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1 Journal of Microbiology and Biotechnology Research Scholars Research Library J. Microbiol. Biotech. Res., 2012, 2 (2): ( ISSN : CODEN (USA) : JMBRB4 Phenotypic detection of Inducible Clindamycin resistance among the clinical isolates of staphylococcus aureus by using the lower limit of inter disk space P. Sreenivasulu Reddy, R.Suresh Department of Microbiology, Narayana Medical College, Nellore, A.P., India ABSTRACT Clindamycin is commonly used in the treatment of serious infections, caused by macrolide (erythromycin) resistant staphylococcus aureus producing wound infections. This is very common among methicillin resistant staphylococcus aureus (MRSA). Routine antibiotic sensitivity tests for clindamycin susceptibility may fail to identify imlsb (inducible MLSB) strains of staphylococcus aureus due to erm genes which are expressed in two important phenotypes one is inducible MLSB another is constitutive MLSB. Total of 150 staphylococcus aureus isolates from different clinical samples were subjected to routine antibiotic sensitivity testing (AST) by Kirby-Bauer disc diffusion technique. All were tested for methicillin resistant by using one microgram oxacillin disc. Erythromycin disc was kept adjacent to clindamycin disc while AST and inducible clindamycin resistance was detected by D- test among the erythromycin resistant isolates as per CLSI guidelines. Out of 150 isolates 77 (51.33%) were MRSA and 73(48.67%) were MSSA. 81(54%) were erythromycin resistant, out of which 31(38.27%) isolates showed inducible clindamycin resistance; 17 (31.48%) showed constitutive resistance and 33 (40.7%) isolates gave negative D-test indicating MS phenotypes. Further it was found that high percentage of both inducible MLSB and constitutive MLSB clindamycin resistance was identified among MRSA isolates. AST by routine disk diffusion method for erythromycin and clindamycin would be misidentified as clindamycin sensitive which results in therapeutic failures. INTRODUCTION Staphylococcus aureus is one among the most common pyogenic bacteriae infecting man. [1] Macrolides, lincosamide and streptogramin B are structurally unrelated but microbiologically having same mode of action. [2,3,] Clindamycin is commonly used for the treatment of serious soft tissue infections produced by staphylococcus aureus due to its excellent pharmokinetic properties and treatment failures were reported during therapy. [4,5] Tremendous use of MLSB antibiotics in serious staphylococcal infections results in emergence of increased number of strains acquiring resistance to MLSB antibiotics. The well established mechanism for such resistance is target site modification by erm genes isolated from variety of bacteria starting from E.coli to some of the species of Treponemes. This resistance is expressed phenotypically in two forms, one is inducible MLSB phenotype another is constitutive MLSB phenotype. Erythromycin disc and clindamycin disc are commonly used while doing antibiotic sensitivity testing (AST) for staphylococcus aureus. Unless we place these antibiotic discs adjacent to each other on the AST testing medium it is difficult to detect inducible resistance to clindamycin by macrolide antibiotic. In those patients, therapy with clindamycin results in the emergence of constitutive erm mutants leading to therapeutic failure. In case of efflux of antibiotic mechanism which is mediated through msra genes, susceptibility pattern for clindamycin and erythromycin are same both in vivo and in -vitro and not leading to therapeutic failure. [16] So it is mandatory to detect such strains for the better outcome of the patient who is on clindamycin therapy. Inducible MLSB strains to clindamycin among the erythromycin resistant staphylococcal isolates is easily done by a simple test ie. D-test according to Frebelkorn et.al. [7] 258

2 MATERIALS AND METHODS The present study was conducted from April 2010 to June A total of 411 samples were collected from inpatients of all clinical departments and types of samples included were pus, sputum, endotrachial tube aspirates, blood and urine. All the samples were processed as per CLSI guidelines. Among the isolates staphylococcus aureus strains were identified selectively by standard techniques. All these isolates were subjected to AST by Kirby-Bauer disc diffusions method on Muller Hinton Agar (MHA) plates using Penicillin (10µg), Ampicillin (10 µg), clindamycin (2 µg), erythromycin (15 µg), tetracycline(30 µg), ciprofloxacin (30µg),cefotaxime (30µg), amoxyclav (30µg) and vancomycin (30 µg). But methicillin resistanc was detected separately by using oxacillin (1 µg) disc on MHA with 2% NaCl followed by incubation at 35 0 C. D-test was performed on all the erythromycin resistant isolates. The isolate suspension for inoculation was prepared from isolated colonies from an overnight growth on nutrient agar plate. Colony was suspended in 0.5ml of sterile saline and turbidity was adjusted to 0.5 Mac Far Lands opacity standards. A sterile swab dipped into the suspension and excess of inoculum was removed by pressing it against the sides of test tube. This swab was used to inoculate bacterial suspension on MHA plate and allowed to dry for five minutes. Clindamycin (2 µg) and erythromycin (15 µg) discs were placed at a distance of 15mm edge to edge. [14] ATCC strain of S.aureus was used as control strain. After over night incubation at standard conditions, flattening of zone of inhibition around the clindamycin in the inter disc place was observed in the D- shape, indicating inducible clindamycin resistance. [6] Following D- test results three different phenotypes are interpreted as follows. 1. Inducible MLSb (imlsb) phenotype: Staphylococci showing sensitive (zone size > 21mm) to clindamycin and resistant (zone size < 13 mm) to erythromycin.[ Figure.1] 2. Constitutive MLSb (cmslb) phenotype: Staphylocccal isolates showing resistance (< 13 14mm zone size) to both erythromycin and clindamycin discs. [Figure.2] 3. MS phenotype staphylococcal isolates showed resistance (< 13mm zone size) to erythromycin and sensitive (> 21mm zone ) to clindamycin disc and negative for D test (No flattening of zone of inhibition for clindamycin). [ Figure.3] 259

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6 RESULTS One hundred and fifty staphylococcal isolates were identified from all four hundred and eleven samples processed (Table.1) comprising 36.5% of all clinical samples. Panel of antibiotics were tested by routine disc diffusion method including erythromycin and clindamycin discs quite apart from each other. Out of one hundred and fifty tested, 81 (54%) were resistant to erythromycin (Table-2). D- test was performed for these isolates and observed that 31 (38.27%) isolates indicating inducible MLSB phenotype(d-test positive) and 33 (40.7%) showed D-test negative suggesting MS phenotype (Table-3). 17 ( 21%) isolates were resistant to clindamycin indicating the percentage of constitutive MLSB phenotype (Table.3). It is further observed that high percentage of both inducible MLSB and constitutive MLSB among MRSA isolates (46.2%, 31.48%) when compared to (22.2%, 0%) MSSA. MS phenotypes were identified more among MSSA (77.8%) when compared to MRSA (22.2%) (Table.3). DISCUSSION The prime step before initiating the antimicrobial therapy of infected individuals is performing the antimicrobial susceptibility testing for clinical isolates to avoid indiscriminate usage of antibiotics on trial and error basis. Empirical treatment for staphylococcal infection is more endangerous due to the emergence of multi drug resistant strains especially MRSA. Commonest antibiotic being preferred while treatment of these staphylococcal infections in case of failure to beta-lactam antibiotics is clindamycin. [2] It has excellent tissue penetration except for the central nervous system. [15] Macrolide induced clindamycin resistance was observed among the clinical isolates of staphylococcus since 1968 which could not be detected by the routine disc diffusion method. [8] From such isolates constitutively resistant mutants are emerged and results in treatment failure with clindamycin in vivo which would be demonstrated on D-test. [9] So before declaring the clindamycin sensitivity among the clinical isolates of staphylococcus aureus it is mandatory to check for inducible resistance. Negative D-test among the erythromycin resistant isolates confirm the sensitivity to clindamycin and possible to choose clindamycin as drug of choice in the treatment of staphylococcal infections. [17] 263

7 The present study showed relatively high number of isolates 81 (54%) resistant to erythromycin. D- test was performed with all erythromycin resistant isolates to detect inducible clindamycin resistance and found 31 (38.27%) positive. 17(31.48%) isolates were shown to have constitutive clindamycin resistance and 33(40.7%) showed real susceptibility to clindamycin which were designated as MS phenotypes. These results were correlated with with the study done earlier by K.M. Mohanasoundaram [10] who reported 34% of imlsb, 19% cmlsb and 30% of MS phenotypes. Out of 81 erythromycin resistant staphylococcal isolates 54 (67%) were MRSA and 27(33%) were MSSA. This observation correlates with S.E. Mshana [13] who reported 61% MRSA and 32% MSSA. In relation to methicillin resistance and D-test positivity it was observed that, 25(46.2%) of MRSA isolates were D-test positive compared to 6(22.2%) among MSSA isolates. MS phenotypes were more (77.8%) among MSSA when compared to MRSA(22.2%). Constitutive clindamycin resistance among the MSSA isolates was detected as 0% in our study which in concordance with the study by Zorgani A et al [11], Nuran delialioglu et al [12] and S.E. Mshana et al [13] who reported 0% of cmlsb phenotype among MSSA. These observations confirm that if we had not been done the D-test, nearly half of the erythromycin resistant Staphyloccus aureus isolates might have been missed and resulting in therapeutic failure with clindamycin. CONCLUSION Treatment of beta-lactamase producing and methicillin resistant staphylococcal infection are ever challenge far clinicians. Keeping the mode of action, adverse reactions and pharmacokinetics in mind of certain antibiotics like vancomycin, clindamycin should be preferred for the treatment of severe and resistant infections. Different studies showed that prevalence inducible clindamycin resistant isolates may differ from place to place. Present study giving an information regarding the presence of high percentage of inducible clindamycin resistance among the erythromycin resistant staphylococci. D- test should be mandatory for all microbiological laboratories before reporting about the clindamycin susceptibility. All D-test positive isolates should not be treated with clindamycin but it is the drug of choice for all D- test negative isolates (MS phenotypes). REFERENCES [1] Ryan KJ. Staphylococci. In: Ryan KJ, Ray CG, editors. Sherris medical microbiology, 4 th ed. New York: McGraw Hill; 2004, [2] Fiebelkorn, K. R., Crawford, S. A., McElmeel, M. L. & Jorgensen, J. H. (2003). J Clin Microbiol 2003; 41: [3] Drincovic D, Fuller, Shore KP, Holland DJ et al. J Antimicrob chemother 2001; 48: [4] George K Siberry GK, Tekle T, Carroll K, Dick J. Clin Infect Dis 2003;37: [5] Lewis JS, Jorgenson JH. Clin. Infect. Dis. 2005; 40: [6] Sureerat Chelae, Varaporn Laohaprertthisarn et al. J Med Assoc Thai 2009; 92 (7): [7] Moreillon P, Que YA, Glauser MP. Staphylococcus aureus (including staphylococcal toxic shock). In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas and Bennett s Principles and practice of infectious diseases. 6 th ed. Philadelphia: Elsevier Churchill Livingstone; 2005, [8] Mc Gehee RF, Barrett F, Finland M. Antimicrob Agents Chemother. 1968;8: [9] Naimi, T. S., LeDell, K. H., Boxrud, D. J., Groom, A. V., Steward, C. D., Johnson, S. K., Besser, J. M., O'Boyle, C., Danila, R. N. Clin Infect Dis 1998,33, [10] Mohanasoundaram K M. Journal of Clinical and Diagnostic Research. 2011,5(1): [11] Zorgani A, Shawerf O, Tawil K, El-Turki E and Ghenghesh KS. Inducible Clindamycin Resistance among Staphylococci isolated from burn patients. LJM : / [12] Nuran Delialioglu, Gonul Aslan, Candan Ozturk, Vildan Baki, Sebahat Sen and Gurol Emekdas. Jpn. J. Infect. Dis. 2005, 58, , [13] S.E.Mshana, E. Kamugisha, M. Mirambo, P. Chalya, P. Rambau, W. Mahalu and E. Lyamuya. Tanzania J of Health Research 2009; 11( 2), [14] National Committee for Clinical Laboratory Standards. Performance standards for antimicrobial susceptibility testing; 12th informational supplement. NCCLS document M100-S14. Wayne, PA: NCCLS; [15] Sivapalasingam S, Steigbigel NH. Macrolides, clindamycin and ketolides. In: Mandell GL, Bennett JE, Dolin R, editors. Mandell, Douglas and Bennett s Principles and practice of infectious diseases. 6 th ed. Philadelphia: Elsevier Churchill Livingstone; 2005, [16] K.R.Fiebelkorn, S. A. Crawford, M. L. McElmeel and J. H. Jorgensen. J. Clin. Microbiol. 2003, 41(10), [17] Roland Leclercq, Clin Infect Dis. 2002, 34 (4):