Microbiology Basics and Applications to Clinical Practice

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1 Microbiology Basics and Applications to Clinical Practice LINDSEY CHILDS-KEAN, PHARMD, MPH, BCPS CLINICAL ASSISTANT PROFESSOR UNIVERSITY OF FLORIDA COLLEGE OF PHARMACY ST. PETERSBURG, FL Disclosures I do not have a vested interest in or affiliation with any corporate organization offering financial support or grant monies for this continuing education activity, or any affiliation with an organization whose philosophy could potentially bias my presentation 1

2 Pharmacist Objectives By the end of the presentation, learners should be able to: Discuss susceptibility testing and how it impacts treatment Compare and contrast the mechanisms of antimicrobial resistance Explain the role of antibiograms in clinical practice Given a patient case, determine which antimicrobial agent (s) would be most effective for treatment Technician Objectives By the end of the presentation, learners should be able to: Discuss susceptibility testing and how it impacts treatment Compare and contrast the mechanisms of antimicrobial resistance 2

3 The time has come to close the book on infectious diseases. We have basically wiped out infection in the United States. -attributed to Dr. William Stewart, United States Surgeon General Spellberg B. Infect Dis Poverty 2013;2:3. If current trends continue unabated, the future is easy to predict. Some experts say we are moving back to the pre-antibiotic era. No. This will be a post-antibiotic era. -Dr. Margaret Chan, Director-General of the World Health Organization World Health Organization

4 Susceptibility Testing Definitions Term Minimum Inhibitory Concentration (MIC) Susceptible (S) Susceptible-Dose Dependent (SDD) Intermediate (I) Resistant (R) Definition Lowest concentration that inhibits bacterial growth MIC at or below concentration expected with usual antibiotic dosing Susceptibility is dependent on dose of antibiotic used MIC at concentration expected with higher antibiotic dosing MIC higher than concentration expected with therapeutic doses Jorgenson J. Clin Infect Dis 2009;49: Clinical and Laboratory Standards Institute Murray PR. Principles and Practices of Infectious Disease. Ch. 17,

5 Gram Positive Organisms Bacilli Bacillus, Listeria, Corynebacterium, Lactobacillus Gram Positive Streptococcus Pairs & Chains Cocci Enterococcus Clusters Staphylococcus Murray PR. Principles and Practices of Infectious Disease. Ch. 17, Gram Negative Organisms Cocci Neisseria, Moraxella Gram Negative Lactose Fermenter E. coli, Klebsiella, Enterobacter, Serratia, Citrobacter Bacilli Lactose Nonfermenter Pseudomonas Morganella, Proteus, Acinetobacter Murray PR. Principles and Practices of Infectious Disease. Ch. 17,

6 Types of Susceptibility Testing Broth Dilution Tests Antimicrobial Gradient Method Disk Diffusion Test Automated Instrument Systems Jorgenson J. Clin Infect Dis 2009;49: Patient Case 44 year old diabetic male presents with foot wound He describes 3 days of erythema and pain at the site of the wound and subjective fever The physician empirically places the patient on clindamycin He had wound cultures taken and preliminary reports show Gram positive cocci in clusters What organism(s) should you suspect at this point? Staphylococcus spp. 6

7 Patient Case Lewis JS. Clin Infect Dis. 2005;40: Patient Case Drug MIC (mg/ml) Interpretati on Benzylpenicillin > 16 R Oxacillin < 2 S Erythromycin > 8 R Clindamycin > 8 R Tetracycline < 1 S Trimethoprim/ Sulfamethoxazole < 10 S Vancomycin 1 S 7

8 WHEN DOES MIC MATTER? Vancomycin For Staphylococcus sp.: Susceptible range: < 2 mcg/ml Clinical failures seen with MICs > 1mcg/mL Soriano A. Clin Infect Dis 2008;46: Murray KP. Clin Infect Dis 2013;56:

9 Cefepime- S-DD Category for Enterobacteriaceae Prior to 2014, Intermediate Category Method Susceptible Intermediate Resistant MIC < 8 mcg/ml 16 mcg/ml > 32 mcg/ml Zone Diameter > 18 mm mm < 14 mm Now, Susceptible-Dose Dependent Category Method Susceptible* Susceptible-Dose Dependent Resistant MIC < 2 mcg/ml 4-8 mcg/ml > 16 mcg/ml Zone Diameter > 25 mm mm < 18 mm *Susceptible based on cefepime dose of 1 gram every 12 hours Clinical and Laboratory Standards Institute Antibacterial Resistance 9

10 Why Do Bacteria Become Resistant to Antibiotics? We are trying to kill them. They are trying to eat and reproduce What would YOU do if someone was trying to kill you while you were trying to eat and/or reproduce? -Stephen B. Brecher, Ph.D. Brecher SB. Microbiology for Stewardship June Types of Bacterial Resistance Intrinsic Extrinsic (acquired) Opal SM. Principles and Practices of Infectious Disease. Ch. 19,

11 Mechanisms of Bacterial Resistance Levy S. Nat Med 2010;10:S Enzymatic Alteration Classic Example: Beta-lactamase Hydrolyze beta-lactam ring Ambler Class Enzyme Type(s) Antibiotic(s) Affected Examples A Penicillinases ESBLs Carbapenemases PCNs, narrow cephs PCNs, most cephs All beta-lactams TEM, SHV, CTX KPC B Carbapenemases All beta-lactams IMP, VIM, SPM, NDM C Cephalosporinases Most cephalosporins AMP C D Oxacillinases ESBLs Carbapenemases PCNs, narrow cephs PCNs, most cephs All beta-lactams OXA PCNs: penicillins, Cephs: cephalosporins, ESBLs: Extended-spectrum beta-lactamases Opal SM. Principles and Practices of Infectious Disease. Ch. 19,

12 Beta-Lactamases Solution: Add beta-lactamase inhibitor Amoxicillin/clavulanic acid Ampicillin/sulbactam Ticarcillin/clavulanic acid Piperacillin/tazobactam Solution: Use beta-lactam that is stable Solution: Use alternate class(es) of antibiotics Opal SM. Principles and Practices of Infectious Disease. Ch. 19, Clinical Pearl Most beta-lactamases produced by anaerobes are inhibited by beta-lactamase inhibitors No need for double anaerobic coverage! Opal SM. Principles and Practices of Infectious Disease. Ch. 19,

13 Other Enzymatic Alteration Aminoglycoside Resistance-Modifying Enzymes Chloramphenicol Acetyltransferase Macrolide Inactivation Tetracycline Inactivation Opal SM. Principles and Practices of Infectious Disease. Ch. 19, Alteration of Target Site Change in site of action of antibiotic Example: Change in ribosomal binding sites Macrolides, Lincosamides, Streptogramins Gram positive bacteria Mediated by erythromycin ribosome methylation (erm) genes Constitutive or inducible Solution: Recognize inducible resistance in micro Solution: Increase concentration of drug Solution: Use alternate antibiotic class Opal SM. Principles and Practices of Infectious Disease. Ch. 19,

14 Other Target Alterations Beta-lactams Fluoroquinolones Aminoglycosides Oxazolidinones Glycopeptides Sulfonamides Opal SM. Principles and Practices of Infectious Disease. Ch. 19, Efflux Membrane transport system removing antibiotic Major mechanism of resistance in Gram negatives to tetracyclines Inner membrane protein produced by tetracyclineresistance determinant (tet) Inducible by low concentrations of tetracyclines Solution: Ensure adequate concentrations Solution: Use alternate antibiotic class Opal SM. Principles and Practices of Infectious Disease. Ch. 19,

15 Other Efflux Macrolides and Streptogramins Beta-lactams Fluoroquinolones Opal SM. Principles and Practices of Infectious Disease. Ch. 19, Decreased Permeability Ceccarelli M. Frontiers in Bioscience 2009;14:

16 Decreased Permeability Not primary mechanism of resistance Present in resistance to many antibiotic classes Beta-lactams Aminoglycosides Macrolides Fluoroquinolones Solution: Use alternate antibiotic class Opal SM. Principles and Practices of Infectious Disease. Ch. 19, Bacteria Can Mix and Match Multi-drug resistant bacteria can have multiple mechanisms of resistance May require unique combinations of antibiotics May require pharmacokinetic/pharmacodynamic optimization Infectious Disease Consult (+ Infectious Disease Pharmacist) Opal SM. Principles and Practices of Infectious Disease. Ch. 19,

17 Antibiograms Basics Summary of susceptibilities of local isolates Should be done at least annually Include only species with at least 30 isolates Only diagnostic cultures (no screening cultures) Only first isolate included Report percent susceptible (%S) Clinical and Laboratory Standards Institute

18 Goal Guide practitioners in selection of appropriate antimicrobials for EMPIRIC management Can also be used to track resistance rates over time Clinical and Laboratory Standards Institute Example Duke University Medical Center Clinical Microbiology Laboratory,

19 Clinical Scenario Your Pharmacy Clinical Coordinator asks you to reevaluate your preferred aminoglycoside for empiric use. He wants to ensure the greatest efficacy against Pseudomonas aeruginosa. Clinical Scenario Duke University Medical Center Clinical Microbiology Laboratory,

20 Clinical Scenario Which aminoglycoside do you recommend to your Clinical Coordinator? A. Amikacin B. Gentamicin C. Tobramycin Patient Case TG is a 32 year old non-pregnant female who presents to clinic with dysuria but is afebrile and denies flank pain. She is able to tolerate oral medication and has no known allergies. She is diagnosed with an uncomplicated urinary tract infection (UTI). The 2010 IDSA Uncomplicated UTI Guidelines state that sulfamethoxazole/trimethoprim is an option if local resistance rate is not greater than 20%. Gupta K. Clin Infect Dis 2011;52:e103-e

21 Patient Case Duke University Medical Center Clinical Microbiology Laboratory, Patient Case Which of the following is TRUE regarding the empiric use of sulfamethoxazole/trimethoprim in this patient? A. Sulfamethoxazole/trimethoprim local resistance is less than 20%; therefore, it is an option. B. Sulfamethoxazole/trimethoprim should not be used empirically as resistance is greater than 20%. 21

22 Patient Case Continued TG s urine culture came back as shown below: Organism: E. coli Drug Ampicillin Cefazolin Cefepime Ceftriaxone Ciprofloxacin Ertapenem Gentamicin Meropenem Nitrofurantoin Sulfamethoxazole/Trimethoprim Interpretation R R R R R S S S S S Patient Case Continued Which of the following is TRUE regarding TG s urine culture? A. Ciprofloxacin is the preferred regimen for this patient. B. Sulfamethoxazole/trimethoprim is an option based on urine culture results. C. The isolate is only susceptible to IV antibiotics. D. Nitrofurantoin is the only oral antibiotic option. 22

23 Summary There are 4 main mechanisms of antibiotic resistance Susceptibility testing is the mainstay of directing antibiotic therapy Antibiograms help guide clinicians in choosing appropriate empiric antibiotic therapy References Brecher SB. Microbiology for Stewardship. Powerpoint presentation. Antimicrobial Stewardship Task Force Monthly Webinar. June 26, Ceccarelli M. Simulating transport properties through bacterial channels. Frontiers in Bioscience 2009;14: CLSI. Analysis and Presentation of Cumulative Antimicrobial Susceptibility Test Data; Approved Guideline-Fourth Edition. CLSI document M39-A4. Wayne, PA: Clinical and Laboratory Standards Institute; CLSI. Cefepime Breakpoint Change for Enterobacteriaceae and Introduction of the Susceptible- Dose Dependent (SDD) Interpretation Category. July Accessed March 30, 2014 at Duke University Medical Center Clinical Microbiology Laboratory. Summary of antimicrobial susceptibility testing February Accessed April 27, 2014 at Gupat K, Heoton TM, Naber KG, et al. International clinical practice guidelines for the treatment of acute uncomplicated cystitis and pyelonephritis in women: a 2010 update by the Infectious Disease Society of American and the European Society for Microbiology and Infectious Diseases. Clin Infect Dis 2011;52:e103-e120. Jorgensen JH, Ferraro MJ. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin Infect Dis 2009;49:

24 References Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges, and responses. Nat Med 2004;10:S Lewis JS, Jorgensen JH. Inducible clindamycin resistance in staphylococci: should clinicians and microbiologists be concerned? Clin Infect Dis 2005;40: Murray KP, Zhao JJ, Davis SL, et al. Early use of daptomycin versus vancomycin for methicillinresistant Staphylococcus aureus bacteremia with vancomycin minimum inhibitory concentration > 1mg/L: a matched cohort study. Clin Infect Dis 2013;56: Murray PR, Witebsky FG. The Clinician and the Microbiology Laboratory. In Mandell, Douglas, and Bennett s Principles and Practice of Infectious Diseases. 7 th ed Opal SM, Pop-Vicas, A. Molecular Mechanisms of Antibiotic Resistance in Bacteria. In Mandell, Douglas, and Bennett s Principles and Practice of Infectious Diseases. 7 th ed Soriano A, Marco F, Martinez J, et al. Influence of vancomycin minimum inhibitory concentration on the treatment of methicillin-resistant Staphylococcus aureusbacteremia. Clin Infect Dis 2008;46: Spellberg B, Taylor-Blake B. On the exoneration of Dr. William H. Stewart: debunking an urban legend. Infect Dis Poverty 2013;2:3. World Health Organization. Antimicrobial resistance in the European Union and the world. March Accessed April 27, 2014 at Question 1 Which of the following susceptibility terms has the following definition: Susceptibility is determined by the antibiotic dose used. A. Susceptible B. Susceptible-Dose Dependent C. Intermediate D. Resistant 24

25 Question 2 Which of the following is NOT a bacterial mechanism of resistance? A. Efflux B. Enzyme Alteration C. Decreased Permeability D. None of the above- all ARE mechanisms of resistance Question 3 Which of the following is the primary goal of an antibiogram? A. Guide empiric antibiotic therapy B. Guide culture result-directed antibiotic therapy C. Determine most cost-effective antibiotic D. None of the above 25

26 Questions? Lindsey Childs-Kean, PharmD, MPH, BCPS 26

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