Antimicrobial Medications

Antibiotics Antimicrobial drugs naturally produced by microorganisms Penicillium species: Penicillins Cephalosporium specis: cephalosporins Streptomyces species: lincosamides, aminoglycosides, tetracyclines, chloramphenicol

Features of antimicrobial drugs Selective toxicity Therapeutic index Antimicrobial action Bactericidal Bacteristatic Spectrum of activity Broad spectrum Narrow spectrum Combination effects Antogonistic Synergistic Additive

Pharmacokinetics: what happens to the drug in the body? Absorption Tissue distribution Metabolism Route of excretion Rate of elimination

Adverse effects Adverse drug reaction Toxic effects Suppression of normal microbiota

Mechanisms of antimicrobial drugs Inhibition of cell wall synthesis Inhibition of protein synthesis Inhibition of nucleic acid synthesis Inhibition of biosynthetic pathways Disruption of cell membrane integrity

Targets of Cell Wall Synthesis

Penicillin Inhibits formation of tetrapeptide side chains

How organisms degrade penicillins

Family of Penicillins Natural penicillins Penicillinase-resistant penicillins Broad-spectrum penicillins Extended-spectrum penicillins Penicillins plus beta-lactamase inhibitors

Family tree of penicillins

β-lactams Penicillins Cephalosporins Carbapenems Vancomycin Bacitracin

Cephalosporins Derived from fungus, Acremonium cephalosporium Chemical structure makes them resistant to beta-lactamase: low affinity for penicillin binding proteins Grouped into first, second, third, and fourth generation cephalosporins

Vancomycin Binds to the terminal amino acids of the peptide chain of NAM molecules, blocks peptidoglycan formation

Antibiotics that inhibit protein synthesis

Oxazolidinones Reversibly bind to the 50S subunit, interfere with initiation of protein synthesis Used for treating gram positive infections resistant to Beta-lactam drugs and Vancomycin Ex: Linezolid

Aminoglycosides Bactericidal Irreversibly bind to 30S ribosome, cause misreading of the mRNA Transported into cells that actively respire (not effective against ananerobes, streptococci, enterococci) Ex: streptomycin, gentamicin, tobramycin

Tetracyclines & Glycylcyclines Bind reversibly to 30S, block attachment of the tRNA to ribosome Actively transported into bacterial cells Effective against gram positive and gram negative Resistance: due to decrease in uptake or increase in excretion Ex: Doxycycline

Macrolides Reversibly bind to the 50S, prevent continuation of protein synthesis Drug of choice for patients allergic to penicillins Not good for Enterobacteriaceae Ex: Erythromycin, Azithromycin Resistance: enzymes that alter drug, decreased uptake

Inhibition of protein synthesis: Chloramphenicol Rare side effect = irreversible bone marrow suppression Banned in food animals Making a come-back in companion animal medicine due to effectiveness against multi-drug resistant staphylococci

Antibiotics that inhibit nucleic acid synthesis Fluoroquinolones Interferes with function of topoisomerase Rifamycins Blocks prokaryotic RNA polymerase from initiating transcription

Antibiotics that inhibit biosynthetic pathways Sulfonamides Trimethoprims

Sulfonamides (sulfa drugs) First synthetic drugs to treat microbial infections Used to treat urinary tract infections (UTIs) Combination of trimethoprim and sulfamethoxazole (TMP-SMZ) example of synergism

Drugs used together inhibit folic acid synthesis

Tests for microbial susceptibility Kirby-Bauer (disk diffusion method)

Tests for microbial susceptibility Minimum Inhibitory Concentration: MIC Grow bacteria in a serial dilution of the antimicrobial being tested Fixed concentration of bacterial cells Observation of turbidity after 16 hrs-24 hrs of growth Lowest concentration of drug that inhibits growth = MIC

Minimum Inhibitory Concentration Manual broth dilution method Automated broth dilution method E-test

Determining the Minimum Inhibitory Concentration (MIC)

Automated MIC

E-test for MIC

Zone size & MIC values Raw data Meaningless without interpretation Correlation of in vitro results with achievable levels of drug concentration in a live patient Correlation with actual clinical outcome

What resistance looks like…

Mutant Prevention Concentration?

Mechanisms of acquired drug resistance Destruction or inactivation of the drug: drug inactivation enzymes Alteration of target molecule (mutation) Decreased uptake: alteration of porins Increased elimination: efflux pumps

Acquiring resistance Spontaneous mutation Gene transfer R plasmids

Emerging antimicrobial resistance

Streptococcus pneumoniae Altered penicillin binding proteins DNA-mediated transformation

Enterococcus species Gram positive enteric cocci; facultative anaerobes; formerly classified as Group D streptococcus Common cause of nosocomial infections Enterococcus faecalis, Enterococcus faecium Intrinsic resistance: Acquired resistance

Mycobacterium tuberculosis Multidrug-resistant M. tuberculosis Resistance to isoniazid & rifampin Extensively drug-resistant M. tuberculosis Resistance to isoniazid & rifampin + 3 or more of the 2nd line drugs

Enterococcus species: intrinsic resistance Low affinity of penicillin binding proteins for many β-lactam antibiotics, esp. cephalosporins Resistance to potentiated sulfonamides (i.e., trimethoprim-sulfa): able to utilize external sources of folate Low permeability for aminoglycosides Treatment with a cell-wall active drug such as ampicillin is synergistic (allows the drug to get into the cell) UNLESS high-level gentamicin resistance is present

Enterococcus species: acquired resistance High-level gentamicin-resistance: plasmid-encoded inactivating enzymes Tetracycine resistance: efflux pumps, ribosomal protection Macrolide resistance: efflux pumps Vancomycin resistance: altered drug binding site on cell wall

Enterobacteriaceae Gram negative enteric rods Intrinsic resistance to many drugs due to outer membrane β-lactamases: enzymatic inactivation of the lactam ring Extended spectrum β-lactamases (ESBL+) Carbapenem-resistance: enzymatic inactivation

Staphylococcus species Staph aureus Staph pseudintermedius Staph schleiferi Methicillin-resistant staph: penicillinase + altered penicillin-binding proteins with low affinity for β-lactam drugs (mecA gene on R plasmid) Vancomycin-resistant staph

Methicillin-resistant staphylococci MRSA: methicillin-resistant Staph aureus drug resistance + increased pathgenicity MRSP: methicillin-resistant Staph pseudintermedius Acquisition of drug resistance is not associated with acquisition of new virulence factors MRSS: methicillin-resistant Staph scheiferi Drug resistance/no new virulence factors Coagulase-negative MRS

Coagulase test Tests for coagulase enzyme = virulence factor produced by Staphylococcus aureus, Staph. pseudintermedius and Staph. schleiferi subspecies coagulans Important in differentiating potentially pathogenic from non-pathogenic species of staphylococci

Coagulase enzymes Bound coagulase (“clumping factor”) – attached to bacterial cell wall Coagulase enzyme + fibrinogen in plasma → fibrin clot surrounding bacteria: prevents antibody and complement binding, prevents phagocytosis, protects from NETs Free coagulase – secreted enzyme Coagulase enzyme + CRF → conversion of prothrombin to thrombin and fibrinogen to fibrin

Coagulase slide test Rabbit plasma + bacteria: agglutination within 1-2 minutes = positive result Detects only bound coagulase False negatives or equivocal results are common Negative or equivocal tests have to be confirmed with tube test

Coagulase tube test Rabbit plasma + bacteria: coagulation of the plasma = thickening OR formation of fibrin clumps or threads Standard practice = read at 4 hrs, if negative recheck at 24 hrs Tests not read at 4 hrs that are negative at > 4 hrs will have to be repeated because early positive results may revert to a negative result

Coagulase negative staph Common isolates from skin cultures Non-pathogenic commensuals Rarely part of mixed population in deep skin/ wound infections (furuncles) Rarely cause bacteremia or other systemic infections in immune-compromised individuals Commonly carry plasmids with mecA gene

MRS: colonization vs. infection Sharing of plasmids + high antimicrobial use Selecting for MRS population Increasing % of staphylococcal isolates from non-lesional skin and nasal mucosa are methicillin resistant

Responsible drug use Use vs misuse of antimicrobial drugs Responsibilities of health care professionals? Responsibilities of patients? Responsibilities of pet owners? Public education Over the counter antimicrobial drugs Developing countries US feed stores

Antimicrobial Stewardship Increasing drug resistance Fewer drugs in development Drugs being developed don’t have novel targets Drugs being developed are broad spectrum “Bad Bugs, No Drugs” task force 10x20 initiative

Antimicrobial Stewardship 4 D’s of antimicrobial therapy Right Drug, Right Dose, De-escelation to pathogen directed therapy Right Duration of therapy Prevent overuse, misuse and abuse Minimize the development of resistance