Microbio Exam 2

Antimicrobic agents

• Functional

• Pharmalogic effects

• Combination effects

• spectrum of effectiveness

• 
  

Functional effects of antimicrobic agents

• capable of killing bacteria

• antimicrobic that kills microbes as its mechanism of action

  • Ex. Vancomycin, daptomycin, penicillin

Bacteriostatic

• Capable of inhibiting growth/ reproduction of bacteria

• antimicrobic that inhibits multiplication/growth as its mechanism of action

• This gives the host immune response time to mount a lethal effect on the bacteria

Bacteriostatic examples meds

• clindamycin

• sulphonamids

• trimethoprim

• tetracylcine

• erthryomycin

Pharmologic effects of antimicrobic agents

• Seletctive toxicity

• therapeutic index

Selective toxicity

• Ability to destroy or inhibit the microbe without damage the human host

  • ideal situation

Therapeutic index (TI)

• Ratio of maximum dose tolerated/ minimum dose required

• The higher the TI, the better for the patient

• TI= max dose tolerated/ min dose required

Combination effects of antimicrobic agents

• Use of more than a single antimicrobic as a treatment strategy

• definitions used for any drug-drug interaction

• Indfference

• additive

• synergy

• antagonism

Indifference

• No additional benefit with combiantion compared to each on alone

• 1x+1x=1x effect

Additive

• Effect of one drug adds to effect of second drug

• 1x+1x=2x effect

Synergy

• Combination produces enhanced effect beyond that of either drug alone

• 1x+1x>2x effect

Antagonism

• Combination effect where one drug conteracts the effect of the otehr

• 1x+1x<1x effect

Spectrum of effectiveness for antimicrobics

• Narrow spectrum

  • Broad spectrum

Narrow spectrum

• Effective specifically against one type of organims

• Ex. Penicllin is more effective against gram positive bacteria tha against Gram negative bacteria

Broad spectrum

• Effective against several types of organisms

• Ex. tetracycline is equally effective against gram positve and negative bacteria

Antimicrobial Agent Sources

• Chemical

• Synthetic

• Microbial

• Antibiotic

Chemical sources

• Chemical with antimicrobic action

  • Ex. heavy metals ex silver

Synthetic Antimicrobial Agents

• Commercially synthesizid and marked

• Allows for mass production of antimicrobic agents

• once the structure of an antimicrobic is known, it can be commercially prepared /synthesized

Microbial sources

• Substance produced by a microbe

Antibiotic source

substance produced by a microbe that has the capacity to kill or inhibit other microbes

Penicliin and alexander Flemming

• Reported existence in 1928

• unable to purify it for use as an antibiotic

• actual photograph of Fleming’s culture plate shows how staphylococci in the region of the penicillium colony were killed by some unknown substance in teh mold

  • That was penicillin

Antibiotic sources

• Molds

• actinomycetes

• Bacteria

Microbes against other microbes

Mold antibiotic examples

• Penicllium

  • causes fruit decay: original source of penicillin

• Cephalosporium

  • wheat fungus: original source of cephalosporins

Actinomycetes antibiotic

• Branching bacteria

• Ex. Streptomyces

  • Soil actinomycete: original source of streptomycin

Bacteria antibiotics

• Ex. Baccilus subtilit

• produces many antibiotics

Mechanisms of Action of Antimicrobics

• Interference with essential metabolites

• Structural integrity

• Inhibition of protein synthesis

• Interference with nucleic acid synthesis

• Disruption of the cell membrane

Inhibition of essential Metabolites

• Enzyme inhibition as an anti-metabolic processes

• Folinic acid

• sulfa drugs

one of the antimicrobics major mechanisms of action

Enzyme inhibition as an anti-metabolic process

• Reaction without inhibitor

  • S+E <> ES <> E+P

• Reaction with inhibitor

  • S+E+I>EI+S

    • Competitve inhibition

• Ex. PABA (substrate) and sulfa drugs (inhibitor) compete for binding to the same site on the enzyme

Folinic acid

• Active form of folic acid

• All cells require folic acid for growth to form purines and pyrimidines

• Bacteria must synthesize their own folic acid from p-aminobenxoic acid (PABA)

  • Mammalian cells don’t because it is in their diet

Sulfa drugs (sufonamides)

• Synthetic antimicrobial agents

• broad specturm

• selective toxicity

  • Toxic for bacteria not mammalian

• block bacteria’s ability to produce a metabolite essential for life

• High TI becasue humans use dietary folic acid and bacteria have to synthesize their own

Sulfa Drugs TI

high tolerance by host/ low dose needed against the bacteria

Sulfa drugs disadvantages

• Bacteriastatic instead of bacteriacidal

  • takes longer to reduce bacterial numbers since immune system is needed

• Insoluble at acid pH

  • Precipitate in urine if used to treat UTIS

  • may lead to kidney damage

• many resistant strains present

Sulfa Drug Advantages

• Readily absorbed into tissue

• can cross BBB

  • ex. can treat brain abcessess

Impairment of structural integrity

• Penicillin

• Cephalosporins

• Monobactam group

  • azetronam

• Carbapenems

• Mechanism of action of some antimicrobics is binding to D-ala of peptidoglycan thereby distrupting cell wall synthesis in bacterial populations that are replicating

Penicillins

• has a B lactam ring

• inhibits synthesis of peptide cross links

• Binding to D-ala of petidoglycan thereby disrupting cell wall synthesis in bacterial populations that are replicating

  • Primarily against gram positive bacteria

B lactam ring

• anti bacterial properties

• some bacteria has evolved to produce an enzyme that cleaves the B righ over

  • Makes antimicrobic ineffective

B lactam antimicrobics

• Cephalosporins

• Monobactams

• carbapenems

• penecillin

Response to B- lactamase production

• Make chemical modifications to existing drugs

• Started with Penicillin G

• Became

  • Ampicillin

  • Amoxicillin

  • Methicillin

  • Oxacillin

  • Nafcillin

  • Cloxacillin

  • Piperacillin

  • Carbenicillin

  • Ticarcillin

  • Mezlocillin

Cephalosporins

• Largest and most diverse family of B-lactam antibiotics

• bacrerialcidal

• Mechanism of action: mimic the structure of the D-ala link in peptidoglycan and bind to active site of penicillin binging proteins

  • Structure has been modified over time in attempts to regain antibacterial properites

  • 5 Generations

1st generation cephalosporins

• First cephalosporings

• against gram positive

• Cephalothin

2nd generation cephalosporins

• Extended spectrum of gram negatives and positive

  • Cefoxitin

3rd generation cephalosporins

• Greatly active against Gram negatives

  • Cefotaxime

• Enhanced activity agaisnt beta-lacatamase producing gram positives

4th generation cephalosporins

• True broad spectrum in action

• cefepime

5th generation

• Only beta-lactam antimicrobic with MRSA and VRSA activity

  • Ceftaroline

  • MRSA and VRSA

Monobactam group

• Aztreonam

  • Only clincially available antimicrobic in this class

  • Structurally related to other B-lactam antibiotics

• Activity spectrum: gram negative rods such as Pseudomonas aeruginosa

  • Most strins are from CF patients are susceptible to aztreonam: FDA approved its use by inhalation

  • ineffective against gram positive

Carbapenems

• inhibits cell wall synthesis similar to penicllin

• B-lactam in antibiotics

• Currently available ones have properties similar to 2nd generation cephalosporins

Carbapenems examples

• meropenem

• imipenem

• cilastatin

Imipenem

• one of the broadest spectrum antibiotics available

• covers

  • enterobacteriaceae, pseudomonas, bacteroides fragilis, most gram positive cocci

B lactam antibiotics TI

• High since effect on mammalian cells is nothing

  • There is no peptidoglycan in cells

B lactam antibiotics activity

• Disrupt cell wall

  • bacterialcidal

B-lactam antibiotics

• varies and is dependent of R-group modifications to antibiotic structure

• Effects of Gram positives> gram negs

B lactam antibiotics disadvantages

1. Hypersensitivity reactions- espto penicillin

   1. act as hapten, attaches to albumin, antigenic, can cause immune problems

2. Microbe resistance

   1. Many bacteria have acquired B-lactamase encoding plasmids

Inhibition of protein synthesis

• Binding to 30S ribosomal subunit reversible

  • Bacteriastatic

    • ex. tetracycline

  • Irreversible

    • bacteriacidal

      • Ex. aminoglycosides

• Binding to 50S ribosomal subunti

  • Bacteriocidal

  • Ex. chloroamphenical or eryhtiomycin

Tetracycline

• From streptomyces

  • An actinomycete

• TI low

  • Readily bind serum proteins, therefore high doses are needed

  • not easily excreted which afects max dose require

  • Low/High

• Structure is four fused rings

Tetracycline Activity

• binds 30s ribosomal subunit in reveresible manner

• inhibits tRNA attachment to mRNA codon

• bacteristatic

• broad spectrum

Tetracycline disadvantages

• plasmid- mediated resistance is common

• Hepatotoxic with continuous high doses

• inactivated by food

• pharamcist recommentation

  • take 1 hour before meals or 2 hours after to prevent binding to food and resultant reduced absorption

Erythromycin

• from streptomyces

• Macrolide antibiotic

  • complete macrocylic lactone ring structure

• Broad spectrum but not highly effetive against some gram negatives

Erythromycin activity

• binds to 50S ribosomal subunit ina. reversible manne r

• prevents peptidyl transfer and transloaction of ribosome

• bacteristatic

Chloramphenicol

• From streptomyces

  • Actinomycete

• Broad spectrum

Chloramphenicol activity

prevent peptidyl transfer and peptide bond formation

• Therefore the full protein can’t form because it is only a Nascent chain at that point

Chloramphenicol disadvantages

• Toxic effect at high doses

• interferes with red blood cell development can cause aplastic anemia

Chloramphenicol Advantages

• Readily diffuses into cells and CNS

  • can cross BBB

• useful against CSF infections

• Effective against serious anaerobic brain abscesses

Aminoglycosides examplesAminoglyco

• Kanamkycin

• tobramycin

• gentamicin

• neomycin

aminoglycosides

• From streptomyces

  • actinomycetes

• Broad spectrum

• Disadvantages

  • Ototoxic, nephrotoxic, ineffective against anaerobic bacteria

Aminoglycosides activity

• Bind 30S subunit in an irreversible manner

• change shape of 30S subunit, cause misreading of mRNA codon, terminate protein synthesis

• bactericidal

General mechanisms of interference

• Disrupt transcription

  • DNA- RNA

• Disrupt replication

  • DNA-DNA

• Break DNA molecule

Drugs that interfere with nucleic acid sythesis

• Rifampin

• quinolones

• metronidazole

Rifampin

• Bactericidal

• Binds to RNA polymerase and prevents transcription

  • no mRNA made

• From strepotomyces

Rifampin disadvantages

• resistance develops quickly

• liver toxicity

• usually used in combo with other drugs

Rifampin spectrum

• Narrow for gram positives

• effective against M. tuberculosis

Quinolones

• Nalidixic acid, ciproflaxin, levofloxacin

• Chemical source

  • fluoroquinolone

• Bactericdal

  • Bins to DNA gyrase

  • action is to prevent replication by inhibiting proper DNA alignment

• Broad spectrum

Metronidazole

• Chemical source

• Bacteriacidal

• narrow specrum

Metronidazole activity

• disrupts DNA by making it unstable

• inactive unless it is in reduced form

• when reduced it is able to incorporate into DNA therby disrupting the DNA and breaking the double helical strands

Metronidazole spectrum

• Narrow because redox potential must be low because reduction products cause dsDNA breakage

• effective against anaerobes and gram negative ones

  • And parasites

Disruption of cell membrane process

• binds to phospholipids

• alter cell permeability

• disrupt osmotic integrity

• Ex

  • Bacitracin, polymyxin

• Microbial source

  • from bacillus

• Bacteriacidal activity

Bacitracin

• limited spectrum- gram pos

• Low TI

  • mammalian cells are affected as well

• Very toxic

• Effective and useful as topical ointments for superficial demrmatologic infections

Polymyxin

• limited sprectrum- effective against gram negative

• low TI

  • mammalian cells affected as well as bacterial by these antibiotics

• Very toxic

• effective and useful as topical ointments for superficial dermatologic infectiosn

Common pathways of bacterial resistance

1. Enzymatic degradation or modification of the antimicrobial agent

2. Decreased uptake or accumulation of the antimicrobial agent

3. altered antimicrobial agent

4. Circumvention of the consequences of antimicrobial action

5. uncoupling of antimicrobial agent- target interactions and subsequent effects on bacterial metabolism

6. Any combo of mechanisms

Intrinsic Resistance

naturally coded and expressed by all or almost all strains of that particular bacterial species

Acquired Bacterial resistancce

• Changes in the bacterial genome through mutation or horizontal gene acquisition may lead to changes in the nature of proteins expressed by microbes

• Changes in proteins may lead to alterations in structural and functional features of the bacteria involved, which may result in change leading to resistance against a particular antibiotic

  • AKA acquired resistance

Enzymatic degradation or modification of antimicrobic agent

• innate production of enzymes that inactivate the drug

• Ex. Chloramphenicol- enzymatic modification leads to decreased uptake

  • Chloramphenicol acetyltransferase

Decreased uptake or accumlation of antimicrobic agent

• Extrusion of the drug by chromosomally encoded active exporters

• ex. Tetracyclines

  • Diminished accumulation bc of efflux system

  • altered or protected ribosomal target

  • enzymatic inactivation

Altered antimicrobial target

• Lack of affinity of a drug for the bacterial target

  • Ex

    • Sulfamides and trimethoprim

      • Altered enzymatic targets that no lnger bind to the antibiotic

    • Rifampin

      • Alters the DNA dependent RNA polymerase

Circumvent consequences of antimicrobial action

inaccessibility of the drug into the bacterial cell

Uncoupled antimicrobic-target interactions example

Macrolides and clidamycin

• Altered ribosomal target

  • Dimminished accumulation and enzymatic modification

Emergence of Antimicrobial resistance causes

Mixing of the bacterial gene pool + selective pressure from excessive antimicrobial use and abuse =survival of the fittest

Emergence of Antimicrobial resistance

1. Emergence of new genes

   1. ex. methicillin- resistant staphylococci, vancomycin resistant enterococci

2. Spread of “ old genes” to new hosts

   1. Ex. penicillin resistant N. gonorrhea

3. Mutations of olf genes resulting in a more potent resistance

   1. Ex. b lactamase mediated resistant to advance cephalosporins E.coli and Klebsiella

4. Emergence of intrinsically resistant opportunisit bacteria

   1. Ex. stenotrophomonas maltophilia

Why susceptibility testing is necessary

• Patient has an infectious process and provider needs to know the best choice of antimicrobics to prescribe

• Microbial resistance to antimicrobics is suspected

  • commonly isolated bacteria tend to develop resistance

• Epidemiology

  • Investigate and track spread of infectious agents through a population

  • antimicrobic resistance pattern can be a supportive identifying characteristic

• Testing new drugs

Common antimicrobic susceptibility tests

• Disk diffusion (kirby Bauer susceptibility test)

• Etest

• Automated methods

• Minimal inhibitory concentration (MIC)

• Minimal bactericidal concentration (MBC)

• MIC and MBC parallel studies

Disk Diffusion test

• determines bacterial sensitivity to a panel of antimicrobics

• aka Kirby Bauer test

• recommended method for intermediate and peripheral labs

• Reccommended by NCCLS-USA

• Most thoroughly described disc diffusion method for which interpretive standards have been developed and supported by lab and clinical data

Disk Diffusion steps

1. standardized suspension of bacteria is swabbed over surface of a specific type of agar plate

   1. results in lawn of frowth

2. Paper discs containing single concentrations of antibacterial agents are placed on inoculated surface

3. plates are incubated

4. diameters of zones of inhibitions of bacterial growth are measured

Reading a Disk diffusion test

• Diameters of zones of inhibition of bacterial growth are measured, recorded, and interpreted as S,R, I

S- disk diffusion method

• Sensitive, susceptible

• microbial growth is inhibited at concentrations safely attainable in pt

R- DDM

• Resistant

• microbial growth is present at concentrations above what is safely attainable in the pt-

I- DDM

• Intermediate

• microbial growth is present at concentrations above the susceptible break point, but may be effective under some conditions

Etest

• manual method of providing susceptibility test

  • Ease of test performance and cost similar to DDM

• est. of antimicrobic density gradient in agar plate

• use of thin plastic test strips impregnated on the underside with conc. gradient of specific antimicrobic agent

• Antimicrobic gradient forms in agar around test strip and gives rise to elliptic area of inhibition

  • Forms after overnight incubation

• MIC is determined as where the growth ellipse intersets the scale of E test strip

Automated method

• Microplate methods

• MIC obtained by use of special instrumentation and reagents

• costly for many locations to perform

• For bacterial ID and MIC assays

  • Card with different biochemicals or antimicrobics into which is suspended the microbe in question

  • Instrument incubates and reads cards

Minimal inhibitory concentration

• MIC

• lowest concentration of antimicrobial agent capable of preventing growth of the microbe

• Tube and microplate methods

Minimal bactericidal concentration

• MBC

  • lowest concentration of antimicrobial agent capable of killing the microbe

MIC and MBC parallel studies

• Bacteriocidal drugs

  • MIC are close to MBC

• Bacteriostatic rugs

  • MIC is lower than MBC