MRSA, ESBLs and Carbapenem Resistance

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

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

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

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

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

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%

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

Gold Standards Detection of mecA gene by PCR Detection of PBP2a

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

Extended-Spectrum-β Lactamase (ESBLs) 16

ESBL-producing isolates can cause nosocomial outbreaks

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

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

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 700603

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.

K. pneumoniae ATCC 700603 (positive control) and E K. pneumoniae ATCC 700603 (positive control) and E. coli ATCC 25922 (negative control) should be used for quality control of ESBL tests

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

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

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)

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

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.

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

Inducible Clindamycin Resistance

Selecting Antimicrobial Agents for Testing andReporting

Test/Report Groups

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