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9/29/14
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
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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
Opal SM. Principles and Practices of Infectious Disease. Ch. 19, 2009.
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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
Opal SM. Principles and Practices of Infectious Disease. Ch. 19, 2009.
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, 2009.
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Other Enzymatic Alteration
Aminoglycoside Resistance-Modifying Enzymes
Chloramphenicol Acetyltransferase
Macrolide Inactivation
Tetracycline Inactivation
Opal SM. Principles and Practices of Infectious Disease. Ch. 19, 2009.
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, 2009.
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Other Target Alterations
Beta-lactams
Fluoroquinolones
Aminoglycosides
Oxazolidinones
Glycopeptides
Sulfonamides
Opal SM. Principles and Practices of Infectious Disease. Ch. 19, 2009.
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
Opal SM. Principles and Practices of Infectious Disease. Ch. 19, 2009.
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Other Efflux
Macrolides and Streptogramins
Beta-lactams
Fluoroquinolones
Opal SM. Principles and Practices of Infectious Disease. Ch. 19, 2009.
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
Fluoroquinolones
Solution: Use alternate antibiotic class
Opal SM. Principles and Practices of Infectious Disease. Ch. 19, 2009.
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, 2009.
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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)
Clinical and Laboratory Standards Institute. 2014.
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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
Clinical and Laboratory Standards Institute. 2014.
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
Duke University Medical Center Clinical Microbiology Laboratory, 2013.
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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
Duke University Medical Center Clinical Microbiology Laboratory, 2013.
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