Antimicrobials

Antibiotics – naturally occurring or synthetic compounds that selectively inhibit microorganisms by targeting a unique property.

often produced by microorganisms (i.e., actinomycetes)

Selective toxicity is achieved by exploiting the differences between the metabolism and structure of the microorganism and the corresponding features of human cells.

Targets of Antibiotics: Cell Wall, Protein Synthesis, Nucleic Acids, Folate, Cell Membrane.

These drugs must achieve adequate concentrations within the body at the site of infection.

Antibiotics are either Bacteriostatic (inhibit growth) or Bactericidal (lethal) to microbes.

Broad spectrum antibiotics are active against several types of microorganisms.

Narrow Spectrum antibiotics are active against one or very few types (e.g., vancomycin against certain gram-p cocci)

Antibiotic Resistance

(1) Reduced Uptake – Ex: gene encoding porin channels in gram-n bugs is mutated as to not allow antibiotics into bug.

(2) Active Efflux – is the process by which channels pump stuff out of the bug. Ex: AcrAB is a pump with low specificity in E. coli. It causes resistance when its transcription is upregulated by a mutation, causing antibiotics to be pumped out of the bug

(3) Target modification – mutation modifies the structure of the enzymes targed by antibiotics.

(4) Enzymatic modification –

Hydrolysis – the bug uses an enzyme to hydrolyze the antibiotic into non-functional components.
Derivatization – This common resistance mechanism against aminoglycosides invovles inactivation of the antibiotic by methylation, acetylation, adenylylation, etc.

(5) Overproduction of Target – targeted enzyme gets overexpressed thus functionally eliminating the antibiotic.

(6) Metabolic bypass of inhibited reaction – rare

(7) Sequestration by protein binding – rare

Cell Wall Inhibitors

(1) b -lactams (penicillins, cephalosporins, monobactams, carbapenems)

Structure/Function

b -lactams act by inhibiting transpeptidases and transglycosylases. Since the stereochemistry of these is similar to D-ala-D-ala, they can bind to the active site of transpeptidase irreversibly and inhibit its activity. They also do the same to transglycosylases (i.e. penicillin binding proteins (PBPs)). Finally, b -lactams activate autolytic enzymes (murein hydrolases) which degrade the peptidoglycan layer. Therefore b -lactams Inhibit cell wall development. b -lactam treated cells die by rupture – without cell wall H₂O rushes in. So b -lactams are bactericidal to actively growing cells only.

(note: some bugs, e.g. S. aureus, are tolerant to the actions of these drugs because the autolytic enzymes are not activated. A tolerant organism is one that is inhibited but not killed by a drug that is usually bactericidal).

Resistance

(a) b-lactamases (enzymatic inactivation) – cleave the four bond ring in the b -lactams. Probably evolved from PBPs.

(b) Alteration of PBPs (target alteration) – either get mutation resulting in ß affinity for b -lactams, or bug acquired a foreign b -lactam resitent PBP (i.e. methicillin resistance in S. aureus).

(2) Glycopeptides (vancomycin, teicoplanin)

Structure/Function

Functional in gram-p bugs only, because it can’t cross gram-n bug cell walls. These bind terminal D-ala-D-ala of peptidoglycan and prevent the transglycosylation reaction. Have become the drug of last resort for infections due to certain organisms such as Staph, Enterococci, and Pneumococci.

Resistance

(a) Bugs can substitute D-lactate or D-serine for D-ala, and vancomycin no longer binds.
(b) Gene clusters vanA, vanB, vanC encode vancomycin resistance. VanA and vanB also encode teicoplanin resistance, they are both found on plasmids in enterococci. Ex: vanA cluster codes for the substitution of the terminal D-ala with D-lactate (as mentioned in #1). If vanA ever transfers to S. aureus this will be a medical disaster.

Protein Synthesis Inhibitors Þ 30S/50S

(1) Aminoglycosides (streptomycin, gentamicin, tobramycin, amikacin, kanamycin, neomycin)

Structure/Function

Aminoglycosides are bactericidal drugs especially useful against gram-n rods. Two modes of action: bind to ribosomal proteins within the 30S subunit of the bacterial ribosome and inhibit protein synthesis, and function to promote misreading of mRNA. The former is more important for bactericidal effects. They also disrupt LPS structure in gram-n bugs. Exhibit synergistic killing in combination with b -lactams.

Resistance

(a) Modification of drugs by plasmid-encoded acetyltransferases (acetylate amino groups), phosphotransferases (phosphorylate hydroxyl groups), and adenyltransferases (adenylate hydroxyl groups). Responsible for the majority of resistance in clincial isolates.

(b) Mutations in 30S ribosomal proteins (Target alteration).

(2) Tetracyclines

Structure/Function

Tetracyclins are a family of antibiotics with bacteriostatic activity (reversible binding) against a variety of gram p/n bugs, mycoplasmas, chlamydiae, and rickettsiae. They inhibit protein synthesis by binding to the 30S ribosomal subunit and blocking the aminoacyl tRNA from entering the A (acceptor) site.

Resistance

(a) Active efflux.
(b) Ribosomal protection by tet M protein. This protein, carried by a conjugative transposon, protects the ribosome from tetracycline by stabilizing the aminoacyl t-RNA (it exhibits homology to elongation factor G (EF-G)).
(c) Enzymatic inactivation in anaerobes. Mechanism unknown.

(3) Chloramphenicol

Structure/Function

Active against broad range of bugs, including gram p/n bugs (including anaerobes).

Bacteriostatic against such bugs as Salmonella typhi, but bactericidal against encapsulated meningitis causing bugs: Haemophilus influenzae, S. pneumoniae, and N. meningitidis. Binds to 50S subunit of the ribosome and inhibits peptidyltransferase reaction.

Resistance

(a) Plasmid encoded resistance via acetyltransferases (i.e. acetylation).
(b) Phosphotransferases (i.e. phosphorylation) – not observed clinically… yet.
(c) Active efflux.

(4) Macrolides (erythromycin, azithromycin, clarithromycin) and Lincosamides (lincomycin, clindamycin)

Structure/Function

Bind to the 23S rRNA within the 50S subunit and block the translocation reaction.

Bacteriostatic actions. Effective against some intracellular pathogens due to the ability concentrate in certain cells.

Resistance

(a) rRNA methyltransferase – ErmF methylates 23S rRNA thereby blocking the binding of the antibiotic. ErmF expression involves translational attenuation, and depends on the presence of erythromycin.
(b) Mutations in 23S rRNA – happens in Mycobacteria only, which have 1 copy of 23S gene.
(c) Macrolide esterase in S. Aureus only.

Inhibitors of nucleic acid synthesis

(1) Quinolones (naladixic acid, ciprofloxacin)

Structure/Function

Quinolones inhibit DNA gyrase by binding to the GyrA subunit and preventing the DNA breakage-reunion reaction mediated by this enzyme. They have a broad spectrum activity.

Resistance

(a) Alterations in DNA gyrase – due to mutations in GyrA or GyrB.
(b) Active efflux (norA gene in staph encodes a quinolone efflux transporter).
(c) Reduced permeability – usually results from a reduction in porins.

(2) Rifampin

Structure/Function

Blocks mRNA synthesis by binding to the b subunit of the RNA polymerase.

Resistance

Results from mutations in the b-subunit (target alteration).

Metabolic inhibitors

(1) Sulfonamides

Structure/Function

Sulfonamides block the synthesis of of tetrahydrofolic acid, which is required in the synthesis of the nucleic acid precursors adenine, guanine, and thymine. They are structural analogs of PABA, and compete with PABA for the active site of dihydropteroate synthetase, an enzyme in the tetrahydrofolic acid pathway. This competitive inhibition can be overcome by an excess of PABA. They are bacteriostatic.

Resistance

Altered dihydropteroate synthetase.

(2) Trimethoprim

Structure/Function

Trimethoprim also inhibits the production of tetrahydrofolic acid, but by a different mechanism. It inhibits the enzyme dihydrofolate reductase. This drug is bacteriostatic, however in a combination with sulfonamides the effect is bactericidal.

Resistance

Due to target alteration by plasmid encoded gene.

Other

(3) Isoniazad (INH)

Structure/Function

INH is a bactericidal drug highly specific for Mycobacterium tuberculosis and other mycobacteria. It may inhibit myolic acid synthesis.

Resistance

(a) Mutations in the katG gene encoding catalase. It is thought that INH is not an active drug but is activated via catalase.
(b) Mutations in inhA, a possible fatty acid biosynthetic gene.

Resistance

Multiple antibiotic resistance (MAR) locus. It was shown that the Tn5 transpon transferred resistance to multiple drugs via the MAR locus. It is thought that this was done by activating various genes including the AcrAB gene and the micF gene. Recall that the AcrAB gene codes for a pump with low specificity in E. coli. It causes resistance when its transcription is upregulated by a mutation, causing antibiotics to be pumped out of the bug. The micF gene is a repressor of porin gene synthesis, resulting in less porins in the outer membrane.

Inducible chromosomal lactamases in gram negatives.