Indian Journal of Medical Microbiology Medknow Publications on behalf of Indian Association of Medical Microbiology ISSN: 0255-0857 EISSN: 1998-3646 Vol. 28, Num. 2, 2010, pp. 124-126

Indian Journal of Medical Microbiology, Vol. 28, No. 2, April-June, 2010, pp. 124-126

Original Article

Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples

*V Deotale, DK Mendiratta, U Raut, P Narang

Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram. Dist Wardha, M.S. - 442 102, India

Correspondence Address: *Department of Microbiology, Mahatma Gandhi Institute of Medical Sciences, Sevagram. Dist Wardha, M.S. - 442 102, India, vijayshree_khairkar@rediffmail.com

Date of Submission: 08-Jun-2009
Date of Acceptance: 21-Sep-2009

Code Number: mb10038

PMID: 20404457

DOI: 10.4103/0255-0857.62488

Abstract

Purpose: Clindamycin is commonly used in the treatment of erythromycin resistant Staphylococcus aureus causing skin and soft tissue infections. In vitro routine tests for clindamycin susceptibility may fail to detect inducible clindamycin resistance due to erm genes resulting in treatment failure, thus necessitating the need to detect such resistance by a simple D test on routine basis.
Materials and Method: 247 Staphylococcus aureus isolates were subjected to routine antibiotic susceptibility testing including oxacillin (1ìg) by Kirby Bauer disc diffusion method. Inducible clindamycin resistance was detected by D test, as per CLSI guidelines on erythromycin resistant isolates.
Results: 36 (14.5%) isolates showed inducible clindamycin resistance, nine (3.6%) showed constitutive resistance while remaining 35 (14.1%) showed MS phenotype. Inducible resistance and MS phenotype were found to be higher in MRSA as compared to MSSA (27.6%, 24.3% and 1.6%, 4% respectively).
Conclusion: Study showed that D test should be used as a mandatory method in routine disc diffusion testing to detect inducible clindamycin resistance.

Keywords: Clindamycin resistance, constitutive MLS _B phenotype, inducible MLS _B phenotype, MRSA, MS phenotype

Introduction

Emergence of methicillin resistance in Staphylococcus aureus has left us with very few therapeutic alternatives available to treat staphylococcal infections. The macrolide-lincosamide-streptogramin B (MLS _B ) family of antibiotics serves as one such alternative, with clindamycin being the preferred agent due its excellent pharmacokinetic properties. ^[1]

However, widespread use of MLS- _B antibiotics has led to an increase in number of staphylococcal strains acquiring resistance to MLS- _B antibiotics. ^[2] The most common mechanism for such resistance is target site modification mediated by erm genes which can be expressed either constitutively (constitutive MLS _B phenotype) or inducibely (inducible MLS _B phenotype). Strains with inducible resistance to clindamycin are difficult to detect in the routine laboratory as they appear erythromycin resistant and clindamycin sensitive in vitro when not placed adjacent to each other. In such cases, in vivo therapy with clindamycin may select constitutive erm mutants leading to clinical therapeutic failure. In case of another mechanism of resistance mediated through msrA genes i.e. efflux of antibiotic, Staphylococcal isolates appear erythromycin resistant and clindamycin sensitive both in vivo and in vitro and the strain does not typically become clindamycin resistant during therapy. ^[3]

This study demonstrates a very simple method of detecting inducible resistance to clindamycin in erythromycin resistant staphylococcal isolates. i.e. D test as described by Fiebelkorn et al. ^[1],[4]

Materials and Methods

The study was conducted from February to May 2008. A total of 247 isolates of Staphylococcus aureus isolated from various clinical specimens like pus, wound swab, aspirates, blood, and sterile fluids were tested. The isolates were first identified as S. auerus by standard biochemical techniques ^[5] and then subjected to susceptibility testing by Kirby Bauer's disc diffusion method on Mueller Hinton agar plates using erythromycin (15ìg), penicillin (10U), ciprofloxacin (5 ìg) and cefoxitin (30 ìg) as per CLSI guidelines. Methicillin resistance was detected using oxacillin (1ìg) on Mueller Hinton agar supplemented with 2% NaCl followed by incubation at 35⁰ C. ^[4]

Those isolates which were erythromycin resistant were further subjected to 'D test' as per CLSI guidelines. Briefly, erythromycin (15 ìg) disc was placed at a distance of 15mm (edge to edge) from clindamycin (2 ìg) disc on a Mueller Hinton agar plate previously inoculated with 0.5 McFarland bacterial suspension. Following overnight incubation at 37 ⁰ C, flattening of zone (D shaped) around clindamycin in the area between the two discs, indicated inducible clindamycin resistance ^[4] [Figure - 1]b. Three different phenotypes were appreciated after testing and interpreted as follows:

1. MS Phenotype - Staphylococcal isolates exhibiting resistance to erythromycin (zone size ≤13mm) while sensitive to clindamycin (zone size ≥21mm) and giving circular zone of inhibition around clindamycin was labelled as having this phenotype [Figure - 1]a
2. Inducible MLS _B Phenotype - Staphylococcal isolates showing resistance to erythromycin (zone size ≤13mm) while being sensitive to clindamycin (zone size ≥21mm) and giving D shaped zone of inhibition around clindamycin with flattening towards erythromycin disc were labelled as having this phenotype [Figure - 1]b.
3. Constitutive MLSB Phenotype - this phenotype was labelled for those Staphylococcal isolates which showed resistance to both erythromycin (zone size ≤13mm) and clindamycin (zone size ≤14mm) with circular shape of zone of inhibition if any around clindamycin [Figure - 1]c.

Results

Two hundred and forty seven Staphylococcal isolates were tested for susceptibility to erythromycin and other antibiotics of the panel by routine disc diffusion testing; 80 (32.4%) of them were erythromycin resistant. These isolates when subjected to D test showed nine (3.6%) isolates resistant to both erythromycin and clindamycin indicating constitutive MLS _B Phenotype; 71 isolates showed clindamycin sensitivity. Out of these, 36 isolates showed positive D test indicating inducible MLS _B phenotype while 35 gave negative D test indicating MS phenotype [Table - 1]. The overall percentage resistance for all three phenotypes was as follows.

Inducible clindamycin resistance - 14.5%

constitutive clindamycin resistance - 3.6%

MS Phenotype - 14.17%

Percentage of both inducible and constitutive resistance was higher amongst MRSA isolates as compared to MSSA [Table - 1].

Discussion

The determination of antimicrobial susceptibility of a clinical isolate is often crucial for optimal antimicrobial therapy of infected patients. This is particularly important considering the increase of resistance and the emergence of multidrug resistant organisms. There are many options available for treatment of MSSA and MRSA infections, with clindamycin being one of the good alternatives. ^[1] However, clindamycin resistance can develop in staphylococcal isolates with inducible phenotype, and from such isolates, spontaneous constitutively resistant mutants have arisen both in vitro testing and in vivo during clindamycin therapy. ^[6] Reporting Staphylococcus aureus as susceptible to clindamycin without checking for inducible resistance may result in institution of inappropriate clindamycin therapy. On the other hand negative result for inducible clindamycin resistance confirms clindamycin susceptibility and provides a very good therapeutic option. ^[7]

In our study we found high percentage of erythromycin resistant isolates [80 (32.4%)]. Amongst them 36 (45%) isolates tested positive for inducible clindamycin resistance by D test while rest of the isolates were negative for D test, out of which 9 (11.25%) were shown to have constitutive clindamycin resistance and 35 (43.75%) showed true sensitivity to clindamycin (MS phenotype). These observations suggest that had D test not been performed, nearly half of the erythromycin resistant isolates would have been misidentified as clindamycin sensitive resulting in therapeutic failure. It was also observed that percentages of inducible resistance and MS phenotype were higher amongst MRSA (27.6% and 24.3% respectively) as compared to MSSA (1.6% and 4%). This was in concordance with a few of the studies reported before - Yilmaz et al.^[6] found inducible resistance of 24.4% in MRSA and 14.8% in MSSA; Gadepalli et al.^[2] showed it to be 30% in MRSA and 10% in MSSA, while Mohamed Rahabar et al.^[8] reported 22.6% in MRSA and 4% in MSSA. Another study from India ^[9] showed very high frequency of inducible resistance (63%) in erythromycin -R, clindamycin sensitive isolates being 74% in MRSA and 45% in MSSA. On the contrary, Schreckenberger et al.^[10] and Levin et al.^[11] showed higher percentage of inducible resistance in MSSA as compared to MRSA, 7-12% in MRSA and 19-20% in MSSA; 12.5% MRSA and 68% MSSA respectively.

Constitutive resistance in our study was seen in 7.3% of MRSA isolates, which is contrary to the only study from India, by Angel et al.,^[12] which did not find it in any of the strains,.

In the light of the restricted range of antibiotics available for the treatment of methicillin-resistant staphylococcal infections and the known limitations of vancomycin, clindamycin should be considered for the management of serious soft tissue infections with methicillin-resistant staphylococci that are sensitive to clindamycin. ^[13] The true sensitivity to clindamycin can only be judged after performing D test on the erythromycin resistant isolates. The prevalence of inducible clindamycin resistance may vary from hospital to hospital. Although we did not study the prevalence of inducible clindamycin resistance in our area, from the current study, we can conclude that there is a fairly high percentage of inducible clindamycin resistance amongst the staphylococcal isolates which shows erythromycin resistance.

Use of D test in a routine laboratory will enable us in guiding the clinicians regarding judicious use of clindamycin in skin and soft tissue infections; as clindamycin is not a suitable drug for D test positive isolates while it can definitely prove to be a drug of choice in case of D test negative isolates.

References

1.	Fiebelkorn KR, Crawford SA, McElmeel ML, Jorgensen JH. Practical disc diffusion method for detection of inducible clindamycin resistance in Staphylococcus aureus and coagulase negative Staphylococci. J Clin Microbiol 2003;41:4740-4.  Back to cited text no. 1  [PUBMED]  [FULLTEXT]
2.	Gadepalli R, Dhawan B, Mohanty S, Kapil A, Das BK, Chaudhry R. Inducible clindamycin resistance in clinical isolates of Staphylococcus aureus. Indian J Med Res 2006;123:571-3.  Back to cited text no. 2  [PUBMED]  [FULLTEXT]
3.	Steward CD, Raney PM, Morrell AK, Williams PP, McDougal LK, Jevitt L, et al. Testing for induction of clindamycin resistance in erythromycin resistant isolates of Staphylococcus aureus. J Clin Microbiol 2005;43:1716-21.  Back to cited text no. 3  [PUBMED]  [FULLTEXT]
4.	Clinical and laboratory standards institute. Performance standards for antimicrobial susceptibility testing; Seventeenth informational supplement. Vol. 27. No.1 Clinical Laboratory Standards Institute; 2007.   Back to cited text no. 4
5.	Kloos WE, Banerman TL. Staphylococcus and Micrococcus, Chapter 22. In: Manual of clinical microbiology. 7 th ed. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Washington DC: ASM Press; 1999. p. 264-82.   Back to cited text no. 5
6.	Yilmaz G, Aydin K, Iskender S, Caylan R, Koksal I. Detection and prevalence of inducible clindamycin resistance in staphylococci. J Med Microbiol 2007;56:342-5.  Back to cited text no. 6  [PUBMED]  [FULLTEXT]
7.	Rodrigues Perez LR, Caierao J, Souza Antunes AL, Alves d'Azevedo P. Use of D test method to detect inducible clindamycin resistance in coagulase negative staphylococci (CoNS). Braz J Infect Dis 2007;11:186-8.   Back to cited text no. 7
8.	Rahabar M, Hajia M. Inducible clindamycin resistance in Staphylococcus aureus: A cross sectional report. Pak J Biol Sci 2007;10:189-92.  Back to cited text no. 8
9.	Ajantha GS, Kulkarni RD, Shetty J, Shubhada C, Jain P. Phenotypic detection of inducible clindamycin resistance amongst Staphylococcus aureus isolates by using lower limit of recommended inter-disk distance. Indian J Pathol Microbiol 2008;51:376-8.   Back to cited text no. 9  [PUBMED]
10.	Schreckenberger PC, Ilendo E, Ristow KL. Incidence of constitutive and inducible clindamycin resistance in Staphylococcus aureus and coagulase-negative staphylococci in a community and a tertiary care hospital. J Clin Microbiol 2004;42:2777-9.  Back to cited text no. 10  [PUBMED]  [FULLTEXT]
11.	Levin TP, Suh B, Axelrod P, Truant AL, Fekete T. Potential clindamycin Resistance in clindamycin-susceptible, erythromycin-resistant Staphylococcus aureus: Report of a clinical failure. Antimicrob Agents Chemother 2005;49:1222-4.  Back to cited text no. 11  [PUBMED]  [FULLTEXT]
12.	Angel MR, Balaji V, Prakash JA, Brahmandathan KN, Mathews MS. Prevalence of inducible clindamycin resistance in gram positive organisms in a tertiary care centre. Indian J Med Micobiol 2008;26:262-4.  Back to cited text no. 12
13.	Rao GG. Should clindamycin be used in treatment of patients with infections caused by erythromycin-resistant staphylococci? J Antimicrob Chemother 2000;45:715.  Back to cited text no. 13  [PUBMED]  [FULLTEXT]

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