1. MRSA, ESBLs and Carbapenem Resistance

4. Methicillin-resistant Staph
MRSA, ORSA,
mecA gene
Codes for a supplemental penicillin-binding protein, PBP2a
Homogeneous expression
Easily detected with standard methods
Heterogeneous expression
More difficult to detect

5. Methods of Detection Disk diffusion Broth dilution MIC
Agar dilution MIC
MRSA chromogenic Agar
Etest
Oxacillin agar screen
Cefoxitin disk diffusion
PBP 2a detection
mecA gene detection

6. Oxacillin Disk Interpretation
Interpretive Criteria (in mm) for Oxacillin Disk Diffusion Tests
Susceptible
Intermediate
Resistant
S.aureus
≥ 13mm
11-12mm
≤10
Co NS
≥18
N/A
≤17mm

7. Cefoxitin Disk for mecA-mediated Oxacillin-Resistance Staph. species
Cefoxitin current recommendation (CX,30)
Replaces oxacillin disk to detect mecA-mediated resistance
Cefoxitin easier to read; better inducer
Read with reflected light

8. Testing Cefoxitin to Predict the Presence of mecA
Cefoxitin zone (mm)
Res Susc
S. aureus 21* 22**
S. lugdunensis
CoNS 24* 25**
* Report as oxacillin resistant
** Report as oxacillin susceptible
CoNS, coagulase-negative staphylococci

9. Performance of Cefoxitin and Oxacillin Disk Tests
Sensitivity Specificity S. aureus fox 98% 100% ox 98% 99% CN-S fox 99% 97% ox 99% 89%

10. Oxacillin-salt agar Screen for S. aureus
Medium
Mueller Hinton Agar + 4% NaCl + 6 ug/ml Oxacillin
Inoculum
Direct Suspension; 0.5 McFarland standard
Use 1 ul loop or swab
Incubation
35C for full 24 hours
Presence of either interpreted as RESISTANT
NOT TO BE USED FOR CoNS

11. Gold Standards
Detection of mecA gene by PCR
Detection of PBP2a

13. Vancomycin Resistant S.aureus
T here is only few report of VRSA
There are some reports reduced susceptiblity of S.aureus to vancomycin.

16. Extended-Spectrum-β Lactamase (ESBLs)16

18. ESBL-producing isolates can cause nosocomial outbreaks

19. Which microorganisms are ESBls positive
K.pneumoniae and K.oxytoca
E.coli
Enterobacter spp
Salmonella spp
Morganell morganii,Proteus mirabilis
Serratia marcescens
Pseudomonas aeruginosa

20. ESBL Reporting Rule The rule The message…
“Strains of Klebsiella spp. E. coli, and Proteus mirabilis that produce ESBLs may be clinically resistant to therapy with penicillins, cephalosporins, or aztreonam, despite apparent in vitro susceptibility to some of these agents.”
The message…
Report “confirmed” ESBL-producing strains as R to all penicillins, cephalosporins, and aztreonam

21. ESBL Phenotypic Confirmatory Test
cefotaxime
cefotaxime/clavulanic acid
ceftazidime
ceftazidime/clavulanic acid
Results:
clavulanic acid restores activity of cefotaxime or ceftazidime or both QC
E. coli ATCC 25922; K. pneumoniae ATCC

23. For disk diffusion testing, a > 5 mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulanic acid versus its zone when tested alone confirms an ESBL-producing organism.

24. K. pneumoniae ATCC 700603 (positive control) and E
K. pneumoniae ATCC (positive control) and E. coli ATCC (negative control) should be used for quality control of ESBL tests

25. If an ESBL is detected, all penicillins, cephalosporins, and aztreonam should be reported as resistant, even if in vitro test results indicate susceptibility.

28. AmpC beta-lactamases
AmpC beta-lactamases differ from ESBL’s in that they are cephalosporinases >50 Enzyme and are resistant to beta-lactamase inhibitors
They hydrolyze the cephamycins (eg. cefoxitin), but not the 4th generation cephalosporins (eg. cefepime)
High-level production of AmpC usually causes resistance to all beta-lactams & monobactam (aztreonam ) except carbapenems and 4th generation cephalosporins (eg. cefepime)
Plasmid-mediated AmpC’s have been detected in organisms such as E .coli, Klebsiella sp, Proteus sp and Salmonella sp

30. KPC
Carbapenems are a class of β-lactam antibiotics with a broad spectrum of antibacterial activity.
The following drugs belong to the carbapenem class and are approved for use by health authorities:
Imipenem (FDA approval 1985)
Meropenem (FDA approval 1996)
Ertapenem (FDA approval 2001, since approved for multiple indications)
Doripenem (FDA Approval 2007)
Panipenem/betamipron (Japanese approval 1993)
Biapenem (Japanese approval 2001)

32. Laboratory Approach to KPC Identification
Reagents
1. 5 ml Mueller Hinton broth (MHB) or 0.85% physiological saline
2. Mueller Hinton agar (MHA)
3. 10 μg meropenem or ertapenem susceptibility disk
4. E. coli ATCC 25922: 18–24hr

34. Carbapenemase production is detected by the MHT when the test isolate produces the enzyme and allows growth of a carbapenem susceptible strain (E.coli ATCC 25922) towards a carbapenem disk.
The result is a characteristic clover leaf-like indentation.

36. MHT Positive Klebsiella pneumoniae ATCC BAA-1705
•MHT Negative Klebsiella pneumoniae
ATCC BAA-1706

39. Inducible Clindamycin Resistance

43. Selecting Antimicrobial Agents for Testing andReporting

45. Test/Report Groups

47. Agents administered by oral route only
The following antimicrobial agents should not be routinely reported for bacteria isolated from CSF
Agents administered by oral route only
1st- and 2nd-generation cephalosporins (except cefuroxime parenteral) and cephamycins
Clindamycin
Macrolides
Tetracyclines
Fluoroquinolones