(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance Medical Intervention – eradicate infecting pathogen Antibiotics – substances that actively inhibit or kill the

organism

Antibiotics maybe:1. Obtained and purified from other microorganisms2. Chemically synthesized

Antimicrobial agents - natural and synthesized substances- play a central role in the control and management

of infectious diseases1. Antibacterial2. Antifungal3. Antiparasitic4. Antiviral

ANTIMICROBIAL ACTION

Principles

Key Steps for antimicrobial agent to inhibit/kill microorganism:1. Must be in active form - Ensured through pharmacodynamic design of

drug2. Must be able to achieve sufficient levels or

concentrations at site of infection- Depends on pharmacokinetic properties of agent3. Remaining steps relate to direct interactions

between agent and bacterial cell.

Pharmacokinetic properties1. Distribution2. Excretion of agent’s metabolites3. Rate of absorption4. Metabolism

** Most targets of antibacterial agents are intracellular.

Basic Steps Required for Antimicrobial Activity1. Active Drug2. Anatomic approximation3. Surface binding (Adsorption)4. Intracellular uptake5. Target Binding6. A. Growth Inhibition

B. Lysis and Death

Anatomic Distribution of Some Common Antibacterial Agents

1. Norfloxacin and Nitrofurantoin- Therapeutic levels generally not achievable at

Serum-Blood2. Gentamicin, Clindamycin, Norfloxacin,

Nitrofurantoin- Therapeutic levels generally not achievable at

CSF3. Vancomycin and Ciprofloxacin

- Moderate to poor therapeutic levels achievable at CSF

4. Urine- Therapeutic levels not achievable at Urine

Bacteriostatic agents – inhibit bacterial growth but do not kill the organism

Bactericidal agents – usually kill target organisms

** Bactericidal agents are more effective against organisms that are more difficult to control in combination with host’s immune system.

** Primary goal is to optimize a drug’s ability to achieve steps for antimicrobial activity.

** Antimicrobial agents are frequently categorized according to their mode of action.

Mode of Action of Antibacterial Agents

Inhibitors of cell wall synthesiso Beta-lactamso Glycopeptides

Inhibitors of Cell Membrane Functionso Lipopeptideso Polymixins

Inhibitors of Protein Synthesiso Binds to 30S

Aminoglycosides Tetracyclines Glycylcyclines

o Binds to 50S Macrolide-Lincosamide-

Streptogramin Oxazolidinone Chloramphenicol

Inhibitors of DNA & RNA Synthesiso Fluoroquinoloneo Ciprofloxacino Metronidazoleo Rifampin

Inhibitors of Folic Acid Synthesiso Sulfonamideso Trimethoprim

A. Inhibitors of Cell Wall Synthesis

1. Beta-Lactams

- Core - four-member, N containing, Beta-lactam ring

- Largest group of antibacterial agents- Lacks toxicity to humans- Beta-lactam ring

key to the mode of action of Beta-lactams

similar to acyl-D-alanyl-D-alanine- Beta-lactam – binds enzyme, inhibiting

transpeptidation and cell wall synthesis- Penicillin-binding proteins (PBPs)

Acyl-D-Alanyl-D-Alanine – normal substrate for synthesis of linear glycopeptide in the bacterial cell wall.

- Types:1. Penicillin

a. Penicillinb. Ampicillin

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistancec. Meziocillind. Piperacillin

2. Cephalosporinsa. Cefazolinb. Cefuroximec. Cefotetand. Cefotaximee. Ceftriaxonef. Ceftazidimeg. Cefepime

3. Carbapenemsa. Meropenemb. Imipenemc. Doripenem

4. Monobactamsa. Aztreonam

Penicillin-binding Proteins (PBP)- Enzymes essential to produce and maintain their

peptidoglycan layer

B-lactam + PBP = cell wall synthesis is halted due to osmotic instability

Spectrum of Activity – type of bacteria against which a particular antimicrobial agent does and does not have activity.

Spectrum of Activity of B-lactams: gram + and gram – bacteria

4 major categories of B-lactamases1. Class A & D

o Serine peptidases2. Class C

o Cephalosphorinases3. Class B

o Requires zinco Metallo-B-lactamases

B-lactamase should be located on:1. Plasmids2. Transposons3. within integrons4. within chromosome

Integron- large cassette region that contains antibiotic

resistance genes

Integrase - required for movement of integrin from one

genetic element to another\

Resistance Mechanisms1. Genetic mutations in PBP coding sequence2. Genetic Recombination3. Overproduction of PBP4. Acquiring new genetic coding sequence

** Acquired type of B-lactam resistance are commonly found in Gram + bacteria

2. Glycopeptides

- Inhibit bacterial cell wall synthesis by binding to end of peptidoglycan

- Interferes with transpeptidation- Interferes with PBP enzymes that incorporate

precursors into growing cell walla. Vancomycin - Levels should be monitored- Potential for toxicityb. Teicoplanin- Approved- Not available in the US

3. Lipoglycopeptides

- Semi-synthetic molecules- Glycopeptides that contain hydrophobic chemical

groups- Increase cell permeability- Cause depolarization of cell membrane potential- Inhibits transglycosylation

a. Oritavancinb. Telavancin

Transglycosylation

- Necessary for cell wall synthesis by complexing with D-alanyl-D-alanine residues

Spectrum of Activity: VISA (Vancomycin Intermediate Staphylococcus aureus.

B. Inhibitors of Cell Membrane Function

1. Lipopeptides

a. Daptomycin- Recently developed- Bind and disrupts cell membrane of Gram +

bacteria- Has potent activity against Gram + cocci,

MRSA,VRSAb. Bacitracin- Inhibits recycling of certain metabolites required

for maintaining peptidoglycan synthesis- Only used as tropical antibacterial agent- Internal consumption is avoided

2. Polymyxins

- Cyclic polypeptide agents that disrupt cell membranes

- Acts as detergents (interacts with phospholipids and increased permeability)

- Disruption results to leakage of macromolecules and ions

- Not equally effective agains all bacteria and more effective against Gram - and poor activity to Gram +

- Pose a risk for toxicity for humans have membranes

- Last resort microbial agents

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance Spectrum of Activity: Gram -, P. aeruginosa,

Acinetobacter

Major Side effects1. Neurotoxicity2. Nephrotoxicity

C. Inhibitors of Protein Synthesis

1. Aminoglycosides and Aminocyclitols

a. Aminoglycosides- Inhibit protein synthesis- Irreversible binding to protein receptors on 30S

ribosomal unit- Major toxicity:

1. Nephrotixicity2. Auditory/Vestibular Toxicity

- Interrupts the following:o Initial formation of protein synthesis

complexo Reading of mRNA codeo Formation of rRNA complex

Gentamycin- Commonly used aminoglycoside

1. Tobramycin2. Amikacin3. Streptomycin

Spectrum of Activity: Not effective against anaerobic bacteria, Effective against aerobic gram – and Staphylococcus aureus.

2. Macrolide-Lincosdamide-Sreptogramin

- Inhibits protein synthesis by binding to 23s rRNA on bacteria 50S ribosomal subunit

- Disrupts pepide chain (blocks translocation reaction

- Bacteriostatic- Can be bactericidal if organism is low and if used

in high concentrations

Spectrum of Activity: Effective against Gram +, mycoplasma, Treponemes, Rickettsiae & Not effective against Gram –

Quinupristin- dalfopristin- Dual streptogramin- Targets 2 sites of 50S ribosomal subunit- Toxicity is generally low

Lincosamides, Clindamycin, Lincomycin- Bind to 50S ribosomal subunit- Prevent elongation- Interfere with peptidyl transfer

Spectrum of Activity: Effective against Gram +

Spectrogramins- Naturally occurring cyclic peptidesa. Quinupristin-daltopristin

- Enter cell through massive diffusion- Bind irreversibly to 50s subunit that induces

conformational change in the ribosomal structure- Alteration of ribosome- Interferes with peptide bond formation that

disrupts elongation- Toxicity:

1. Low toxicity2. Localized phlebitis – major complication of IV

infusion

Spectrum of Activity: Effective agains Gram + and some Gram -

3. Ketolides

- Consist of:1. Chemical derivatives of erithromycn A2. Other macrolides

- Bind to 23s rRNA of 50S ribosomal subunit- Inhibits protein synthesis

4. Telithromycin

- Maintain activity agains most macroled-resistant gram +

- Does not induce common macrolide resistance mechanism

- Effective against respiratory pathogens & intracellular organisms

- Low toxicity- Major side effects:

1. Gastrointestinal symptoms

Spectrum of Activity: Effective against gram +, some gram -, mycoplasma, mycobacteria, chlamydia, rickettsia, Francisella tularensis

5. Oxazolidinones

- Represented by linezolid- Linezolid

o Synthetic agento Inhibits protein synthesis by interactive

with 23s rRNA in 50S ribosomal subunito Interfere with binding of tRNA for

formylated-methionine- Low toxicity- Gastrointestinal symptoms

Spectrum of Activity: Effective against Gram +, mycobacteria

6. Chloramphenicol

- Inhibits addition of amino acid to growing peptide chain

- Reversibly bind to 50S ribosomal subunit- Inhibits transpeptidation- Highly active- Uses has dwindled because of drug toxicity and

development of new effective and safer drugs- Bone marrow toxicity- Major side effect: Aplasia

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistance

Spectrum of Activity: Effective against Gram – and Gram +

7. Tetracyclines

- Broad spectrum bacteriostatic- Inhibit protein synthesis by binding reversibly to

30S ribosomal subunit

- Toxicity:o Upper gastrointestinal effectso Cutaneous phototoxicityo Photoallergic immune reaction

Spectrum of Activity: Effective against Gram – and Gram +, Intracellular pathogens (Rickettsia and Chlamydia), Protozoa, N. gonorrhoeae, Mycoplasma, Spirochetes

8. Glycylglycines

- Semi-synthetic tetracycline derivatives- Tigecycline

o 12st agent of glycylglycines approved for clinical use

o Inhibits protein synthesis by binding reversibly to 30S ribosomal subunit

o Has advantage of being refractory to most common tetracycline resistance

o Most common effects are: Nausea Vomiting Diarrhea

D. Inhibitors of DNA and RNA Synthesis

1. Fluoroquinolones

- Simply quinolones- Derivatives of Nalidixic acid

o Ciprofloxacino Ofloxacin

- Bind and interfere with DNA gyrase enzymes regulating DNA supercoiling

- Also inhibit topoisomerase IV- Topoisomerase IV

o Unlinks DNA after replication- Potent bactericidal agents- Toxicity:

o Tendinitiso Rupture of Achilles tendon

Spectrum of Activity: Effective against gram – (topoisomerase is targeted) and gram + (DNA gyrase is targeted)

2. Metronidazole

- Antibacterial activity is related to the presence of nitro group in chemical structure

- Nitro groupo Reduced by nitroreductase in bacterial

cytoplasm

o Generates: Autotoxic compounds Free radicals

- Activation requirementso Reduction under low redox potentials

(anaerobic)- Low toxicity- Side effects: Mild gastrointestinal symptoms

Spectrum of Activity: Effective against Anaerobic, Microaerophilic gram – and Protozoans (Trichomonas, Giardia, Entamoeba histolytica)

3. Rifamycin

- Include Rifampin (Rifampicin)- Semisynthetic- Bind to the enzyme

o DNA dependento RNa polymerase

- Inhibits RNA synthesis- Does not effectively penetrate oputer membrane

of gram (-)- Spontaneous mutation- Rifampin are usually used in combination with

other agents- Side effects:

o Gastrointestinal symptomso Hypersensitivity reactions

E. Inhibitors of Other Metabolic Processes

1. Sulfonamides

- Disrupts folic acid pathway- Bind to dihydropteroate synthase- Moderate toxicity- Side effects:

o Vomitingo Nauseao Hypersensitivity reactions

- Antagonistic for other medications

Spectrum of Activity: Effective against Gram – and Gram +

2. Trimethoprim

- Targets folic acid pathway- Inhibits dihydrofolate reductase- Usually combined with sulfonamide

(sulfamethoxazole) to simultaneously attack 2 targets

- Mild toxicity- Adverse side effects:

o Person with AIDS develop side effects more often

3. Nitrofurantoin

- Consists of nitrogroup on heterocyclic ring- Mechanism is diverse and multifaceted- Used to treat uncomplicated UTI- Side effects:

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistanceo Gastrointestinal infections or symptoms

Diarrhea Nausea Vomiting

o Chronic pulmonary conditiono Irreversible pulmonary fibrosis

Spectrum of Activity: Effective against Gram – and Gram +

MECHANISMS OF ANTIBIOTIC RESISTANCE

Principles

Resistance requires: interruption or disturbance of one or more steps

Result is:o Partial loss of effectivenesso Complete loss of effectiveness

Aspects

1. Biologic vs. Clinical2. Environmentally-mediated3. Microorganism-mediated

A. Biologic vs. Clinical Resistance

Biologic resistance- Changes that result in observed reduced

susceptibility to a particular agent

Clinical resistance- Most laboratory methods used to detect

resistance

S. pneumoniae- Inhibited by penicillin

B. Environmentally Mediated Antimicrobial Resistance

Antimicrobial Resistance1. Drug2. Microorganisms3. Environment

**These three factors coexist

Environmentally mediated resistance- Resistance directly resulting from physical or

chemical changes of environment- Alters:

o Microbial agento Microorganisms normal physiological

response

Environmental factors1. pH2. anaerobic atmosphere3. cation concentration4. thymidine content

pH- Erythromycin & Aminoglycosides

o Low pH; Low effect of agent- Tetracycline

o High pH; Low effect of agent

Aminoglycoside – mediated shutdown of bacterial protein synthesis

- Requires intracellular uptake- Driven by oxidative process- No oxygen, low uptake- Affected by Ca++ and Mg++

o P. aeruginosa – has negative net charge in cell membrane

o Cations compete with aminoglycoside for negative charged binding sites

o High cations, low effect of agent

Presence of Metabolites- Enterococci

o Uses thymine and exogenous folic acid to circumvent activities of sulfonamides and trimethoprim

o Absence of metabolites, full susceptibility to antibiotics

C. Microorganism Mediated Resistance- Antimicrobial resistance that results from

genetically encoded traits of the microorganisms

2 subcategories of Organism based resistancea. Intrinsic/inherentb. Acquired

Intrinsic Resistance- Results from the normal genetic, structural or

physiologic state of microorganism- Considered as natural inherited characteristic- Resistance pattern may be predictable- Intrinsic resistance profiles are also useful

markers to aid identification of certain bacteria

Intrinsic Resistance MechanismAnaerobic bact vs.

aminoglycosidesLack of ocidative metabolism

Enterococci vs. aminoglycosides

Gram + vs. Aztreonam Lack of PBPEnterococci vs.

cephalosporinGram – vs. Vancomycin Lack of uptake

P. aeruginosa vs. Sulfonamides

Klebsiella spp vs. ampicillin

Production of enzymes

Stenotrophomonas maltophilia vs. imipenem

Aerobic bact vs. metronidazole

Inability to anaerobically reduce to active form

Lactobacilli and Leuconostoc sp. vs.

Lack of appropriate cell wall precursor target

(Finals) MICROBIOLOGY / Principles of Antimicrobial Action and Resistancevancomycin

Acquired Resistance- Results from altered cellular physiology and

structure caused by changes in microorganism genetic makeup

- Trait associated with specific strains of particular organism

- Unpredictable- May be acquired by:

o Genetic mutationo Acquisition of geneso Combination of mutational and gene

transfer events

COMMON PATHWAYS FOR ANTIMICROBIAL RESISTANCE

1. Enzymatic degradation or modification of antimicrobial agent

2. Decreased uptake or accumulation of antimicrobial agent

3. Altered antimicrobial agent4. Circumvention of the consequences of

antimicrobial action5. Uncoupling of antimicrobial agent-target

interactions and subsequent effects on bacterial metabolism

6. Any combination of mechanisms 1 to 5

Resistance mechanisms against:1. B-lactams

a. Enzymatic destruction- B-lactamase

b. Altered target- Mutational changes in PBP

c. Decreased uptake- Porin channels change in number

2. Glycopeptidesa. Altered target

- Alteration in molecular structure b. Target overproduction

- Excess peptidoglycan3. Aminoglycoside

a. Enzymatic modification- Modifying enzymes

b. Decreased uptake- Porin channels change in number

c. Altered target- Mutational changes in ribosomal

binding site4. Quinolones

a. Decreased uptake- Alterations in the outer membrane

b. Altered target- Changes in DNA gyrase subunits

5. Macrolidesa. Efflux

- Pumps drugs out of cellb. Altered target

- Enzymatic alteration

Factors contributing to the emergence and dissemination of antimicrobial resistance among bacteria1. Emergence of “new” genes2. Spread of “old” genes to new hosts3. Mutations of “old” genes resulting in more potent

resistance4. Emergence of intrinsically resistant opportunistic

bacteria