Research & Reviews: Journal of Medical and Health Sciences
MenuISSN: 2319-9865
ISSN: 2319-9865
Smita Sood*
Department of Laboratory Medicine (SRL Ltd.), Fortis Escorts Hospital, Jaipur, India.
Received: 24/08/2013 Accepted: 26/09/2013 Revised: 09/09/2013
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A major concern regarding the use of Clindamycin for Staphylococcal infections is the possible presence of inducible resistance to clindamycin. In vitro routine tests for clindamycin susceptibility may fail to detect inducible clindamycin resistance thus necessitating the need to detect such resistance by a simple D test on routine basis .The present study was conducted to determine the incidence of inducible clindamycin resistance in Staphylococcal isolates in an urban hospital in North west India. A total 129 Staphylococcal strains (Staphylococcus aureus and Coagulase Negative staphylococci) isolated from various clinical specimens were subjected to disc-approximation method (D-test) to detect inducible clindamycin resistance with standard erythromycin and clindamycin discs.15.50% inducible Clindamycin resistance phenotype, 20.93% constitutive Clindamycin resistance phenotype and 3.10% MS phenotype was detected among 129 clinical isolates of staphylococci. In case of a Staphylococcal infection, inducible MLSBi resistance in Staphylococcus aureus strains (40%) was found to be lesser than in CONS strains (60%). This study indicates the importance of D test to differentiate inducible clindamycin resistant isolates of Staphylococci so as to facilitate the optimal treatment of patients.
Inducible clindamycin resistant, staphylococcus, Rajasthan
Staphylococcus aureus and Coagulase Negative Staphylococci are recognized as causing nosocomial and community acquired infections in every region of the world. The resistance to antimicrobial agents among Staphylococci is an increasing problem [1].
The Macrolide-Lincosamide-Streptogramin B (MLSB) family of antibiotics is commonly used in the treatment of Staphylococcal infections. Clindamycin a protein synthesis inhibitor is an attractive option for use in the scenario of increasing drug resistance among the Staphylococci especially for skin and soft tissue infections and as an alternative in Penicillin allergic patients. This drug has excellent tissue penetration, requires no renal dosing adjustments and has a good oral absorption. All these factors make it convenient for outpatient prescription or as follow-up after intravenous therapy [2]. However, one important issue with Clindamycin treatment is the risk of clinical failure during therapy.
The MLS family of antibiotics has three different mechanisms of resistance: target site modification, enzyme antibiotic inactivation and macrolide efflux pumps [3]. Macrolide antibiotic resistance in Staphylococcus aureus and Coagulase negative Staphylococci (CONS) may be due to an active efflux mechanism encoded by msr A (macrolides streptogamin resistance) genes , conferring resistance to macrolides and type B streptogamin only [4] or may be due to ribosomal target modification, affecting macrolides, lincosamides, and type B streptogamin (MLSB resistance). Erm (erythromycin resistant methylase) genes are responsible for encoding enzymes that confer inducible or constitutive resistance to MLS agents via methylation of the 23S rRNA and reducing binding by MLS agents to the ribosome [5].
Antimicrobial susceptibility data are important for the management of infections, but false susceptibility results may be obtained if isolates are not tested for inducible Clindamycin resistance. This resistance missed by using standard susceptibility test methods such as standard broth-based or agar dilution susceptibility tests. The inducible MLSB resistance can be detected by a simple test known as Disk approximation test or D test. Low level of Erythromycin is an inducer of MLSBi phenotype and this is the basis of performing D test [6]. Failure to identify inducible MLSB resistance may lead to clinical failure of Clindamycin therapy. Conversely, labeling all erythromycin-resistant staphylococci as Clindamycin resistant prevents the use of Clindamycin in infections caused by truly Clindamycin-susceptible Staphylococcal isolates.
The present study was thus designed to investigate the prevalence of Erythromycin – induced Clindamycin resistance in Staphylococci isolated from various clinical specimens at an urban hospital in North West India.
129 isolates of Staphylococci comprising of Staphylococcus aureus and Coagulase negative Staphylococci were obtained from various clinical samples received in the Microbiology lab of an urban hospital in Northwest India over a period of 8 months (October 2008-May 2009). Duplicate isolates from the same patient were not included. Isolated bacteria were identified by the conventional microbiological methods including colony morphology, Gram’s stain, Slide and Tube Coagulase test [7].
Methicillin resistance was determined by Cefoxitin (30 mcg) disc diffusion test according to the recommendations of CLSI (Clinical Laboratory Standards Institute) [8]. Isolates were initially screened for resistance to Erythromycin using disc diffusion testing. The isolates found resistant to Erythromycin were further screened for inducible Clindamycin resistance. A 0.5 Mc Farland equivalent suspension of organism was inoculated onto a Mueller Hinton Agar plate as described in the CLSI recommendations. Clindamycin (2 mcg) and Erythromycin (15 mcg) discs were placed 15 – 20 mm apart from the center on Mueller Hinton Agar. Plates were analyzed after 18 hours of incubation at 37 degree C.
Interpretations of zone of diameters were as follows:
• Erythromycin sensitive ER-S>=23mm
• Erythromycin intermediate sensitive 14-22mm
• Erythromycin resistant ER-R<=13mm
• Clindamycin sensitive CL-S>=21 mm
• Clindamycin intermediate sensitive 15-20mm
• Clindamycin resistant CL-R<=14mm
If Erythromycin zone is <=13mm and Clindamycin zone is >=21mm and both in inhibition zones have a circular shape the organism was considered as negative for inducible Clindamycin resistance on D-testing and were defined as showing MS phenotype.
If Erythromycin zone is <=13mm and the Clindamycin zone is >=21mm with a D shaped inhibition zone around Clindamycin the organism was considered as positive for inducible Clindamycin resistance on D testing and were defined as showing inducible Clindamycin resistance (MLSBi).
Strains where resistance to both Erythromycin and Clindamycin were observed were defined as showing constitutive MLSB resistance (MLSBc).
Quality control of the Erythromycin and Clindamycin discs were performed with Staphylococcus aureus ATCC 25923. In house strains of Staphylococcus aureus showing D-test positive repeatedly was used as a positive control for inducible Clindamycin resistance.
Among the 129 Staphylococcal isolates studied, 43 (33.33%) were Staphylococcus aureus and 86 (66.66%) were Coagulase negative Staphylococci (CONS).
Table- 1 shows the categorization of the Staphylococcal isolates along with their specimen source. Among the 129 Staphylococcal isolates, 10 (7.75%) were Methicillin resistant Staphylococcus aureus (MRSA) and 33 (25.58%) were Methicillin sensitive Staphylococcus aureus, 45 (34.88%) were Methicillin sensitive Coagulase negative Staphylococci (MSCONS) and 41 (31.78%) were Methicillin resistant Coagulase negative Staphylococci (MRCONS).
Table 1: Frequency distribution of Staphylococci isolated from various clinical samples
Of the 129 clinical Staphylococcal isolates 51 (39.53%) showed Erythromycin resistance. Of the Erythromycin resistant Staphylococcal isolates 20 (15.50% of total isolates) belonged to MLSBi phenotype and showed inducible Clindamycin resistance, 27 Staphylococcal isolates (20.93% of total isolates) showed constitutive resistance to Clindamycin. Resistance to Erythromycin and susceptibility to Clindamycin with no flattening of Clindamycin inhibition zone i.e. MS phenotype was observed in 4(3.10% of total) isolates. Resistance phenotypes of the isolates are provided in Table 2.
Table 2: Distribution of various resistance phenotypes in Staphylococcal species
The isolation rates of MLSBi phenotypes from clinical samples are provided in Table 3. Among the MLSBi isolates 60% were CONS and 40% Staphyloccocus aureus.
Table 3: Distribution of MLSBi isolates in various clinical specimens
The increasing frequency of Staphylococcal infections among patients and changing patterns in antimicrobial resistance have led to renewed interest in the use of Clindamycin therapy to treat such infections [9]. Clindamycin is a good alternative for treatment of both Methicillin – resistant and susceptible Staphylococci [10]. Clindamycin resistance can develop in Staphylococcal isolates with the inducible phenotype, and spontaneous constitutively resistant mutants have been selected from such isolates both in vitro and in vivo during Clindamycin therapy [11].
In this study we found a total of 8 (18.60%) Staphylococcus aureus and 12 (13.95%) CONS isolates with Erythromycin resistance and Clindamycin sensitive phenotype which demonstrated inducible resistance. Overall the results indicate a high incidence 20 (15.50%) of inducible Clindamycin resistance. Inducible clindamycin rates of 23.6% in Staphylococci isolated from various clinical samples in Nasik, Maharashtra has been reported [12]. Pal et al. in their previous study conducted in the same region have also demonstrated inducible clindamycin resistance to be 23.48% among the staphylococci isolated from various clinical samples [13]. Another recent study from Mumbai reports 34.66% of Erythromycin resistant Staphylococci to exhibit inducible clindamycin resistance [14]. Similar to our findings a study from an urban hospital in Dhaka also reported MLSBi to be 20 % [15].
Studies have reported that the incidence of constitutive and inducible MLSB phenotype is higher in MRSA [16,17]. Our results demonstrate that in case of a Staphylococcal infection inducible MLSBi resistance in Staphylococcus aureus strains (40%) is lesser than in CONS strains (60%). Lim et al (2006) also reported that inducible Clindamycin resistance strains more prevalent in CONS [18].
We found 15.50% Staphylococcal isolates with inducible Clindamycin resistance phenotype, 20.93% with constitutive Clindamycin resistance phenotype and 3.10% with MS phenotype. 60% of Methicillin sensitive Staphylococcus aureus (MSSA) and 62.5% of Methicillin resistant Staphylococcus aureus (MRSA) of Erythromycin resistant and clindamycin susceptible strains exhibited inducible Clindamycin resistance. 35.71% Methicillin sensitive Coagulase negative staphylococcus (MSCONS) and 29.16% Methicillin resistant Coagulase negative staphylococcus (MRCONS) with Erythromycin resistant and Clindamycin susceptibility exhibited inducible clindamycin resistance. Gadepalli et al (2006) have reported 26.5% of the S.aureus isolates to exhibit constitutive MLSBc phenotype, 21% to exhibit inducible MLSBi phenotype and 12% to exhibit MS phenotype [19]. No constitutive resistant phenotype was reported by Angel et al (2008) in their study of prevalence of inducible Clindamycin resistance in Gram positive organisms from South India [20].
This study demonstrates that the D shape of Clindamycin zone adjacent to Erythromycin disc in a conventional disc diffusion test can serve to detect S. aureus or CONS strains with inducible resistance to Clindamycin. As the D test is simple, inexpensive and easy to perform it can be included as a part of routine antibiotic susceptibility testing. Additionally it can be used to survey the MLSB resistance of Staphylococci strains from specific geographical regions/hospitals. However the confirmation of erm gene in Staphylococcal isolates with a positive D test would asset in standardization of the test.