1. Antibiotic Resistance and Its Relationship to Antibiotic Use
Antibiotic Stewardship
Curriculum
Developed by:
Vera P. Luther, M.D.
Christopher A. Ohl, M.D.
Wake Forest School of Medicine
With Support from the Centers for Disease Control and Prevention
Targeted for 1st year medical students for use during their microbiology or fundamental infectious diseases or pharmacology (antibiotic) blocks.

2. Objectives
Define antibiotic susceptibility, antibiotic resistance and breakpoint
List four methods for determining antibiotic susceptibility
Discuss factors that contribute to antibiotic resistance
List five bacterial resistance mechanisms and the antibiotic classes each affects
Understand the clinical implications of antibiotic resistance for Staphylococcus aureus, Streptococcus pneumoniae and gram-negative organisms

3. Outline Introduction Key Terms Susceptibility Testing Methods
Factors Contributing to Antibiotic Resistance
Mechanisms of Resistance
Clinical Examples
Conclusion

4. Introduction
Since the first use of antibiotics in the 1930s and 1940s, bacteria quickly adapted and developed mechanisms to escape their effects
Over the following decades, new antibiotics were developed to overcome resistance
Since the 1990s, new antibiotic development has fallen sharply while bacterial resistance continues to increase
Antibiotic resistance is responsible for countless human deaths and billions of dollars in healthcare expenses
Understanding antimicrobial resistance, how bacteria become resistant to antibiotics and the factors that contribute to resistance will be extremely important for us to preserve these important drugs for the future.
Antimicrobial resistance is not a new phenomenon; however, the current magnitude of the problem and the speed with which new resistance phenotypes have emerged elevates the public health significance of this issue to a crisis level

5. Resistance Beyond Typical Bacteria
Introduction
Resistance Beyond Typical Bacteria
Imidazole-resistant Candida spp.
Multidrug-resistant tuberculosis
Multidrug-resistant malaria
Anti-viral resistant influenza
The problem of antimicrobial resistance is far-reaching. Although this presentation focuses on antibiotic resistance in typical bacteria, it is important to recognize viruses and fungi are also quite capable of becoming resistant. Here are a few examples of resistance in other pathogens, including fungi, protozoa, and viruses

6. Outline Introduction Key Terms Susceptibility Testing Methods
Factors Contributing to Antibiotic Resistance
Mechanisms of Resistance
Clinical Examples
Conclusion

7. Key Terms
Antibiotic = A drug that kills or inhibits the growth of microorganisms
Resistant = Somewhat arbitrary designation that implies that an antimicrobial will not inhibit bacterial growth at clinically achievable concentrations
Susceptible = Somewhat arbitrary designation that implies that an antimicrobial will inhibit bacterial growth at clinically achievable concentrations

8. Key Terms
MIC = Minimal inhibitory concentration. Lowest concentration of antimicrobial that inhibits growth of bacteria. Commonly used in clinical lab
MBC = Minimal bactericidal concentration. Concentration of an antimicrobial that kills bacteria. Used clinically only in special circumstances
Breakpoint = The MIC that is used to designate between susceptible and resistant. Arbitrarily set by a committee

9. Outline Introduction Key Terms Susceptibility Testing Methods
Factors Contributing to Antibiotic Resistance
Mechanisms of Resistance
Clinical Examples
Conclusion

10. Minimum Inhibitory Concentration
MIC (Minimal inhibitory concentration), the lowest concentration of antimicrobial that inhibits growth, is an important concept. This test is fundamental to susceptibility testing and determining resistance in bacteria. This slide shows such testing done in test tubes, where the far left tube with a concentration of 100mcg/mL is given through serial dilutions to 0.4 mcg/mL. A standard amount of bacteria is added to each tube and incubated overnight. The lowest concentration of antibiotic showing no bacterial growth (seen as cloudiness) is the minimal inhibitory concentration: in this case 6.25 mcg/mL.
MIC = 6.25 mcg/mL

11. Well Plate for MIC Testing
Automated Methods
Well Plate for MIC Testing
Many Labs Use Automated Testing
In practice, manual antimicrobial susceptibility testing is done in micro-titer plates with 48 or 64 wells. One bacterium can be tested on each plate with each row having serial dilutions of a distinct antibiotic. Thus, several antibiotics can be tested on one plate. Other advantages include less volume of reagent and bacteria are needed. These plates can be reduced in size and used in automated testing machines that photometrically measure growth for each antimicrobial concentration well and determine an MIC within 6-8 hours. This method is now used in most hospital clinical laboratories because of reduced labor, costs, and decreased time to results. The antimicrobial susceptibilities can be determined at the same time the organism is identified.

12. Other Methods for Determining Susceptibility
E-test®
Kirby-Bauer
Disk Diffusion
In addition to broth-dilution, other methods for determining antimicrobial susceptibility include Kirby-Bauer disk diffusion, agar dilution and E-test. E-test and agar dilution can determine MICs directly while Kirby-Bauer disk diffusion is done by measuring the diameter of growth inhibition and comparing to a chart. This test can determine only susceptible vs resistant and not an MIC. These methods are used less-often in most hospital clinical laboratories because of increased labor, costs, and increased time to results. The methods are still used for special MIC testing.
Agar dilution

13. Concept of Breakpoint to Determine Susceptibility
EXAMPLE: Susceptibility testing for a single isolate of Pseudomonas aeruginosa
Antibiotic
MIC
Breakpoint
Susceptibility
Ampicillin
>16 8
Resistant
Gentamicin 2 4
Susceptible
Cephalothin
N/A
Cefepime 32
Cefotaxime 16
16/32
Intermediate
Ceftazidime
Aztreonam
Ciprofloxacin
Amp/Sulbactam
Meropenem
4/8
Pip/tazo
32-64/128
-Breakpoint for intermediate resistance for meropenem is 4 and for piperacillin/tazobactam (pip/tazo) 32
-Pip/tazo is the better choice between the two
-Ciprofloxacin is a poor choice even though the MIC is lowest of the three
Determining the minimal inhibitory concentration of an antibiotic for a specific bacterial strain does not tell you if the antibiotic is susceptible or resistant. MICs are relative while susceptible and resistant are absolute determinations. For any bacteria/antimicrobial pair, susceptible vs resistant is determined by comparing the MIC to an arbitrarily designated “breakpoint”. If the MIC is lower than the breakpoint, the bacterium is considered susceptible. If it is higher, it is considered resistant. Breakpoint numbers are designated by committees of microbiologists and antimicrobial experts. Data on antimicrobial clinical efficacy, pharmacokinetics and achievable antibiotic concentrations in serum and tissue are used to determine this number.
Because susceptibility is determined by comparing the MIC to the breakpoint and the breakpoint is different for each antibiotic, one cannot compare MICs between different antibiotics to determine efficacy or potency of antibiotic. This example shows this clearly for a Pseudomonas aeruginosa clinical isolate.

14. Outline Introduction Key Terms Susceptibility Testing Methods
Factors Contributing to Antibiotic Resistance
Mechanisms of Resistance
Clinical Examples
Conclusion

15. Antibiotic Use Leads to Antibiotic Resistance
Inpatient
Agriculture
Sources of antibiotic resistant bacteria are where antibiotics are used. Most of the resistant pathogens of significance arise in the acute or long-term care facility and the outpatient clinics. Depending on the source, different pathogens are pressured and transmitted. This talk focuses on antimicrobial use in humans. However, it is important to note that almost one half of all of the antibiotic used in North America are used in agriculture. This pressures resistance in food-borne pathogens such as salmonella, campylobacter, and less importantly enterococcus.
Outpatient

16. Reasons for Antibiotic Overuse : Conclusions from 8 Focus Groups
Patient Concerns
Want clear explanation
Green nasal discharge
Need to return to work
Physician Concerns
Patient expects antibiotic
Diagnostic uncertainty
Time pressure
Antibiotic Prescription
Barden L.S. Clin Pediatr 1998;37:665

17. Antibiotic Use Leads to Antibiotic Resistance
Resistant bacteria or their genetic determinates are selected when colonizing or infecting bacteria are exposed to antibiotics
Resistant bacteria can then be transmitted between patients
Highest risk patients:
Immunocompromised
Hospitalized
Invasive devices
(central venous catheters)
Important concept is that antibiotics are the only drug that have direct public health consequences for persons other than the one who received the antibiotic

18. Outline Introduction Key Terms Susceptibility Testing Methods
Factors Contributing to Antibiotic Resistance
Mechanisms of Resistance
Overview
Specific Examples
Antibiotic Degrading Enzymes
Decreased Permeability
Efflux Pumps
Target Alterations
Clinical Examples
Conclusion

19. Cycle of Antibiotic Resistance Acquisition: Bacterial Selection
Selection by Drug X
Under increasing antibiotic selection pressure:
Bacteria resistant to a particular drug are selected and replicate
Different antibiotics select different bacteria but can select resistant phenotypes to other drugs as well
This results in multidrug-resistant (MDR) organisms and increases their total number
Replication
Selection by Drug Y
Reference: Norberg et al. Nature Communications, 2011; 2: 272
Replication
Resistance to:
Drug X
Drug Y
Drug Z

20. Antibiotic Mechanism of Action
Linezolid
Daptomycin
Daptomycin
Daptomycin
Daptomycin
Daptomycin
Daptomycin

21. Mechanisms Of Antibiotic Resistance
Bacteria are capable of becoming resistant through several mechanisms
One or many mechanisms may exist in an organism
Multidrug-resistant bacteria often have multiple mechanisms
Genes encoding resistance may exist on plasmid or chromosome
Alteration in Target Molecule
Decreased
Permeability

22. Mechanisms of Resistance
Antibiotic Degrading Enzymes
Sulfonation, phosphorylation, or esterifictation
Especially a problem for aminoglycosides
β-lactamases
Simple, extended spectrum β-lactamases (ESBL), cephalosporinases, carbapenemases
Confer resistance to some, many, or all beta-lactam antibiotics
May be encoded on chromosome or plasmid
More potent in gram-negative bacteria
Examples: S. aureus, H. influenzae, N. gonorrhoeae, E. coli, Klebsiella sp., Enterobacter sp., Serratia sp., other enteric bacteria, anaerobes

23. Extended Spectrum -lactamases
-lactamases capable of hydrolysing extended spectrum cephalosporins, penicillins, and aztreonam
Most often associated with E. coli and Klebsiella pneumoniae but spreading to other bacteria
Usually plasmid mediated
Aminoglycoside, ciprofloxacin and trimethoprim-sulfamethoxazole resistance often encoded on same plasmid
Has become a significant resistance determinate in acute and long-term care facility enteric pathogens
ESBLs hydrolyze a number of different compounds, especially broad-spectrum cephalosporins, including penicillins. Usually susceptible to carbapenem antibiotics unless the organism also encodes a carbapenemase
ESBLs are most often associated with E coli and Klebsiella
Other pathogens can produce ESBLs as well
Many different types of ESBLs (more than 170 species) have been described 23

24. Class A Carbapenemases
Most common in Klebsiella pneumoniae (KPC)
Also seen in E. coli, Enterobacter, Citrobacter, Salmonella, Serratia, Pseudomonas and Proteus spp.
Very often with multiple other drug resistance mechanisms, resistance profile similar to ESBL but also carbapenem resistant
Became problem in New York City first in and is being increasingly recognized in Mid-Atlantic US.
Spreading across species to other gram-negatives and enterobacteriaceae
Emerging in long-term care facilities 24

25. Mechanisms of Resistance
Decreased Permeability
Pseudomonas spp.
Affects many antibiotics including carbapenems
Efflux Pumps
Pseudomonas spp. (multiple antibiotics)
Tetracyclines
Macrolides

26. Mechanisms of Resistance
Target Alteration
DNA gyrase
Fluoroquinolones
Many gram-negatives, S. pneumoniae
Penicillin-binding protein
Methicillin-resistant S. aureus (MRSA)
Penicillin-resistant S. pneumoniae
Gram positive cell wall
Vancomycin
Enterococcus spp.

27. Mechanisms of Resistance
Target Alteration (cont’d)
Ribosome
Tetracyclines
Macrolides
S. pneumoniae, Staphylococcus sp., N. gonorrhoeae, enteric gram-negative rods

28. Outline Introduction Key Terms Susceptibility Testing Methods
Factors Contributing to Antibiotic Resistance
Mechanisms of Resistance
Clinical Examples
Staphylococcus aureus
Streptococcus pneumoniae
Escherichia coli
Conclusion

29. Illustrative Case 1
A 50 y.o. female with type 2 diabetes mellitus was admitted for an elective total knee replacement. Postoperative day 4: fever to 39ºC and a gray, purulent wound discharge
Gram stain of the exudate showed neutrophils and gram positive cocci in clusters
She was started on IV cefazolin. After two days of therapy she remained febrile and her wound showed little improvement

30. Case 1
Gram
Stain

31. Illustrative Case 1 (cont’d)
Wound cultures yielded S. aureus resistant to penicillin, methicillin, all cephalosporins, erythromycin, tetracycline, gentamicin and ciprofloxacin
The wound was débrided and she was started on IV vancomycin with improvement
After 4 days of vancomycin she was discharged on oral trimethoprim/sulfamethoxazole for 2 weeks
3 months later she experienced a recurrence of symptoms after initial improvement and was diagnosed as having a deep prosthetic joint infection due to MRSA

32. Increase in MRSA Prevalence in US Comparison to Other Drug-Resistant Organisms
There has been some encouraging news, though, that in the last few years invasive infections due to hospital-acquired MRSA are starting to decrease (
It is important to note that MRSA is a problem in both the community-setting as well as the hospital setting.
Wenzel et. al. ICHE 2008;29;1012

33. Surgical Site Infections Impact of Resistance on Clinical Outcomes
Unadjusted
MSSA (N=165)
MRSA (N=121)
P Value
Mortality, 90-day
6.7%
20.7%
P<.001
Length of stay: median days
After surgery
14 (7-25)
23 (12-38)
After infection
10 (4-17)
15 (7-30)
P=.001
Purpose of slide: Antibiotic Resistance increases healthcare costs and mortality. This is not specific to surgical site infections, but this slide provides a good illustration of increased costs & mortality due to MRSA vs MSSA surgical site infections.
The occurrence of MRSA surgical site infection (SSI) has a significant impact on clinical and economic outcomes, notably an increase in postoperative mortality. A cohort study of patients undergoing surgery at a large academic medical center and a regional community hospital from January 1, 1994 to November 30, 2000 prospectively identified those who developed SSI caused by S aureus to assess the impact of methicillin resistance.
Mortality during the 90-day postoperative period was significantly higher in patients with SSI due to MRSA (20.7%) compared with patients with MSSA (6.7%) (OR 3.6; 95% CI ; P<.001).
In a multivariate analysis adjusted for other predictors of mortality (age, The American Association of Anaesthetists (ASA)* score, and duration of surgery), the independent impact of MRSA on 90-day mortality remained significant (OR 3.4; 95% CI ; P=.003).
The median LOS after surgery was significantly longer (P<.001) for patients with MRSA (23 days; range 12-38) vs patients with MSSA (14 days; range 7-25). Similarly, patients with MRSA had a significantly longer LOS after documentation of infection (15 days; range 7-30) than patients with MSSA (10 days; range 4-17) (P=.001). However, after adjusting for other predictors of longer hospital stay, the association between MRSA and LOS was not significant (P=.11).
*The American Association of Anaesthetists (ASA) score subjectively categorizes patients into five subgroups by preoperative physical fitness:
I - A completely healthy patient
II - A patient with mild systemic disease
III - A patient with severe systemic disease that is not incapacitating
IV - A patient with incapacitating disease that is a constant threat to life
E - Emergency case 
Engemann JJ, Carmeli Y, Cosgrove SE, et al. Adverse clinical and economic outcomes attributable to methicillin resistance among patients with Staphylococcus aureus surgical site infection. Clin Infect Dis. 2003;36:
Adjusted* mortality for MRSA (P=0.003)
*Adjusted for other predictors of mortality: age, physical status, duration of surgery.
Engemann JJ, et al. Clin Infect Dis. 2003;36:

34. Glycopeptide Resistant S.aureus
Glycopeptide Intermediate Resistance:
First reported in Japan
Vancomycin MIC 8μg/mL
Still uncommon
All with prolonged vancomycin use due to persistent S.aureus infections
Glycopeptide High Level Resistance:
First report in Michigan in June 2002
Vancomycin MIC >128 ug/ml
Diabetic with peripheral vascular disease and chronic renal failure
Resistance determinant acquired from Vancomycin-resistant Enterococcus (VRE)
Very uncommon
MMWR 2002; 51:565-7
MMWR 2000;48:1165-7
Smith TL et al. NEJM 1999;340:

35. Illustrative Case 2
67 y.o. man with chronic lymphocytic leukemia admitted with sudden onset high fever, rigors, pleuritic chest pain and productive cough
Physical exam and chest x-ray confirmed pneumonia
Started on IV azithromycin

36. Illustrative Case 2 (cont)
Gram stain showed numerous neutrophils and sheets of lancet shaped gram positive diplococci
After 48 hours the patient was still febrile and developed progressive respiratory tract failure
Blood culture from admission yielded S. pneumoniae resistant to penicillin, ceftriaxone, erythromycin and clindamycin
Gram stain

37. 2009 S. pneumoniae Susceptibility CDC ABC surveillance Network
Invasive Isolates (Meningitis, bacteremia, etc.)

38. Macrolide-Resistant S. pneumoniae Prevalence is Increasing in US
While Penicillin, cephalosporin and fluoroquinolone resistance has decreased in the US, likely due to introduction of the conjugate pneumococcal vaccine and prudent antibiotic use macrolide resistant continued to increase in the later 2000s. One possible reason being that macrolide use, especially azithromycin, continued to increase in the US from 2000 to 2005
Prevalence is increasing, but incidence is decreasing. We have seen a huge drop in incidence with PCV7 and expect further decreases with PCV13.
Jenkins S. et al Emerg Infect Dis. 2009;5:1260
Hicks L et. Al. Emerg Infect Dis. 2010;16:896

39. Decreased Susceptibility of S
Decreased Susceptibility of S. pneumoniae to Fluoroquinolones (FQRSP) in Canada Relationship of Resistance to Antibiotic Use
While fluoroquinolone resistance in S. pneumonia was feared 10 years ago the proportion of isolates resistant to this class has not increased much above 1% in the United States, perhaps, in part, due to nonuse of fluoroquinolones in children where S. pneumonia infections are common. As illustrated in this study fluoroquinolone resistance does seem to be correlated with amount of use of this agent.
No reduced susceptibility in children
FQRSP prevalence higher in the elderly and in Ontario
Highest FQ use in the elderly and in Ontario
Chen et. al., NEJM 1999;341:233-9

40. Illustrative Case 3
45 y.o. female seen in clinic for urinary urgency, frequency and dysuria. Her urinalysis is positive for leukocyte esterase and white blood cells. A urine gram stain was performed and showed 2+ white blood cells and many gram-negative rods
Urine culture reveals E. coli, which is resistant to ciprofloxacin and trimethoprim-sulfamethoxazole, but is sensitive to ceftriaxone

41. Community-Acquired Resistant E. Coli
Mostly UTIs
Young healthy women in addition to the elderly
10-20% now resistant to fluoroquinolones
30-50% resistant to trimethoprim-sulfamethoxazole
CTX-M β-lactamases becoming more common
Cause cephalosporin resistance

42. Outline Introduction Key Terms Susceptibility Testing Methods
Factors Contributing to Antibiotic Resistance
Mechanisms of Resistance
Clinical Examples
Conclusion

43. Conclusion
Inappropriate and excessive use of antibiotics is a major factor contributing to emerging antibiotic resistance
Determinants of resistance are selected for by antibiotic use
Multiple mechanisms exist for bacteria to become resistant to antibiotics
Antibiotic resistance is a problem in outpatient and inpatient settings and is a factor in a wide variety of infections
Antibiotic resistance continues to emerge as a serious threat to public health