DOI: 10.5958/j.2319-5886.2.2.

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International Journal of Medical Research & Health Sciences

www.ijmrhs.com
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Volume 2 Issue 2 April - June

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Coden: IJMRHS

Copyright @2013

ISSN: 2319-5886
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Received: 4 Feb 2013

Research article

Revised: 2 Mar 2013

Accepted: 7 Mar 2013

DETECTION OF INDUCIBLE CLINDAMYCIN RESISTANCE AMONG CLINICAL ISOLATES OF STAPHYLOCOCCI FROM A RURAL TERTIARY CARE HOSPITAL Makam Sri Rangakumar Manjunath1, Vijaya Rayapu2, *Dhandapany Senthil Pragash2, Ivvala Anand Shaker3, Sandeep Kasukurthy4 Technician C National Institute of Virology, Bangalore, India Department of Microbiology, 3Department of Biochemistry, Melmaruvathur Adhiparasakthi Institute of Medical Sciences, Melmaruvathur, 603319, India. 4 Research Assistant, RMRC (ICMR), Port Blair, Andaman & Nicobar Island, India.
2 1

*Corresponding author email: drsenthiledu@gmail.com

ABSTRACT

Clindamycin has been used successfully to treat pneumonia and soft tissue infections caused by methicillin-resistant Staphylococcus aureus. However, Clinical failure of clindamycin therapy has been reported due to multiple mechanisms that confer resistance to Macrolide, Lincosamine and Streptogramin antibiotics. Methods: A total of 96 staphylococcal isolates from different clinical specimens were tested for inducible clindamycin resistance by the disk-diffusion induction test using erythromycin (15g) disk and clindamycin (2g) disc placed 15mm (edge to edge). Results: Inducible clindamycin resistance was detected in 47.22% of methicillin-resistant Staphylococcus aureus isolates and in 21.67% of methicillin-sensitive Staphylococcus aureus isolates. In our setting, clindamycin is safe and effective agent to treat both systemic sand localized Staphylococcal infections, but we recommend that staphylococci isolates, particularly methicillin-resistant Staphylococcus aureus, are tested by the D-test routinely to avoid treatment failure. Keywords: Inducible clindamycin resistance, Methicillin resistant Staphylococcus aureus, D test. INTRODUCTION Staphylococcus aureus are recognized to be caused nosocomial & community acquired infections in every region of world.1 Skin & Soft infections are common manifestation of Staphylococcus diseases in many community outbreaks.2 Antibiotic resistance in Staphylococcus aureus has become an ever increasing problem. Large outbreaks of infection by Methicillin Resistant Staphylococcus aureus (MRSA) were recorded in many hospitals in Australia, USA, Britain and Ireland. Many of these outbreaks appear to have been caused by a single epidemic strain that was transferred between hospitals by the movement of patients.3 Clindamycin is a frequent choice for treating both MRSA & MSSA infections particularly for skin & soft tissue infections and as an alternative in the penicillin allergic patients.4
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The Macrolide Lincosamine Streptogramin-B (MLSB) family of antibiotics is commonly used in the treatment of Staphylococcus infections. 1 Erythromycin & Clindamycin are separate antimicrobial agents their mechanisms of action (inhibition of protein synthesis) and mechanism of resistance are similar. 5 However, one important issue in clindamycin treatment is the risk of clinical failure during therapy. Therapeutic failures caused by MLSB inducible resistance are being more commonly reported.1 MLSB resistance can be detected by a simple test known as disk approximation test or D-test.4,6 Erythromycin are the most effective inducer of inducible MLSB resistance, Antimicrobial susceptibility data are important for the management of infections but false susceptibility result may be obtained if isolates are not tested for inducible clindamycin resistance (iMLSB).7 Hence this study was conducted to know the occurrence of inducible clindamycin resistance (iMLSB) among MRSA and MSSA in this region.
MATERIALS AND METHOD

The present study was carried out in the department of Microbiology, P.E.S. Institute of Medical Sciences & Research, Kuppam. Institutional Ethical Committee clearance was obtained for the study. A total of 96 consecutive, non-repetitive isolates of Staphylococcus aureus from various clinical samples such as urine, pus, sputum, suction tips, stool, blood, body fluids, vaginal swabs etc., were collected for the study over a period of 6 months (January 2009 to June 2009) from hospitalized patients. The study protocol was explained and informed consent was obtained from the particpants of the study. The isolates were first identified as Staphylococcus aureus by standard biochemical techniques8 and methicillin resistance was tested by Kirby Bauer disk diffusion method using Oxacillin disc (1g) obtained from Hi-media laboratories, on Muller Hinton agar plates containing 4% Sodium chloride. The plates were

incubated and isolates were considered methicillin resistant if the zone of inhibition was 10mm or less. After making a lawn culture of the isolated Staphylococcus, erythromycin (15g) disk was placed 15mm (edge to edge) from a clindamycin (2g) disc.1 The plates were incubated at 37oC and the results read. Interpretation of the test was done as stated below: I. MS phenotype - Staphylococcal isolate exhibited resistance to erythromycin (zone size 13 mm) while sensitive to clindamycin (zone size 21 mm) and giving circular zone of inhibition around clindamycin was labelled as having this phenotype. II. Inducible MLSB (iMLSB) phenotype Staphylococcal isolate showed resistance to erythromycin (zone size 13 mm) while being sensitive to clindamycin (zone size 21 mm) and giving D -shaped zone of inhibition around clindamycin with flattening towards erythromycin disc was labelled as having this phenotype. III. Constitutive MLSB phenotype - this phenotype was labeled for those Staphylococcal isolates, which showed resistance to both erythromycin (zone size 13 mm) and clindamycin (zone size 14 mm) with circular shape of the zone of inhibition if any around clindamycin. Quality control strains: Control strains used for disk diffusion test were: 1. Staphylococcus aureus ATCC 25923 (used for routine quality control).9, 10 2. In house strains of Staphylococcus aureus showing D-test positive repeatedly was used as positive control for inducible Clindamycin resistance.
RESULTS

Out of 96 Staphylococcus aureus, 37.5% were MRSA & 62.5% were MSSA. Inducible Clindamycin resistance among Staphylococcus
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aureus was 31.25%. Among MRSA, 47.22% were D-test positive and 27.78% were D-test negative. In MSSA 21.67% were D-test positive and 40% were D-test negative (TABLE: 1). 8(22.22%) isolates in MRSA & 23(38.33%) isolates in MSSA were sensitive to both Erythromycin & Clindamycin (TABLE: 1). The distribution of D-test positive MRSA and MSSA among various clinical samples is shown in Table: 2.

Highest number 44(45.83%) of Staphylococcus isolates was from pus sample, showing inducible resistance of 16.67% in MRSA & 8.33% in MSSA (TABLE: 2).Highest number 40(41.66%) of Staphylococcus isolates were from department of surgery, 33.33% of MRSA & 46.66% of MSSA (TABLE: 3).

Table.1: Distribution of inducible Clindamycin Resistance in Staphylococcus aureus*

Staphylococcus aureus MRSA MSSA Total No of Isolates 36 (37.5%) 60 (62.5%) 96(100%) D-Test positive (E-R;Cd-S) with D-shape 17 (47.22%) 13 (21.67%) 30(31.25%) D-test negative (E-R; Cd-S) 10 (27.78%) 24 (40%) 34(35.41%) Sensitive to (E-S; Cd-S) 8 (22.22%) 23 (38.33%) 31(32.30%) Resistant to (E-R; Cd-R) 1 (2.78%) _ 1(10.41%)

D-Test positive (E-R; Cd-S) with D-shape

D-Test positive (E-R;Cd-S) with D-shape

Table.2:Specimen wise distribution of inducible Clindamycin resistance in Staphylococcus aureus* Sample No of MRSA MSSA Isolates D-test negative (E-R; Cd-S) D-test negative (E-R; Cd-S) Resistant to (E-R; Cd-R) Resistant to (E-R; Cd-R) _ _ _ _ _ _ _ resistant
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Sensitive to (E-S; Cd-S)

Pus Urine Sputum Throat swab Suction tip Plural fluid Total

44(45.8%) 6(16.6%) 25(26.0%) 4(11.1%) 11(11.4%) 2(5.5%) 6(6.25%) 6(6.2%) 4(4.1%) 2(5.5%) 2 (5.5%) 1(2.7%)

4(11.1%) 2 (5.5%) 1 (2.7%) 1 (2.7%) 1(2.7%) 1(2.78%) 10(27.7%)

6(16.6%) 1(2.7%) 1 (2.7%) _ 1 (2.7%) _ _ _ _ _ _ _

5 (8.3%) 3(5%) 2(3.33%) 1(1.67%) 1(1.67%) 1(1.67%)

10(16.6%) 12(20%) 7(11.6%) 3(5%) 2(3.3%) 1(1.6%) 1(1.6%) 8(13.3%) 2(3.3%) _ 1(1.67%) _ 23(38.3%)

90(100%) 17(47.2%)

8(22.2%) 1(2.7%)

13(21.6%) 24(40%)

*E-Erythromycin, Cd-Clindamycin, R-Resistant, S-Sensitive, MRSA-Methicillin Staphylococcus aureus, MSSA Methicillin resistant Staphylococcus aureus

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Sensitive to (E-S; Cd-S)

Table.3:Department wise distribution of inducible Clindamycin resistance in Staphylococcus aureus

Sample No of Isolates D-Test positive (E-R; Cd-S) with D-shape D-test negative (E-R; Cd-S) MRSA D-Test positive (E-R; Cd-S) with D-shape MSSA D-test negative (E-R; Cd-S)

Resistant to (E-R; Cd-R)

Surgery Medicine Ortho OBGY Paed ENT Total

40(41.66%) 6(16.67%) 4(11.11%) 22(22.91%) 4(11.11%) 2 (5.55%) 11(11.45%) 2 (5.55%) 2 (5.55%) 12(12.5%) 5(5.20%) 6(6.25%) 96(100%) 2 (5.55%) 1 (2.78%) 1 (2.78%) _ 2 (5.55%) 1 (2.78%)

1(2.78%) 1(2.78%) 3 (8.33%) _ _ _

3 (5%)

11(18.33%) 14(23.3%) 5 (8.33%) 2 (3.33%) 1 (1.67%) 1 (1.67%) _ 23(38.3%)

2(3.33%) 6 (10%) 2(3.33%) 3 (5%) 3 (5%) 2 (3.33%)

3 (8.33%) 1 (2.78%) _ _ _

1(1.67%) 1 (1.67%) 2(3.33%) 1 (1.67%)

17(47.2%) 10(27.8%) 8(22.22%) 1(2.78%) 13(21.6%) 24 (40%)

E-Erythromycin, Cd-Clindamycin, R-Resistant, S-Sensitive, MRSA-Methicillin Staphylococcus aureus, MSSA Methicillin resistant Staphylococcus aureus

resistant

Fig 1: Staphylococcal isolate showing resistance to erythromycin disc while being sensitive to clindamycin disc and giving D-shaped zone of inhibition around clindamycin with flattening towards erythromycin disc.

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Resistant to (E-R; Cd-R)

Sensitive to (E-S; Cd-S)

Sensitive to (E-S; Cd-S)

DISCUSSION

Staphylococcus aureus infections are an important cause of morbidity in hospitals and the community. The spectrum of diseases produced by this organism varies from toxin mediated phenomenon to pyogenic infection either primary or post operative both in the community as well as in hospitals. Staphylococcus aureus has been known to acquire resistance to most antibiotic including the penicillinase resistant ones like methicillin. Drug selection is important in the treatment of MRSA infections. Inducible clindamycin occurs in the presence of strong methylase inducers like Macrolide antibiotics such as Erythromycin or Azithromycin. MLSB resistant strains are seen sensitive falsely in the presence of a weak inducer like clindamycin and Quinupristin.4, 11 A major concern with regard to use of clindamycin for staphylococcal infections is the possible presence of inducible resistance to clindamycin. Also the incidence of clindamycin resistance varies by geographical area and therefore local statistics are crucial to guide empiric therapy.12 The occurrence rate of inducible Clindamycin resistance in the present study is 47.2% in MRSA & 21.67% in MSSA. Earlier study in the same hospital during 2004 revealed 19% Inducible Clindamycin resistance in MRSA & 19.2% in MSSA respectively.13 In 2008, Ajantha GS et al 9 observed D-test positives in 74% of MRSA & 45% of MSSA isolates. In 2003, Delialioglu N et al 14 from turkey reported that 5.4% D-test positive in MRSA & 10.7% D-test positive in MSSA. In 2004, it has been reported that 12.31% in MRSA & 68% in MSSA isolates by Levin TP et al.15 Similarly in the study conducted by Matthew V.N.OSulivan et al 200516 reported 27.12% in MRSA & 9.5% in MSSA isolates. It was also observed that percentages of inducible resistance and constitutive clindamycin resistance were higher amongst MRSA as compared to MSSA. This was in concordance with few of the studies reported before.1 Some studies have shown a very high frequency of inducible resistance MRSA.9 On the contrary,

Schreckenberger et al and Levin TP et al have shown a higher percentage of inducible resistance in MSSA as compared to MRSA.12,15 Clindamycin, is a semi synthetic derivative of Lincosamine, has excellent tissue penetration like lung, pleural fluid & bile but it is poorly penetrated in CSF & meningitis and crosses the placenta & enters fetal tissue. Clindamycin distributes well into bones & it is an effective antibiotic in treating Osteomyelitis. It is a rapid oral absorption, no requirement of dosage adjustment in the presence of renal disease.17 Staphylococcus showing resistance to Macrolide, Lincosamine, Streptogramin-B (MLSB) antibiotics generally involved drug inactivation, target site modification, impermeability (or) efflux mechanism. 18 Clindamycin resistance can develop in staphylococcal isolates with inducible phenotype, and from such isolates, spontaneous constitutive resistance mutants have arisen both in vitro and in vivo during clindamycin therapy.1 Reporting Staphylococcus aureus as susceptible to clindamycin without checking for inducible resistance may result in institution of inappropriate clindamycin therapy.19 Because of the emergence of resistance to multiple antibiotics among staphylococci the therapeutic options available for clinicians are limited. A therapeutic decision is not possible with-out the relevant clinical and microbiological data. The increasing frequency of MRSA with in vitro inducible clindamycin resistance raises a concern of clindamycin treatment failures.20 Therefore, using in vitro erythromycin resistance as a surrogate marker for all MLS antibiotics, and thereby avoiding them as a treatment option, would be inappropriate. A therapeutic decision is not possible without the relevant antibiotic susceptibility data. This is where the D test becomes significant.21

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CONCLUSION

Due to the emergence of resistance to antimicrobial agents, the accurate drug susceptibility data of the infecting microbe are essential for deciding the therapeutic option. The Erythromycin-Clindamycin disc approximation test or the D test is a simple, reliable method to detect Clindamycin resistance in erythromycin resistant isolates. The D test is an easy to perform test which can enable us in guiding the clinicians regarding the judicious use of clindamycin in skin and soft tissue infections. 186
ACKNOWLEDGEMENT

The author Makam Sri Rangakumar Manjunath is very thankful to the Principal and to the Management of P.E.S. Institute of Medical Sciences & Research, Kuppam for their generous support and encouragement during the period of study. REFERENCES 1. Gurdal Yilmaz, Kemalettin Aydin, Serap Iskender, Rahmet Caylan and Ifthar Koksal. Detection Of Inducible Clindamycin Resistance Among Staphylococci. J Med Microbiol. 2007;56:342-45. 2. Fokas S, Tsironi M, Kalkani M, and Dionysopouloy M. Prevalence of Inducible Clindamycin Resistance in MacrolideResistant Staphylococcus Spp. Clin Microbiol Infect. 2005;11:337-40. 3. Gearald L.Mendell, John E. Bennett and Raphael Dolin, editors. Principles and practice of infectious diseases. 6th edition: Churchill Livingstone Inc; 2005;1:396-11. 4. Fiebelkorn KR, Crawford SA, Mc Elmeel ML and Jorgensen JH. Practical Disk Diffusion Method for Detection of Inducible Clindamycin Resistance in Staphylococcus Aureus & Coagulase negative Staphylococci. J Clin Microbiol. 2003; 41(10):4740-44. 5. Mallick SK, Basaks, Bose S Inducible clindamycin resistance in Staphylococcus Aureus-A Therapeutic Challenge. Journal of

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