microbiology basics and applications to clinical …9/29/14 1 microbiology basics and applications...

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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

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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

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“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. 2012.

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Susceptibility Testing

Definitions

Term Definition

Minimum Inhibitory Concentration (MIC) Lowest concentration that inhibits bacterial growth

Susceptible (S) MIC at or below concentration expected with usual antibiotic dosing

Susceptible-Dose Dependent (SDD) Susceptibility is dependent on dose of antibiotic used

Intermediate (I) MIC at concentration expected with higher antibiotic dosing

Resistant (R) MIC higher than concentration expected with therapeutic doses

Jorgenson J. Clin Infect Dis 2009;49:1749-1755. Clinical and Laboratory Standards Institute. 2013. Murray PR. Principles and Practices of Infectious Disease. Ch. 17, 2009.

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Gram Positive Organisms

Gram Positive

Bacilli Bacillus, Listeria,

Corynebacterium, Lactobacillus

Cocci

Pairs & Chains

Streptococcus

Enterococcus

Clusters Staphylococcus

Murray PR. Principles and Practices of Infectious Disease. Ch. 17, 2009.

Gram Negative Organisms

Gram Negative

Cocci Neisseria, Moraxella

Bacilli

Lactose Fermenter

E. coli, Klebsiella,

Enterobacter, Serratia,

Citrobacter

Lactose Non-fermenter

Pseudomonas

Morganella, Proteus,

Acinetobacter

Murray PR. Principles and Practices of Infectious Disease. Ch. 17, 2009.

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Types of Susceptibility Testing

  Broth Dilution Tests

  Antimicrobial Gradient Method

  Disk Diffusion Test

  Automated Instrument Systems

Jorgenson J. Clin Infect Dis 2009;49:1749-1755.

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.

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Patient Case

Lewis JS. Clin Infect Dis. 2005;40:280-285

Patient Case

Drug MIC (mg/mL)

Interpretation

Benzylpenicillin > 16 R

Oxacillin < 2 S

Erythromycin > 8 R

Clindamycin > 8 R

Tetracycline < 1 S

Trimethoprim/ Sulfamethoxazole

< 10 S

Vancomycin 1 S

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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;46:193-200. Murray KP. Clin Infect Dis 2013;56:1562-1569.

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Cefepime- S-DD Category for Enterobacteriaceae

  Prior to 2014, Intermediate Category

  Now, Susceptible-Dose Dependent Category

Method Susceptible Intermediate Resistant

MIC < 8 mcg/mL 16 mcg/mL > 32 mcg/mL

Zone Diameter > 18 mm 15-17 mm < 14 mm

Method Susceptible* Susceptible-Dose Dependent Resistant

MIC < 2 mcg/mL 4-8 mcg/mL > 16 mcg/mL

Zone Diameter > 25 mm 19-24 mm < 18 mm

*Susceptible based on cefepime dose of 1 gram every 12 hours

Clinical and Laboratory Standards Institute. 2013.

Antibacterial Resistance

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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 2013.

Types of Bacterial Resistance

  Intrinsic

 Extrinsic (acquired)

Opal SM. Principles and Practices of Infectious Disease. Ch. 19, 2009.

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Mechanisms of Bacterial Resistance

Levy S. Nat Med 2010;10:S122-129.

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


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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


Clinical Pearl

  Most beta-lactamases produced by anaerobes are inhibited by beta-lactamase inhibitors

  No need for double anaerobic coverage!


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Other Enzymatic Alteration

  Aminoglycoside Resistance-Modifying Enzymes

  Chloramphenicol Acetyltransferase

  Macrolide Inactivation

  Tetracycline Inactivation


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


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Other Target Alterations

  Beta-lactams

  Fluoroquinolones

  Aminoglycosides

  Oxazolidinones

  Glycopeptides

  Sulfonamides


Efflux

  Membrane transport system removing antibiotic

  Major mechanism of resistance in Gram negatives to tetracyclines   Inner membrane protein produced by tetracycline-

resistance determinant (tet)

  Inducible by low concentrations of tetracyclines

  Solution: Ensure adequate concentrations

  Solution: Use alternate antibiotic class


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Other Efflux

 Macrolides and Streptogramins

 Beta-lactams



Decreased Permeability

Ceccarelli M. Frontiers in Bioscience 2009;14:3222.

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Decreased Permeability

  Not primary mechanism of resistance

  Present in resistance to many antibiotic classes   Beta-lactams

  Aminoglycosides

  Macrolides


  Solution: Use alternate antibiotic class


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)


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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)


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Goal

  Guide practitioners in selection of appropriate antimicrobials for EMPIRIC management

  Can also be used to track resistance rates over time


Example

Duke University Medical Center Clinical Microbiology Laboratory, 2013.

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Clinical Scenario

  Your Pharmacy Clinical Coordinator asks you to re-evaluate your preferred aminoglycoside for empiric use.

  He wants to ensure the greatest efficacy against Pseudomonas aeruginosa.

Clinical Scenario


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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-e120. .

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Patient Case


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%.

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Patient Case Continued

  TG’s urine culture came back as shown below:

Organism: E. coli Drug Interpretation

Ampicillin R

Cefazolin R

Cefepime R

Ceftriaxone R

Ciprofloxacin R

Ertapenem S

Gentamicin S

Meropenem S

Nitrofurantoin S

Sulfamethoxazole/Trimethoprim 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.

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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, 2013.

  Ceccarelli M. Simulating transport properties through bacterial channels. Frontiers in Bioscience 2009;14:3222-3238.

  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; 2014.

  CLSI. Cefepime Breakpoint Change for Enterobacteriaceae and Introduction of the Susceptible- Dose Dependent (SDD) Interpretation Category. July 2013. Accessed March 30, 2014 at http://community.clsi.org/micro/wp-content/uploads/sites/15/2013/07/Cefepime-BP-Change-for-Enterobacteriaceae_-Intro-of-SDD-For-Labs.pdf

  Duke University Medical Center Clinical Microbiology Laboratory. Summary of antimicrobial susceptibility testing 2012. February 2013. Accessed April 27, 2014 at http://clinlabs.duke.edu/DukeMicrobiology/Documents/ANTIBIOGRAM2012.pdf

  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;49:1749-1755.

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References   Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges, and responses.

Nat Med 2004;10:S122-129.

  Lewis JS, Jorgensen JH. Inducible clindamycin resistance in staphylococci: should clinicians and microbiologists be concerned? Clin Infect Dis 2005;40:280-285.

  Murray KP, Zhao JJ, Davis SL, et al. Early use of daptomycin versus vancomycin for methicillin-resistant Staphylococcus aureus bacteremia with vancomycin minimum inhibitory concentration > 1mg/L: a matched cohort study. Clin Infect Dis 2013;56:1562-1569.

  Murray PR, Witebsky FG. The Clinician and the Microbiology Laboratory. In Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 7th ed. 2009.

  Opal SM, Pop-Vicas, A. Molecular Mechanisms of Antibiotic Resistance in Bacteria. In Mandell, Douglas, and Bennett’s Principles and Practice of Infectious Diseases. 7th ed. 2009.

  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;46:193-200.

  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 2012. Accessed April 27, 2014 at http://www.who.int/dg/speeches/2012/amr_20120314/en/

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

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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

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Questions?

Lindsey Childs-Kean, PharmD, MPH, BCPS [email protected]