Antimicrobial agents: mechanisms of action and resistance

Global Sorun: Antibiyotik Direnci (WHO, 2011, Dünya Sağlık Günü Teması)

Antibiotic resistance challenge

Antibiotic resistance challenge

Antibiotic resistance challenge

Temel Terminoloji

• Antibiyotik: Canlı organizmalardan elde edilmiş (geçmişte; günümüzde sentetik dahil) ve (genellikle) bakteriyel infeksiyonların tedavisinde kullanılan maddelerdir.

• Antimikrobiyal: Bakteri, mantar, protozoon, virus gibi mikroorganizmaların üremesini durduran (statik) veya öldüren (sidal) maddelerdir. (Dezenfektan; cansız nesnelere veya vücut-dışı kullanılan) – Antibakteriyel, antiviral, antifungal, antiparazitik

Temel Terminoloji

• Duyarlı: İnfeksiyon bölgesi için önerilen doz kullanıldığında, antimikrobiyal ajanın genellikle erişilebilir konsantrasyonları ile bakteriyel izolatlar inhibe edilir.

• Dirençli (doğal dirençli, kazanılmış dirençli): İnfeksiyon bölgesi için önerilen doz kullanıldığında, antimikrobiyal ajanın genellikle erişilebilir konsantrasyonları ile bakteriyel izolatlar inhibe edilmez; ve/veya; inhibe olduğu zon çapı veya MİK değeri, spesifik direnç mekanizmalarının olabileceği kırılma-noktası aralığına (örn. beta-laktamazlar) düşer ve ajanın bakteriyel izolata karşı etkinliği klinik olarak tedavi araştırmalarında güvenilir olarak gösterilememiştir.

• Orta duyarlı: Ajanın genellikle erişebildiği kan ve doku MİK düzeylerine karşı duyarlı izolatlara kıyasla klinik yanıtın daha az olduğu bakteriyel izolatlar için kullanılır. Droğun fizyolojik olarak konsantre olduğu kompartmanlarda etkili olabilir (örn. idrarda kinolon ve beta-laktamlar). Ayrıca, dar farmakotoksisite marjini olan droglar için, küçük, kontrol edilemeyen teknik faktörlerin, sonuç yorumunda majör uyumsuzluğa neden olmaması için bir tampon aralığıdır.

• Çoklu-dirençli (MDR): ≥ x2 kimyasal sınıf; farklı tanımlamalar var!• Aşırı-dirençli (XDR): XDR-TB (R ≥INH+RMP+FQ+AG) • Pan-drog dirençli (PDR): tartışmalı tanım önerileri var!

Temel Terminoloji

Outline General features of antimicrobials Classification of antimicrobials based on

mechanisms of effect and resistance mechanisms Inhibition of cell-wall synthesis Increase in cell-membrane permeability Inhibition of protein synthesis Inhibition of nucleic acid synthesis Antimetabolites

Terminology

Past, Present, and Future

• Topical antiseptics were ineffective against systemic bacterial infections.• In 1935, the dye prontosil was shown to protect mice against systemic

streptococcal infection and to be curative in patients suffering from such infections.

• It was soon found that prontosil was cleaved in the body to release p-aminobenzene sulfonamide (sulfanilamide), which was shown to have antibacterial activity.

• This first "sulfa" drug started a new era in medicine; chemotherapy

Past, Present, and Future

• Compounds produced by microorganisms (antibiotics) were discovered that inhibit the growth of other microorganisms

• Alexander Fleming was the first to realize the mold Penicillium prevented the multiplication of staphylococci.

• Streptomycin and the tetracyclines were developed in the 1940s and 1950s • Aminoglycosides, semisynthetic penicillins, cephalosporins, quinolones, and other

antimicrobials followed. • All these antibacterial agents greatly increased the range of infectious diseases that

could be prevented or cured. • Although the development of new antibacterial antibiotics has lagged in recent

years, some new classes of agents have been introduced: ketolides (e.g., telithromycin), glycylcyclines (tigecycline), lipopeptides (daptomycin), streptogramins (quinupristin-dalfopristin), and oxazolidinones (linezolid).

• Unfortunately, with the introduction of new chemotherapeutic agents, bacteria have shown a remarkable ability to develop resistance.

• Thus antibiotic therapy is only one weapon, against infectious diseases. • As resistance to antibiotics is not predictable, physicians must rely on local

surveillance (epidemiological) data and guidelines for the initial selection of empirical therapy.

Antibiotics

Toxic for the microorganismal agent In host,

non-toxic, or tolerable toxicity

The effects of this selective toxicity in microorganism; Inhibition of growth (bacteriostatic), or Death (bactericidal)

Bacteriostatic effect is sufficient in most of the patients

After inhibition of growth, immune system eliminates the bacteria

However; In immunodeficient patients, or In serious infections such as,

endocarditis, meningitis, etc. bactericidal effect is required

Antibiotics

Antibiotics Usually, the effect of antibiotics on

bacteria occur by more than one mechanism An antimicrobial agent that distrupts the cell-

wall synthesis may also distrupt the protein or nucleic acid synthesis at certain concentration level

However, usually, one of these mechanisms is more important than others

Mechanisms of antimicrobial effect

Inhibition of cell-wall synthesis Increase in cell-membrane permeability Inhibition of protein synthesis Inhibition of nucleic acid synthesis Antimetabolites

Cell-wall in bacteria

Cell-wall in bacteria

Antimicrobials that act by cell-wall inhibition

Beta-lactams; Penicillins;

Natural penicillins; penicillin G, penicillin V Aminopenicillins; ampicillin, amoxicillin Anti-stafilococcal penicillins; methicillin, nafcillin, oxacillin Anti-pseudomonal penicillins; carbenicillin, ticarcillin, ureidopenicillins ( piperacillin) β-Lactam with β-lactamase inhibitor (combination)

ampicillin-sulbactam, amoxicillin-clavulanate, ticarcillin-clavulanate, piperacillin-tazobactam

Cephalosporins and Cephamycins; First generation (narrow spectrum) ; cephalexin, cephalothin, cefazolin, cephapirin,

cephradine Second generation (expanded-spectrum); cefaclor, cefuroxime Expanded-spectrum cephamycins ; cefotetan, cefoxitin Third generation (broad spectrum); cefixime, cefotaxime, ceftriaxone, ceftazidime,

cefoperazon Fourth generation (extended spectrum); cefepime, cefpirome

Monobactams; aztreonam Carbapenems; imipenem, meropenem, ertapenem

Glycopeptides; vancomycin, teicoplanin Bactericidal for bacteria in exponential growth

Effective on Gr (+)s Some Gr (+)s are resistant intrinsically

Lactobacillus Pediococcus Leuconostoc

Phosfomycin inactivates the enzyme UDP-N-actetylglucosamine-3-

enolpyruvyltransferase NAM production from NAG is blocked in the cytoplasm and cell-wall

synthesis is impaired Ethionamide Bacitracin Isoniazid

Antimicrobials that act by cell-wall inhibition

Cycloserine; Analogue of D-alanine D-alanine-D-alanine bond is prevented; so,

production of cell-wall precursor is avoided Highly toxic Only for use in the treatment of resistant M.

tuberculosis infections

Antimicrobials that act by cell-wall inhibition

Antimicrobials that act by cell-wall inhibition

Cell-wall synthesis is organized by; Transpeptidase, Carboxypeptidase, Endopeptidase,

These enzymes can also bind beta-lactam antibiotics, so;

also called PBPs (penicillin-binding proteins)

In a bacterium that grows; Antibiotics are bound to PBPs Otolytic enyzmes are released Cell-wall cannot be produced

Antimicrobials that act by cell-wall inhibition

Numerous PBPs can be found in a bacterium

These enzymes are classified as PBP-1, PBP-2, PBP-3, etc., respectively, based on the order of their molecular weights

If a PBP with a mid-range weight is discovered later on, it is named as PBP-1a, PBP-1b, etc.

Affinity to PBPs differs among beta-lactam antibiotics

Therefore, efficacy of distinct beta-lactam antibiotics on distinct bacteria are different

Most significant effect of beta-lactams are on transpeptidases

Usually, beta-lactams with an affinity to larger PBP molecules are more potent

Antimicrobials that act by cell-wall inhibition

Aminoglycosides (30S); streptomycin, kanamycin, gentamicin, tobramycin, amikacin

Tetracyclines (30S); tetracycline, doxycycline, minocycline

Macrolides (50S); erythromycin, azithromycin, clarithromycin, spiramycin, roxithromycin

Ketolides (50S); telithromycin

Lincosamide (50S); clindamycin, lincomycin

Chloramphenicol (50S) Streptogramins (50S);

quinupristin-dalfopristin Oxazolidinone (50S);

linezolid Fusidic acid

Antimicrobials that act by inhibition of protein synthesis

By binding to bacterial ribosomes; inhibition of protein synthesis mismatch reads in mRNA codons; incorporation of

wrong aa’s in polypeptides; non-functional proteins mis-reading as a stop codon; termination before a

completed synthesis of a protein

Aminoglycosides

They pass bacterial membrane in two steps In the first step, energy is not required In the second step, energy is required

Their effect is bactericidal Inhibiton of protein synthesis, and Disruption of cytoplasmic membrane

structure

Aminoglycosides

• Active transport with energy and oxygen is inhibited by;– cations like Ca ve Mg,– in anaerobic conditions, – in low pH, – in high osmolarity,

• Therefore, activity is decreased; • in anaerobic conditions of abcesses, or • acidic and hyperosmolar environment of urine

Aminoglycosides

in Gr (-) bacteria, they can enter through the porin chanells in cell-wall by passive diffusion

bind reversibly to the 30S ribosomal subunits, thus block the binding of aminoacyl-transfer RNA (tRNA) to the 30S ribosome-mRNA complex peptide chain is not elongated

Bacteriostatic However, aluminum, calcium, magnesium, iron in the

nutritional uptake, causes chelation with and inactivates tetracycline

Tetracyclines

Chloramphenicol

• by binding reversibly to the peptidyl transferase component of the 50S ribosomal subunit, blocks peptide elongation

• Bacteriostatic

• by their reversible binding to the 23S rRNA of the 50S ribosomal subunit, which blocks polypeptide elongation (through blocking tRNA molecule)

• Bacteriostatic

Macrolides

Lincosamides, Streptogramins:– blocks protein elongation by binding to the 50S ribosome (same site with macrolides)

Mupirocin:– inhibits isoleucine t-RNA synthetase that integrates isoleucin and tRNA

• inhibits bacterial tRNA synthesis, and • protein synthesis

Fucidic acid:– acts as a bacterial protein synthesis inhibitor by preventing the turnover of elongation

factor G (EF-G) from the ribosome

Quinolones nalidixic acid fluoroquinolones

ciprofloxacin, levofloxacin, ofloxacin, norfloxacin, pefloxacin, levofloxacin, moxifloxacin

Rifampin Metronidazole

Antimicrobials that act by inhibition of nucleic acid synthesis

Mechanism of effect; inhibits the enzymes (topoisomerase type II

(gyrase) or topoisomerase type IV) that have functions in;

DNA replication DNA recombination DNA repair

Therefore, inhibits nucleic acid synthesis The DNA gyrase-A subunit is the primary quinolone target in gram-negative

bacteria, whereas topoisomerase type IV is the primary target in gram-positive bacteria

Quinolones

Quinolones

• Bactericidal • There may be other mechanisms effective • The differences in efficacy of distinct quinolones

caused by differences in binding to enyzme-DNA complexes

Rifampicin (Rifampin)

• binds to DNA-dependent RNA polymerase and inhibits the initiation of RNA synthesis

• Bactericidal• Similar enzymes in mammalian cells are less

sensitive to rifampicins

Antimetabolites

• Sulfonamides• Trimethoprim• Dapsone (sulfons)

Antimicrobials that act by an increase in cell-membrane permeability

• Polymyxins and Colistin– act like cationic detergents and damage

cytoplasmic membrane by interacting with the phospholipids and incresing the permeability

– also damage the cell-wall lipopolisaccarides in Gr (-) bacteria

Mechanisms of action

Mechanisms of action

Mechanisms of action

Antimicrobial consumption

Antimicrobial spectrum

Range of activity of an antimicrobial against bacteria

Broad spectrum: inhibits a wide variety of gram-positive and gram-negative bacteria

Narrow spectrum: active against a limited variety of bacteria

Antimicrobial spectrum

Penicillins

Cephalosporins and Cephamycins

Other beta-lactams

Resistance to beta-lactams

• Three general mechanisms: • (1) prevention of the interaction between the antibiotic and the target PBP, • (2) modification of the binding of the antibiotic to the PBP, and • (3) hydrolysis of the antibiotic by β-lactamases.

• (1) seen only in gram-negative bacteria (particularly Pseudomonas species), because they have an outer membrane that overlies the peptidoglycan layer.

• Penetration of β-lactam antibiotics into gram-negative rods requires transit through pores in the outer membrane.

• Changes in the proteins (porins) that form the walls of the pores can alter the size or charge of these channels and result in the exclusion of the antibiotic.

• (2) modification of the β-lactam antibiotic binding to the PBP. • (a) an overproduction of PBP (a rare occurrence), • (b) acquisition of a new PBP (e.g., methicillin resistance in Staphylococcus aureus),

or • (c) modification of an existing PBP through recombination (e.g., penicillin

resistance in Streptococcus pneumoniae) or a point mutation (penicillin resistance in Enterococcus faecium).

Resistance to beta-lactams

• Three general mechanisms: • (1) prevention of the interaction between the antibiotic and the target PBP, • (2) modification of the binding of the antibiotic to the PBP, and • (3) hydrolysis of the antibiotic by β-lactamases.

• (3) Finally, bacteria can produce β-lactamases that inactivate the β-lactam antibiotics.• More than 200 different β-lactamases have been described. • Some are specific for penicillins (i.e., penicillinases), cephalosporins (i.e.,

cephalosporinases), or carbapenems (i.e., carbapenemases), whereas others have a broad range of activity, including some that are capable of inactivating most β-lactam antibiotics.

• Unfortunately, simple point mutations in the genes encoding these enzymes have created β-lactamases with activity against all penicillins and cephalosporins.

• These β-lactamases are referred to as extended-spectrum β-lactamases (ESBLs) and are particularly troublesome because they are encoded on plasmids that can be transferred from organism to organism.

Resistance to Vancomycin

• Some organisms are intrinsically resistant to vancomycin: • Leuconostoc, Lactobacillus, Pediococcus, and Erysipelothrix • some species of enterococci; Enterococcus gallinarum, Enterococcus

casseliflavus • Some species of enterococci (particularly Enterococcus faecium and

Enterococcus faecalis) have acquired resistance to vancomycin. • resistance genes, primarily vanA and vanB, can be carried on plasmids

• More importantly, the gene for vancomycin resistance can be transferred to S. aureus in laboratory.

• a transposon on a multiresistance conjugative plasmid has been transferred in vivo from E. faecalis to a multiresistant S. aureus.

• The transposon then moved from the E. faecalis plasmid and recombined and integrated into the S. aureus multiresistance plasmid.

Resistance to Isoniazid, Ethionamide, Ethambutol, and Cycloserine

• Resistance to these four antibiotics results primarily from reduced drug uptake into the bacterial cell or alteration of the target sites.

Resistance to Aminoglycosides

• Resistance to the antibacterial action of aminoglycosides can develop in one of four ways:

• (1) mutation of the ribosomal binding site, • (2) decreased uptake of the antibiotic into the bacterial cell, • (3) increased expulsion of the antibiotic from the cell, or • (4) enzymatic modification of the antibiotic.

• The most common mechanism of resistance is enzymatic modification of aminoglycosides.

• This is accomplished by the action of phosphotransferases (APHs; seven described), adenyltransferases (ANTs; four described), and acetyltransferases (AACs; four described) on the amino and hydroxyl groups of the antibiotic.

• The other mechanisms by which bacteria develop resistance to aminoglycosides are relatively uncommon.

Resistance to Tetracyclines

• Resistance to the tetracyclines mey be due to; • decreased penetration of the antibiotic into the bacterial cell, • active efflux of the antibiotic out of the cell, • alteration of the ribosomal target site, or • enzymatic modification of the antibiotic.

• Mutations in the chromosomal gene encoding the outer membrane porin protein, OmpF, can lead to low-level resistance to the tetracyclines, as well as to other antibiotics (e.g., β-lactams, quinolones, chloramphenicol).

• Researchers have identified a variety of genes that control the active efflux of the tetracyclines from the cell in different bacteria. This is the most common cause of resistance.

Resistance to Macrolides

• Resistance to macrolides most commonly stems from the methylation of the 23S ribosomal RNA, preventing binding by the antibiotic.

• Other mechanisms of resistance include inactivation of the macrolides by enzymes (e.g., esterases, phosphorylases, glycosidase) or mutations in the 23S rRNA and ribosomal proteins.

Resistance to Quinolones

• Resistance to the quinolones is mediated by chromosomal mutations in the structural genes for DNA gyrase and topoisomerase type IV.

• Other mechanisms include overexpression of efflux pumps that actively eliminate the drug and decreased drug uptake caused by mutations in the membrane permeability regulatory genes.

• Each of these mechanisms is primarily chromosomally mediated.

Antibiotic combinations

To broaden the spectrum for empirical therapy or treatment of polymicrobial infections

To prevent the emergence of resistant microorganisms

To achieve a synergistic killing effect

Antibiotic combinations

A good example is the treatment of tuberculosis

A)Synergism B)Antagonism