special antibiotic containing lactone macrocyclic circle consists of 12-22 carbon atom binding to one or more sugars. Narrow spectrum & toxic to the heart. Antibiotic of this group: Erythromycin: the most important in macrolides. Mainly bacteriostatic, but bactericidal if applied in high concentration. First choice in treatment of Mycoplasma pneumoniae, L. pneumophila, diphteria, and pertussis. Also used as an alternetive to infection of strept. Group A and patients allergic to penicillin.

The linkomycines (lincomycin dan clindamycin). Second spectrum of this drug = erythromycin, although there is no chemical relationship. Both are active against strept. group A, & staph. Producing penicillinase. Clindamycin: lincomycin derivative absorbed better compared to lincomycin. Activity > to anaerobic bacterial infection mainly Bacteroides fragilis. Because of its spectrum, low toxicity, and clinical efficay is most suitable to replace penicillin.

Griseofulvin: specifically for treating fungi with chitinous cell wall Not useful for bacteria and fungi with cell walls composed of cellulose, exp. Yeasts.

F. Antibacteria that inhibit metabolite intermediates

Most of bacteria need folic acid self synthesized using PABA (para-amino-benzoic acid) as basic material. Antibiotic group that inhibit folic acid synthesis and interferes with purine and pyrimidine metabolism are antibiotics with similar structures to PABA, such that folic acid synthesis is disrupted because PABA is replaced by the antibiotic.

Antibacteria that belong to this group are:

Sulphonamides: sulfadiazine, sulfafurazole, sulfamethoxazole, sulfoxazole, sulfamethoxazole Bacteriostatic with broad spectrum. Well reabsorbed in the intestine in adequate amount in the blood after preoral treatment.

Minor features of sulphonamides: Second administration could result in hypersensitivity reaction. High dosis administration: cause haemotological abnormality & crystal formation in genitourinary tract. Causes: an. Haemolitic because of its toxic effect to the liver. Toxic to kidney.

Trimetoprim: structure is similar to hydrofolic acid able to bind to dihydrofolate reductase enzyme needed for folat metabolism. Sulphamethoxazole-trimetoprim (cotrimoxazole): combination synergistic Frequently used for ISK and bacterial gastroenteritis.

Other drug similar to PABA

Sulphones Sulphon derivative known as Dapsone: Treatment of infection by genus Mycob., commonly used for treating leprae. Toxic reaction in dapson administration: an. haemolytic, peripheral neuropathy, dermatitis, and eritema nodosum. P-Aminosalicylic acid (PAS). PAS activity is specifically against M. tbc Bacteriostatic: similar structure to PABA invivo could be inhibited by PABA.

BACTERIAL RESISTANCE TO ANTIBACTERIAL DRUG

A.
B. C.

Intrinsic Resistance Mutational Resistance Acquisition of Resistance genes

Intrinsic resistance Related to bacterial structure: bacterial cell wall permeability. Bacterial natural immunity is carried by genes inside the chromosome. Exp. Immunity of P. aeroginosa to antibiotic. Resistance caused by mutation (Mutational resistance) Bacterial resistance is the result of chromosomal mutation bacteria previously sensitive to antimicrobial drug becomes resistant.

Mutations

Acquisition of resistance genes Bacteria resistance occur as the result of: - movement of plasmid carrying resistance gene ( R ) from other bacteria already resistant to a particular antibiotic. - formation of a new chromosomal gene.

MECHANISM OF BACTERIAL RESISTANCE TO ANTIBIOTIC

Antimicrobial inactivation by enzym activity (Enzymatics inactivation) Resistance occur by change in bacterial cell wall permeability. Change in target molecule. Bacteria alters synthetic pathway utilized by antimicrobials. Antimicrobials is actively excreted out of bacterial cell. Occurrence of tolerance

A. Inactivation of antimicrobials by enzyme activity (Enzymatics inactivation)

Penicillin inactivation: hydrolysis of lactam ring by -lactamases. Aminoglycoside inactivation: alteration caused by acetyltransferase and phophorylase, nucleotidases activities unable to bind to ribosome. Chloramphenicol inactivation: chloramphinecol acetyltransferases have similar activity mechanism to aminoglycoside transferases.

B. Resistance by change in cell wall permeability.

Porin: outer membrane protein specific to Gram negative bacteria plays a role in entrance of hydrophilic antimicrobials. Porin mutation: antimicrobial transportation into the cell is hindered bacteria becomes resistent to multiple antimicrobials (multi-resistant) Lipopolysaccharide (LPS): inhibit the entrance of hydrophilic antimicrobials through the cell wall. Mutant containing small amount of polysaccharide capsules & small amount of LPS will be more permeable to many antimicrobials.

Membr. transport proteins. Mutation of membrane transport protein causes bacterial resistance to tetracycline as the result of reduction of transportation into the cell. Electron transport. The entrance of aminoglycosides into the cell is dependent on electron transport into oxygen. Thats why aminoglycoside is not effective against anaerobic bacteria & facultative bacteria in anaerobic environment, like abscess.

D. Change in target molecule.

Antimicrobial targets could be: Cytoplasm: penicillin-binding protein (PBP), Membr. Cytoplasm: ribosom 30S atau ribosom 50S. Change in target molecule reduced bacterial affinity towards antibacterial. Example Change of 30S ribosome bacterial resistance to aminoglyside. Change of 50S ribosome bacterial resistance to macrolide Change of DNA gyrase: bacterial resistance to quinolone.

E. Bacteria changes synthetic pathway utilized by antimicrobials.

The formation of mutant enzyme change in synthetic pathway. Exp. Bacteria resistant to vancomycin produce an enzyme with low affinity to vancomycin.

F. Antimicrobials are actively excreted from the bacteria. Bacterial resistance to tetracycline is caused by the formation of a new transport protein which could actively excreted antimicrobials out of bacterial cell. G. Occurrence of tolerance With the lost of outer membrane permeability & autolytic enzyme inactivation, changes of bactericidal substance into bacteriostatic substance would occur.

THE DANGER OF IRRATIONAL ANTIMICROBIAL USE

Increase in strains resistant ( R) to antibody. Disease suffered by patients gets more severe. Increase in mortality. Increase in infection. Increase in treatment cost.

ANTIBACTERIAL SENSITIVITY TESTING

Sensitivity testing: to understand bacterial sensitivity towards a particular antibiotic. There are two methods frequently used to observe sensitivity to antibiotic, which are: Tube Dilution Method Diffusion Method

Diffusion Method: only to see resistance Diffusion Method is recommended by the National Committee of Clinical Laboratory Standard (NCCLS) United States.

Tube Dilution Method : to determine MIC & MBC MIC : minimum concentration of antibacterials which could still inhibit the growth of bacteria. MBC :minimum concentration of antibacterials which could still kill bacteria.