J. Commun. Dis. 44(2) 2012 : 97-102 Practical disk diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus at a tertiary care hospital: Implications for clinical therapy Saxena Sonal*, Singh Trishla* and Dutta Renu* (Received for publication January 2012) Abstract The increase in incidence of variety of infections due to Staphylococcus aureus and especially methicillin resistant Staphylococcus aureus has highlighted on the need for better agents to treat such infections. Also the resistance to antimicrobial agents among staphylococci is an increasing problem. The macrolide-lincosamide-streptogramin (MLS ) groups of antibiotics are commonly used in the treatment of staphylococcal infections with clindamycin being the preferred agent. This study demonstrates a simple phenotypic method (D-test) for detecting inducible clindamycin resistance in erythromycin resistant strains of staphylococcus aureus & coagulase negative Staphylococcus. A total of 181 erythromycin resistant staphylococcal isolates were obtained from various clinical specimens. Among the Staphylococcus aureus isolates, 38(25.4%) were methicillin resistant Staphylococcus aureus (MRSA) and 112(74.6%) were methicillin sensitive staphylococcus aureus (MSSA). Constitutive resistance was seen in 56(37.3%) of isolates. Of these 18(47.4%) were MRSA and 38(33.9%) were MSSA. Inducible resistance was seen in 25(16.6%) isolates, in which 11(28.9%) were methicillin resistant Staphylococcus aureus & 14(12.6%) were methicillin sensitive staphylococcus aureus. It is likely that the true percentage of clindamycin resistance is being * Department of Microbiology, Lady Hardinge Medical College, New Delhi. Correspondence to: Dr. Sonal Saxena, Flat No. 6, Deluxe Apartments, -5, Vasundhara Enclave, Delhi-110096, Mobile : 9811349053 E-mail: sonalsaxena3@rediffmail.com
98 Saxena Sonal et al underestimated, since testing for inducible resistance is not being routinely performed. So the test should be done routinely in all the labs to avoid therapeutic failures. Key words : Clindamycin, D-test, Inducible resistance, MLS Resistance, MRSA INTRODUCTION The increase in the occurrence of methicillin resistant Staphylococcus aureus (MRSA) and emergence of Vancomycin intermediate Staphylococcus aureus (VISA)/Vancomycin resistant Staphylococcus aureus (VRSA) has left fewer options for the treatment of infections caused by Staphylococcus aureus. The macrolide-lincosamide-streptogramin (MLS ) groups of antibiotics are commonly used in the treatment of staphylococcal infections with clindamycin being the preferred agent. Good oral absorption makes 1 it an important option in outpatient settings. Resistance to Macrolide-lincosamide- Streptogramin antibiotics can occur due to drug interaction, target site modification or efflux mechanisms. Target site modification mediated by erm genes is the most common mechanism. The erm (erythromycin ribosome methylase) genes encode enzymes that cause methylation of A2058 residue located in the conserved domainv of 23s rrna which results in formation of phenotype of resistance pattern known as MLS. This resistance can be expressed either 2,8,9 constitutively or inducibly. It is difficult to detect inducible clindamycin resistance in the lab as these strains can appear sensitive to clindamycin & resistant to erythromycin. However, therapeutic failures are common as the therapy with clindamycin may select constitutive erm mutants. The detection of such inducible resistance requires the disc of erythromycin & clindamycin to be placed adjacent to one another. Practical disk diffusion methods have been described for detection of inducible clindamycin resistance. This method has been standardized recently by Clinical Laboratories Standard Institute (CLSI M100-3 S15 2005) and is known as the D-tese. The present study was designed to detect the prevalence of inducible clindamycin resistance in clinical isolates of Staphylococcus species. MATERIALS AND METHODS A total of 150 strains of erythromycin resistant Staphylococcus aureus and 31 strains of erythromycin resistant coagulase negative Staphylococcus isolated from various clinical samples in our laboratory were used in this study. Duplicate isolates were not included. The isolates were identified using conventional microbiological methods including colony morphology, Gram stain, catalase and coagulase tests. Cefoxitin 30 µg disc (Hi-Media; India Ltd) was used to detect 4 MRSA as per CLSI recommendations. Antimicrobial susceptibility to c1indamycin and erythromycin was determined by disc diffusion methods as per CLSI guidelines and zone of inhibition was measured. For erythromycin staphylococcal isolates exhibi-
Practical disk diffusion method for detection of inducible clindamycin resistance... 99 A C Figure 1 : Disc diffusion test for inducible clindamycin resistance (A) Constitutive MLS phenotype () Inducible MLS phenotype (C) MS phenotype. ting zone of inhibition <13mm were taken as resistant while those >21mm were taken as sensitive. For clindamycin zone of inhibition <14mm were taken as resistant while those >2lmm were taken as sensitive. The isolates - were further studied for inducible clinda- 3 mycin resistance as per CLSI guidelines. A 0.5 McFarland equivalent suspension of organism was inoculated on to Mueller Hinton agar plates. Clindamycin (2 µg, Hi- Media; India Ltd) and Erythromycin (15µg, Hi-Media;India Ltd) discs were placed 15mm apart edge to edge. Staphylococcus aureus ATCC 25923 was used as quality control. Plates were analyzed after 18 hours incubation. Complete circular zone of inhibition was taken as negative D test, while blunting of clindamycin zone adjacent to erythromycin was interpreted as positive D test. Erythromycin resistant and clindamycin resistant strains were interpreted as constitutive MLS phenotypes, while erythromycin resistant, clindamycin sensitive and D test positive strains were interpreted to have inducible MLS (imls ) phenotypes. Erythromycin resistant, clindamycin sensitive and D test negative strains were interpreted as MS 8 phenotypes (Figure 1). RESULTS A total of 181 erythromycin resistant staphylococcal isolates were obtained from various clinical specimens. Out of 181 isolates, 150 were Staphylococcus aureus & 31 were coagulase negative staphylococcus. Among the Staphylococcus aureus isolates, 38(25.4%) were methicillin resistant Staphylococcus aureus (MRSA) and 112(74.6%) were methicillin sensitive staphylococcus aureus (MSSA). Constitutive resistance was seen in
100 Saxena Sonal et al Table 1 : Resistance in Staphylococcus aureus Phenotype MRSA(%) MSSA(%) TOTAL(%) ER-R, CL-R 18(47.4) 38(33.9) 56(37.3) ER-R, CL-S, D+ 11(28.9) 14(12.6) 25(16.6) ER-R, CL-S, D- 9(23.7) 60(53.5) 69(46) 38(25.4) 112(74.6) 150 Key: ER-R: Erythromycin resistant, CL-R: Clindamycin resistant, CL-S: Clindamycin sensitive, D+: D test positive, D-: D test negative. Table 2 : Resistance in Coagulase negative staphylococcus species Phenotype MRSA(%) MSSA(%) TOTAL(%) ER-R, CL-R 3(30) 9(42.8) 12(38.7) ER-R, CL-S, D+ 3(30) 5(23.8) 8(25.8) ER-R, CL-S, D- 4(40) 7(33.4) 11(35.5) 10(32.3) 21(67.7) 31 Key: ER-R: Erythromycin resistant, CL-R: Clindamycin resistant, CL-S: Clindamycin sensitive, D+: D test positive, D-: D test negative. 56(37.3%) of isolates. Of these 18(47.4%) were MRSA and 38(33.9%) were MSSA. Inducible resistance was seen in 25(16.6%) of isolates, in which 11(28.9%) were methicillin resistant Staphylococcus aureus & 14(12.6%) were methicillin sensitive staphylococcus aureus (Table 1). Among the coagulase negative staphylococcus isolates, 10(32.3%) showed methicillin resistance while 21 (67.7%) were methicillin sensitive. Constitutive resistance was seen in 12(38.7%) isolates. Of these 3(30%) were methicillin resistant and 9(42.8%) were methicillin sensitive. 8(25.8%) isolates exhibited inducible resistance, 3(30%) were methicillin resistant and 5(23.8%) were methicillin sensitive (Table 2). DISCUSSION Accurate detection of antimicrobial resistance in a microbe is an essential factor. In determining appropriate therapeutic regimens. The increase in frequency of staphylococcal infections among patients and changes in antimicrobial resistance patterns have led to renewed interest in the use of 5 clindamycin therapy. Clindamycin is a useful drug in the treatment of skin and soft tissue infections and serious infections caused by staphylococcal species and anaerobes. It is a good alternative for the treatment of both methicillin resistant and methicillin sensitive staphylococcal infections. It has excellent tissue penetration (except central nervous
Practical disk diffusion method for detection of inducible clindamycin resistance... 101 system) and accumulates in abscesses and no renal dosing adjustments are required. Good oral absorption makes it an important option in outpatient therapy or as follow up after intravenous therapy. It is of particular importance as an alternative antibiotic in 6 penicillin allergic patients. ut ignorance regarding inducible clindamycin resistance has led to therapeutic failures. Inducible clindamycin resistance in Staphylococci cannot be detected by using in-vitro susceptibility tests such as broth or agar 11 dilution. Also, if the discs of Erythromycin & clindamycin are not placed in adjacent positions, this phenotype will be easily missed. Hence D test offers a reliable method for detecting imls phenotype. Our study has detected 16% Staphylococcus aureus & 26% coagulase negative staphylococcus to possess inducible clindamycin resistance. Amongst Staphylococcus aureus, 29% of methicillin resistant Staphylococcus aureus & 12% of methicillin sensitive staphylococcus aureus showed this phenotype. These findings correlate well with 6,7,8,12 7 various authors. Gadepalli et al detected inducible clindamycin resistance to be 30% in methicillin resistant Staphylococcus aureus & 10% in methicillin sensitive staphylococcus 6 aureus. Yilmaz et al found inducible resistance in 24% of methicillin resistant Staphylococcus aureus & 14.8% in methicillin sensitive staphylococcus aureus. Similar 12 findings were reported by Hamilton Miller from Korea and Delialioglu et al from 12,13 Turkey. Characteristically, each report from different regions has shown a different pattern of resistance. Indian reports on inducible clindamycin-resistance are few. The incidence of resistance is highly variable with regard to geographic locality, hence the local data regarding the inducible clindamycin resistance is helpful in guiding anti staphylococcal therapy & therapeutic failures 7 can be avoided. Implications for therapy: One of the major concerns with usage of Clindamycin therapy is the possible presence of inducible Clindamycin resistance. Uncertainty regarding reliability of susceptibility reports & confusion regarding inducible resistance can lead to avoidance of Clindamycin therapy by clinicians. Clindamycin therapy can be safely 13 given in non MLSbi infections. Also, it has been reported that constitutive resistance to Clindamycin prevents inhibition of toxin production & fails to inhibit growth of Staphylococcus aureus. However, the same is not clear for inducible resistance. Inducible resistance phenotypes can clearly appear to be resistant to erythromycin but susceptible to clindamycin, if the discs of erythromycin & clindamycin are not placed adjacent to each 14 other. CLSI recommends that clinical laboratories where methicillin resistant Staphylococcus aureus is routinely encountered must consider routine testing & reporting for inducible Clindamycin resis- 15 tance using D test. However, if it has to be used for MLSi producing isolates close follow up & monitoring for failure or relapse is needed. To conclude, none reporting of cases of methicillin resistant Staphylococcus aureus treated with Clindamycin are needed to better understanding of the role of this compound in organisms with varying MLS resistant phenotypes.
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