Chapter 13 - Concepts in Antimicrobial Therapy

Antimicrobial Susceptibility Testing (AST)Presented By Madeeha Naz1Antimicrobial drugsAntimicrobial drugs are chemicals used to prevent and treat microbial infections.

Antimicrobial drugs can be classified as antibacterial, antifungal, antiviral, or antiparasitic depending on the type of microbe the drug targets.

Origin & Classification of Antimicrobial DrugsThe development of antimicrobial drugs began in the late 1800's with Paul Erlich, a German scientist, who discovered that arsenic compounds were an effective treatment for syphilis. Unfortunately, these compounds were also highly toxic to the patients they were being used to treat. In 1928 Sir Alexander Fleming accidentally stumbled upon the discovery of the wonder drug, penicillin. As he was inspecting a plate of Staphylococcus aureus contaminated with the mold Penicillium, Fleming noticed that the mold had inhibited the growth of the bacterial colonies. He later isolated the compound responsible for this inhibition and named it penicillin.Classification of Antimicrobial drugsbased upon how they are made.based upon their spectrum or range of effectiveness.based upon their primary site of action.Classification based upon how they are made.

Antimicrobial drugs can be naturally-derived, semi-synthetic or synthetic.

Naturally-derived antibiotics.

Penicillin, derived from the fungus Penicillium.Cephalosporin comes from the fungus Cephalosporium.Bacitracin and polymyxin which are produced by the soil bacteria, Bacillus licheniformis and Bacillus polymyxa

Semi-synthetic drugs

Isolated from natural sources, but then chemically modified in a laboratory to make the drugs more effective, longer lasting, easier to administer, and less toxic to the patient.

For example,ampicillin and methicillin are semi-synthetic drugs derived from penicillin.

Synthetic drugs

compounds that are completely artificial and are synthesized in a laboratory. Synthetic drugs tend to have high toxicity to both the microbe and the human host.Classification based upon their spectrumBroad-spectrum drugs are effective against many types of microbes and tend to have higher toxicity to the host.(Tetracycline)

Narrow-spectrum drugs are effective against a limited group of microbes and exhibit lower toxicity to the host. (Penicillin)

Classification based upon their primary mode of action.Antibacterial drugs regulate microbial growth and division by: 1) inhibiting cell wall synthesis;2) disrupting the plasma membrane;3) blocking protein synthesis;4) disrupting metabolic pathways; 5) inhibiting nucleic acid (DNA and RNA) synthesis.Cell wall inhibitors block the synthesis and repair of the bacterial cell wall and include drugs such as penicillin, cephalosporin, vancomycin, bacitracin.

Plasma membrane inhibitorsa drug that disrupts the bacterial plasma such as Polymyxin.

Protein synthesis inhibitors

include those that target the 50S ribosomal subunit (chloramphenicol, erythromycin, and clindamycin) and those that target the 30 ribosomal subunit (aminoglycosides, tetracyclines, and streptomycin).

Nucleic acid inhibitors block replication and transcription and include drugs such as ciprofloxacin (DNA) and rifampin (RNA).

Metabolic inhibitors prevent folic acid synthesis and include sulfa drugs and trimethoprim.

Drug ResistanceMicrobes develop drug resistance due to genetic mutations and by acquiring genes from other microbes via processes like conjugation, transformation, and transduction.

Humans contribute to the development and spread of drug resistance by: 1) not using the appropriate drug for a particular type of infection; 2) not finishing medication or not taking it properly; 3) using antimicrobial drugs when they are not needed. When an antimicrobial drug is present and the majority of drug-sensitive cells die, the drug-resistant cells left behind will be allowed to grow unchecked until they outnumber the drug-sensitive cells.

Why is antimicrobial resistance a global concernfail to respond to the standard treatment, resulting in prolonged illness and greater risk of death.patients remain infectious for a longer time, increasing the risk of spreading resistant microorganisms to others.becoming untreatable and uncontrollableresistant to first-line medicines, more expensive therapies must be used.increases health-care costs and the economic burden to families and societies.Some types of bacteria have developed multiple-drug resistant forms. For example, Staphylococcus aureus has developed resistance to penicillin, methicillin(MRSA: Methicillin-resistant Staphylococcus aureus), and vancomycin (VRSA: Vancomycin-resistant Staphylococcus aureus).DefinitionsMinimum inhibitory concentration(MIC)Lowest concentration of an antimicrobial agent that visibly inhibits the growth of the organism.

Minimum bactericidal concentration (MBC)Lowest concentration of the antimicrobial agent that results in the death of the organism.19Definitions Susceptible SInterpretive category that indicates an organism is inhibited by the recommended dose, at the infection site, of an antimicrobial agentIntermediate IInterpretive category that represents an organism that may require a higher dose of antibiotic for a longer period of time to be inhibitedResistant RInterpretive category that indicates an organism is not inhibited by the recommended dose, at the infection site, of an antimicrobial agent.20Reasons and Indications for Antimicrobial Susceptibility Testing (AST)GoalOffer guidance to physician in selecting effective antibacterial therapy for a pathogen in a specific body sitePerformed on bacteria isolated from clinical specimens if the bacterias susceptibility to particular antimicrobial agents is uncertain21Factors to Consider When Testing is WarrantedBody site of infection

Susceptibility not performed on bacteria isolated from body site where they are normal floraEx. Susceptibility for E. coli is NOT performed when isolated from stool, but IS performed when isolated from blood22Factors to Consider When Testing is WarrantedPresence of other bacteria and quality of specimenEx. Two or more organisms grown in a urine specimenHost statusImmunocompromised patientsAllergies to usual antimicrobials23Selecting Antimicrobial Agents for Testing and ReportingClinical & Laboratory Standards Institute (CLSI)Develop standards, methods, QC parameters, and interpretive criteria for sensitivity testingIf necessary, can alter the breakpoints of the SIR ( susceptible, intermediate, resistant) based on emerging resistance

24Selecting Antimicrobial Agents for Testing and Reporting There are approximately 50 antibacterial agentsFollow CLSI recommendationsEach laboratory should have a battery of antibiotics ordinarily used for testingDrug formulary decided by medical staff, pharmacists, and medical technologists 25Selection of Antimicrobial Agents Generally, labs choose 10-15 antibiotics to test susceptibility for GP organisms and another 10-15 for GN organismsToo many choices can confuse physicians and be too expensivePrimary objectiveUse the least toxic, most cost-effective, and most clinically appropriate agentsCheap in cost, broader-spectrum agents

26Ideal antimicrobial agentAn ideal antimicrobial medication should be: 1) highly toxic to the microbe; 2) non-toxic to the host; 3) not interfere with the ability of the host to fight other diseases; and 4) not lead to the development of drug resistance.Methods of Performing ASTAgar dilution methodBroth macrodilution / Tube dilutionBroth microdilutionDisk diffusion methodGradient diffusion method (E-Test)28Standardization of Antimicrobial Susceptibility TestingInoculum PreparationUse 4-5 colonies NOT just 1 colonyInoculum Standardizationusing 0.5 McFarland standard

29Methods of Performing ASTAgar DilutionDilutions of antimicrobial agent added to agarGrowth on agar indicates MICBroth macrodilution/Tube Dilution TestsTwo-fold serial dilution series, each with 1-2 mL of antimicrobial Too expensive and time consumingMicrodilution Testsplastic trays with dilutions of antimicrobials30Disk Diffusion/ Kirby- BauerProcedureUse a well-isolated, 18-24 hour old organismTransfer organism to a brothEither tryptic soy/sterile salineEnsure a turbidity of 0.5 McFarlandInoculate MH agar by swabbing in three different directions Lawn of growth Place filter paper disks impregnated with anitmicrobial agents on the agarInvert and incubate for 16-18 hours at 35-37 oC in non-CO2

31Disk Diffusion/ Kirby-Bauer During incubation, drug diffuses into agarDepending on the organism and drug, areas of no growth form a zone of inhibitionZones are measured to determine whether the organism is susceptible, intermediate, or resistant to the drug.

32E- test/ Gradient Diffusion MethodMIC on a stickPlastic strips impregnated with antimicrobial on one sideMIC scale on the other sideRead MIC where zone of inhibition intersects E strip scale

33Automated Antimicrobial Susceptibility Test Methods

Detect growth in microvolumes of broth with various dilutions of antimicrobialsDetection via photometric, turbidimetric, or fluorometric methodsTypesBD PhoenixMicroscan WalkawayTREK SensititreVitek 1 and 2

34Automated Antimicrobial Susceptibility Test Methods

AdvantagesIncreased reproducibilityDecreased labor costsRapid resultsSoftwareDetects multi-drug resistancesESBLsCorrelates bacterial ID with sensitivityDisadvantagesCost35Quality Control in Susceptibility TestingReflects types of patient isolates & range of susceptibilityFrequency of quality control depends on method, CLSI, or manufacturerReference strains of QC materialAmerican Type Culture Collection(ATCC) E. coli ATCC* 25922S. aureus ATCC* 25923

36The SuperbugsOrganisms resistant to previously effective drugsMRSA methicillin-resistant Staphylococcus aureusmecA gene codes for a PBP that does not bind beta-lactam antibioticsResistant to oxacillinVancomycinVRE Enterococcus speciesVISA/VRSA- Staphylococcus aureus

37The Superbugs: The Beta-LactamasesGram negative rods that have genes on chromosomes that code for enzymes against certain antimicrobialsESBLs-extended spectrum beta lactamaseResistant to extended spectrum cephalosporins, penicillins, aztreonamExamples: E. coli, KlebsiellaCarbapenemases (CRE)Klebsiella pneumoniaeResistant to penicillins, cephalosporins, carbapenems, and aztreonamCephalosporinasesAmpC enzymeinducibleSPACE organisms38Controlling the SuperbugsLabs Role

Recognize and report isolates recovered from clinical specimens

Methods for identification include automated systems and screening agars

39Controlling the SuperbugsRole of Health Care Workers/FacilitiesHand hygiene with the use of alcohol-based hand rubs or soap and water after patient care/ performing lab procedure.Contact precautions for patients identified as colonized or infected with a superbug.Healthcare personnel education about the methods of transmission, contact precautions, and proper use of hand hygiene Minimization of invasive devices (catheters, etc.)Proper administration of antimicrobial agents where therapy is selected for susceptible organisms for the proper duration.

40Referenceshttp://www.biomerieux-diagnostics.com/servlet/srt/bio/clinical-diagnostics/dynPage?doc=CNL_CLN_PRD_G_PRD_CLN_22http://www.cdc.gov/std/gonorrhea/lab/diskdiff.htmhttp://www.who.int/drugresistance/Antimicrobial_Detection/en/index.htmlKiser, K. M., Payne, W. C., & Taff, T. A. (2011). Clinical Laboratory Microbiology: A Practical Approach . Upper Saddle River, NJ: Pearson Education.Mahon, C. R., Lehman, D. C., & Manuselis, G. (2011). Textbook of Diagnostic Microbiology (4th ed.). Maryland Heights, MO: Saunders.Murray, P. R. (2013,May). Carbapenem-resistant Enterobacteriaceae: what has happened, and what is being done. MLO, 45(5), 26-30.