Year : 2008 | Volume
: 51 | Issue : 3 | Page : 376--378
Phenotypic detection of inducible clindamycin resistance among Staphylococcus aureus isolates by using the lower limit of recommended inter-disk distance
GS Ajantha, Raghavendra D Kulkarni, Jeevan Shetty, C Shubhada, Pavithra Jain
Department of Microbiology, SDM College of Medical Sciences and Hospital, Sattur, Dharwad - 580 009, Karnataka, India
G S Ajantha
Department of Microbiology, SDM College of Medical Sciences and Hospital, Dharwad - 580 009, Karnataka
Context: Clindamycin is one of the important alternative antibiotics in the therapy of Staphylococcus aureus, particularly in methicillin-resistant S. aureus (MRSA) infections. Inducible clindamycin resistance (iMLS B - inducible Macrolide-Lincosamide-Streptogramin B resistance) is a critical factor in antimicrobial susceptibility testing. Aims: To know the rate of inducible clindamycin resistance among clinical isolates of Staphylococcus aureus in our hospital by Disk approximation test (D-test) using the average recommended inter-disk distance and comparing the results with that of D-test using the lower limit of recommended inter-disk distance. Materials and Methods: A total of 51 erythromycin-resistant and clindamycin-susceptible S. aureus isolates were subjected to disk approximation testing with 21 ± 1 mm and 15 mm edge-to-edge distance between the clindamycin and erythromycin disks. Statistical Methods: Z-test levels. Results: Among 51 erythromycin-resistant and clindamycin-susceptible S. aureus isolates, 25 (49%) were recorded as inducible clindamycin resistant by D-test with 21 ± 1 mm edge-to-edge distance between the clindamycin and erythromycin disks. When we re-tested all the 51 strains by D-test with 15 mm inter-disk distance, we identified 14% more iMLS B strains previously reported as D-test negative. Z-test for MRSA indicates that 15 mm edge-to-edge distance has significant advantage. Conclusions: Since the incidence of inducible clindamycin resistance is high (63% in our study), accurate identification of inducible clindamycin resistance is important to prevent therapeutic failure in infections caused by these strains. We suggest the use of D-test with 15 mm edge-to-edge inter-disk distance for detecting iMLS B .
|How to cite this article:|
Ajantha G S, Kulkarni RD, Shetty J, Shubhada C, Jain P. Phenotypic detection of inducible clindamycin resistance among Staphylococcus aureus isolates by using the lower limit of recommended inter-disk distance.Indian J Pathol Microbiol 2008;51:376-378
|How to cite this URL:|
Ajantha G S, Kulkarni RD, Shetty J, Shubhada C, Jain P. Phenotypic detection of inducible clindamycin resistance among Staphylococcus aureus isolates by using the lower limit of recommended inter-disk distance. Indian J Pathol Microbiol [serial online] 2008 [cited 2020 Aug 11 ];51:376-378
Available from: http://www.ijpmonline.org/text.asp?2008/51/3/376/42515
Staphylococcus aureus is one of the most common pyogenic bacteria infecting man.  S. aureus is known for acquiring antimicrobial resistance promptly after the introduction of new antibiotics.  Clindamycin, a protein synthesis inhibitor, is a frequent therapeutic option for staphylococcal infections, particularly for skin and soft-tissue infections and as an alternative in the penicillin-allergic patients.  It has excellent tissue penetration except for the central nervous system.  The drug accumulates in abscesses and no renal dosing adjustments are needed. Good oral absorption makes it convenient for outpatient prescription or as follow-up drug after intravenous therapy.  However, resistance to this drug is again a problem. Staphylococcal resistance to clindamycin may be inducible (iMLS B - inducible Macrolide-Lincosamide-Streptogramin B resistance) or constitutive. It is noted that treatment of patients harboring iMLS B Staphylococci with clindamycin leads to the development of constitutive resistance, subsequently leading to therapeutic failure.  Inducible MLS B resistance can be detected by a simple test known as Disk approximation test or D-test.  The ideal inter-disk distance between the antibiotics is yet not clear and Clinical and Laboratory Standards Institute (CLSI) recommends a range of 15 to 26 mm disk separation.  The aim of the present study was to evaluate the efficacy of using the lower limit of recommended inter-disk distance to detect inducible clindamycin resistance in S. aureus and also to know the prevalence of inducible clindamycin resistance among S. aureus isolates from our Hospital.
Materials and Methods
A total of 324 consecutive, nonduplicate S. aureus isolates were recovered in our laboratory from clinical samples of patients with active infections like boils, folliculitis, wound infections, cellulitis, abscesses, pneumonia, osteomyelitis, bacteremia and urinary tract infections These isolates were tested for antimicrobial susceptibility by disk diffusion method on Mueller-Hinton agar plates (Hi-Media Laboratories Pvt. Ltd.) as per CLSI guidelines.  Staphylococcus aureus ATCC 25923 was used for quality control. Methicillin resistance was determined by disk diffusion method using 1µg oxacillin (Hi-Media Laboratories Pvt. Ltd.) disks according to CLSI guidelines.
Fifty-one of the 324 S. aureus isolates were erythromycin resistant and clindamycin susceptible (ER/CS). For these isolates, disk approximation testing was performed using 2µg clindamycin disks (Hi-Media Laboratories Pvt. Ltd., Mumbai) and 15-µg erythromycin disks (Hi-Media Laboratories Pvt. Ltd., Mumbai). Disks were placed 21 ± 1 mm apart edge-to-edge (i.e., mean of the recommended range). Tests showing flattening of the clindamycin zone adjacent to the erythromycin disk ['D' shape] were classified as D-test positive.  For the detection of inducible clindamycin resistance, recommended edge-to-edge inter-disk distance is between 15 and 26 mm. Recently, higher sensitivity has been reported with 15 mm inter-disk distance.  We, therefore, re-tested all the 51 strains of ER/CS Staphylococcus aureus with 15 mm edge-to-edge distance.
Data of inter-disk distance analyses were analyzed using Z-test levels.
Out of 324 S. aureus isolates in our study [Table 1], 215 were methicillin susceptible (MSSA) and 109 were methicillin resistant (MRSA). Of these, 51 strains were erythromycin resistant but clindamycin susceptible. Among these strains, 25 (MSSA-7 and MRSA-18) were recorded as D-test positive. We re-tested all the 51 strains of ER/CS S. aureus by D-test with 15 mm inter-disk distance. On re-testing, we could detect seven more iMLS B strains previously reported as D-test negative. Among these additional iMLS B strains, two were MSSA and the remaining five were MRSA. Thus, with 15 mm inter-disk distance, a total of 32 strains were found to be iMLS B strains (63%).
This study shows a high frequency (63%) of iMLS B resistance among S. aureus isolates with an erythromycin-resistant and clindamycin-susceptible phenotype. Inducible MLS B phenotypes were higher in MRSA (74%) as compared to MSSA (45%). The incidence of constitutive and iMLS B resistance varies by geographic region and even from hospital to hospital. The frequency of iMLS B resistance ranges from 7% to 94%. 
Thirty-two out of 51 ER/CS S. aureus isolates were from superficial infections. Of the 51 ER/CS isolates, 29 were isolated from patients admitted to our hospital, while 22 were from outpatient cases. Of the total 32 iMLS B isolates, 19 (59.4%) were recovered from indoor patients, while 13 (40.6%) belonged to the outpatient group which may roughly be considered as an indicator of iMLS B strains in hospital-associated and community-acquired infections, respectively.
Clindamycin is frequently used in skin and soft tissue infections, especially outpatient cases and after intravenous therapy in hospitalized patients.  However, in our study, 21 (65.6%) of 32 ER/CS isolates from superficial infections showed inducible clindamycin resistance. The remaining 11 iMLS B isolates were from deep infections like bacteremia, osteomyelitis, urinary tract infections and pneumonia.
The laboratories and also clinicians must be aware of the local prevalence of iMLS B isolates. Considering the high prevalence of clindamycin resistance among the clinical isolates, we feel that the laboratories should routinely test S. aureus strains for iMLS B . As the D-test is simple, inexpensive and easy to perform, it can be included as a part of routine antibiotic susceptibility testing. The benefit of routine D-testing is that we can clearly identify those strains that remain susceptible to clindamycin despite macrolide resistance.
In one of the recently published reports, 15 mm edge-to-edge distance between the disks increased the sensitivity of detection of iMLS B strains. The authors confirmed the results with multiplex PCR using detection of at least one erm gene as a gold standard and have reported 15 mm disk-to-disk distance to be 100% specific and sensitive.  It is also important to note that the reduction in the inter-disk distance to 15 mm does not give any false-positive results. ,
On re-testing of our isolates with the lower limit of disk-to-disk distance, we could identify 14% more iMLS B strains, which were previously labeled as clindamycin susceptible by D-test carried out with 21 mm inter-disk distance. In our set-up, it was not possible to compare the results with the gold standard (detection of erm genes). Use of MIC for confirmation of the inducible clindamycin resistance also is not practicable because of the inability of the standard broth or agar dilution tests for the detection of iMLS B . ,,
The Z-test analysis showed that the use of 15 mm edge-to-edge distance for the detection of iMLS B in MRSA had significant advantage. Though more iMLS B strains of Staphylococcus aureus as well as MSSA were detected by D-test with 15 mm edge-to-edge inter-disk distance, it was not statistically significant. O'Sullivan et al have reported a major error rate using 22 mm distance (12.3%) and it was higher for MRSA isolates (18.2%) than for MSSA (3.5%). In the present study also, the false-negative results were more with MRSA (5 out of 7) as indicated by the Z-test analysis. Thus, the use of 15 mm inter-disk distance may be more useful and recommended particularly for MRSA strains.
In conclusion, we suggest the use of D-test with 15 mm edge-to-edge inter-disk distance for detecting iMLS B as a routine. The wide range of edge-to-edge distance between the disks recommended may have to be evaluated further, as a narrow range may reduce the error rate of identifying iMLS B strains accurately.
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