Inducible Clindamycin Resistance (2): Methodology
Specimen:
Pure culture of Staphylococcus sp. (or beta hemolytic streptococci) on blood agar should be used for the D test. The procedure should not be used directly with clinical specimens.
Reagents and Materials:
1. Pure culture of test organism on agar plate incubated 16-24 hours
2. Mueller-Hinton agar, 15 x 100 or 15 x 150 mm plates or Tryptic Soy- 5% sheep blood agar plate
3. 5 mL Mueller-Hinton broth (MHB) or sterile physiologic saline (PSS) in 16 x 125 mm tubes
4. 0.5 McFarland standard
5. Cotton swabs
6. QC strains
7. Erythromycin 15 ug disk
8. Clindamycin 2 ug disk
Procedure:
A. Standard inoculation using Mueller-Hinton broth (MHB)
1. Remove tube of MHB or PSS, disks, and agar plates from refrigerator one to two hours before use so that they may equilibrate to room temperature.
2. Inoculum Preparation
a. Using 1 swab, transfer growth from the surface of the 16-18 hour BAP to the tube of MHB or PSS to equal turbidity of a 0.5 McFarland standard.
b. Vortex well.
c. Use within 1-5 minutes of preparation of suspension.
3. Inoculation
a. Dip a sterile cotton swab into the MHB inoculum and express the excess fluid out by rotation against the side of the tube.
b. Swab the entire surface of the MH agar plate 3 times in 3 different planes. If the surface is visibly wet, be sure the inoculum is dry before adding disks. This should be done within 15 minutes.
4. Application of disks
a. Apply erythromycin and clindamycin disks to agar approximately 15-mm apart using forceps measuring with a ruler from the edge of one disk to the edge of the other. Gently press against disk to be sure it is in complete contact with the agar.
5. Incubate plates within 1-5 minutes of inoculation at 35C in ambient air for 16-20 hours.
Reading and Interpretation:
1. Positive for inducible clindamycin resistance:
Blunting of the zone forming a "D" shaped zone around clindamycin indicates inducible resistance to clindamycin (see photo below). Report the clindamycin with the interpretation of "Inducible clindamycin resistance".
Add the comment :
"This S. aureus is presumed to be resistant based on the detection of inducible clindamycin resistance. Clindamycin may still be effective in some patients".
2. Negative for inducible clindamycin resistance:
No flattening of the clindamycin zone. Report MIC value and report as clindamycin susceptible. Add the comment to the test of the report "This S. aureus does not demonstrate inducible clindamycin resistance in vitro".
Quality Control:
Quality control of the D test should be performed when testing patient isolates.
Notes:
This procedure may also be used to detect inducible clindamycin resistance in beta-hemolytic streptococci.
References:
1. Jorgensen JH, Crawford SA, McElmeel, and Fiebelkorn KR. 2004. Detection of Inducible Clindamycin Resistance of Staphylococci in Conjunction with Performance of Automated Broth Susceptibility Testing. Journal of Clinical Microbiology, 42 (4): 1800-01.
2. Sutcliffe J., A-Tait-Kamradt, L. Wondrack. 1995. Streptococcus pneumoniae and Streptococcus pyogenes resistance to macrolides but sensitive to clindamycin: a common resistance pattern mediated by an efflux system. Antimicrobial Agents Chemother. 40 (8) : 1817-24.
3. Mechanisms of resistance to macrolides and lincosamides: nature of the resistance elements and their clinical implications. 2002. Clin. Infect. Dis. 34: 482-492.
4. Fiebelkorn, K. R., S. A. Crawford, M. L. McElmeel, and J. H. Jorgensen. 2003. Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci. J Clin Microbiol. 41:4740-44
5.Frank, A. L., J. F. Marcinak, P. D. Mangat, J. T. Tjhio, S. Kelkar, P. C. Schreckenberger, and J. P. Quinn. 2002. Clindamycin treatment of methicillin-resistant Staphylococcus aureus infections in children. Pediatr Infect Dis J. 21:530-34.
6. Jorgensen, J. H., S. A. Crawford, M. L. McElmeel, and K. R. Fiebelkorn. 2003. Practical method for detection of inducible clindamycin resistance of staphylococci in conjunction with performance of automated susceptibility testing. Abstract #D241, ICAAC, Chicago, IL.
7. Sanchez, M. L., K. K. Flint, and R. N. Jones. 1993. Occurrence of macrolide-lincosamidestreptogramin resistances among staphylococcal clinical isolates at a university medical center. Is false susceptibility to new macrolides and clindamycin.
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