Detection of inducible clindamycin resistance among Staphylococcal isolates from different clinical specimens in western IndiaN Pal, B Sharma, R Sharma, L Vyas
Department of Microbiology, SMS Medical College, Jaipur, India
Background: Macrolide (MLS B ) resistance is the most widespread and clinically important mechanism of resistance encountered with Gram-positive organisms. Resistance may be constitutive (cMLS B phenotype) or inducible (iMLS B phenotype). The iMLS B phenotypes are not differentiated by using standard susceptibility test methods, but can be distinguished by erythromycin-clindamycin disk approximation test (D-test) and demonstration of resistance genes by molecular methods. Aims: To demonstrate in vitro inducible clindamycin resistance (iMLS B ) in erythromycin-resistant (ER) and clindamycin-susceptible (CLI-S) clinical isolates of Staphylococci spp., and interpretation of susceptibility tests to guide therapy. Materials and Methods: Eight hundred and fifty-one isolates of Staphylococci spp. were recovered from various clinical specimens. All the Staphylococcal spp. were identified by conventional microbiological methods including colony morphology, Gram stain, catalase, slide coagulase and tube coagulase. Antibiotic susceptibility testing was performed by Kirby Bauer disc diffusion method. Erythromycin-resistant isolates were examined for inducible clindamycin resistance (iMLS B ) by using double disk approximation test (D-test) at 15 mm disk separation. Results: The Staphylococci spp. isolated were 379 S. aureus [31.60% methicillin-resistant S. aureus (MRSA), 12.92% methicillin-sensitive S. aureus (MSSA)] and 472 coagulase-negative Staphylococci (CNS) [37.60% methicillin-resistant coagulase-negative Staphylococci (MRCNS), 17.86% methicillin-sensitive coagulase-negative Staphylococci (MSCNS)]. Four hundred and thirty (50.52%) Staphylococcal spp. isolates showed erythromycin resistance. Constitutive resistance was demonstrated in 202 (46.97%), inducible clindamycin resistance (iMLS B ) in 101 (23.48%), and non-inducible (MS) in 127 (29.53%). Two distinct induction phenotypes, D (18.13%) and D + (5.34%) were observed. All iMLS B isolates were susceptible to linezolid and vancomycin while 78.78% to ciprofloxacin. Conclusions: Fifty percent of Staphylococcal spp. were ER among which 23.48% were iMLS B phenotypes. Eighty-seven per cent of iMLS B phenotypes were observed to be methicillin-resistant. The high frequency of methicillin resistance isolates (87.12%) with in vitro inducible clindamycin resistance at our institute raises concern of clindamycin treatment failures with methicillin-resistant infections. So we recommend that microbiology laboratories should include the D-test for inducible resistance to clindamycin in the routine antibiotic susceptibility testing.
Keywords: D-test, inducible clindamycin resistance, Staphylococcus aureus
Staphylococcus aureus and coagulase-negative Staphylococci (CNS) infections have become common among both hospitalized and non-hospitalized patients. The emergence of resistance to antimicrobial agents among Staphylococci is an increasing problem.
The development of resistance in Staphylococcus species to macrolide, lincosamide and streptogramin B has limited the use of these antibiotics. Macrolide resistance may be due to enzymes encoded by a variety of erm genes-MLS B phenotype and may be constitutive (cMLS B phenotype) or inducible (iMLS B phenotype). Another mechanism is active efflux pump encoded by the mrs A gene (MS phenotype). The MS and iMLS B phenotypes are indistinguishable by using standard susceptibility test methods, but can be distinguished by erythromycin-clindamycin disk approximation test (D-test) and demonstration of resistance genes by molecular methods. ,
The aim of this study was to determine the rate of inducible clindamycin resistance in both methicillin-resistant and susceptible strains of Staphylococcus in our hospital as data describing iMLS B prevalence among Staphylococcus isolates in this region is unknown.
Eight hundred and fifty-one isolates of Staphylococci spp. were recovered from blood, cerebrospinal fluid, pus, sputum, tracheal aspirate, nasal swab, urine and other specimens received in the Department of Microbiology over a period of nine months from August 2008 to April 2009.
All the Staphylococcal species were identified by conventional microbiological methods including colony morphology, gram stain, catalase, slide coagulase and tube coagulase test.
Antibiotic susceptibility testing was performed by Kirby Bauer disc diffusion method. Antibiotic discs used were ampicillin (10 μg), amoxiclav (20/10 μg), cefepime (30 μg), ceftriaxone (30 μg), cephotaxime (30 μg), cefoperazone-sulbactam (75/30 μg), cephalexin (30 μg), ciprofloxacin (5 μg),) doxycycline (30 μg), erythromycin (15 μg), linezolid (30 μg), netilmicin (30 μg), piperacillin-tazobactum (100/10μg) and vancomycin (30μg).
Methicillin resistance was detected by agar screen test. Inoculum was prepared by suspending organisms from 24-h culture in sterile saline and adjusting the turbidity to 0.5 McFarland. A sterile cotton swab was dipped into the bacterial suspension; spot inoculated on Mueller-Hinton agar plate (supplemented with 4% NaCl containing 6 μg of oxacillin per ml). The plates were incubated at 35 0 C for 24 h for Staphylocccus aureus and 48 h for coagulase-negative Staphyloccci (CNS). Growth of even a single colony is indicative of resistance. 
Disk approximation testing (D-test) was performed for each isolate according to Clinical and Laboratory Standards Institute (CLSI) method.  A 0.5 McFarland suspension was prepared in normal saline for each isolate and inoculated on Mueller-Hinton agar plate. Clindamycin (CLI)-2 μg and erythromycin (ER)-15μg disks were placed 15 mm apart edge to edge manually. Plates were incubated at 35 0 C for 24 h and zone diameters were recorded. Induction test categories were interpreted as given in [Table 1]. 
A total of 851 Staphylococcal spp. isolates were included, of which 379 were S. aureus [31.60% methicillin-resistant S. aureus (MRSA), 12.92% methicillin-sensitive S. aureus (MSSA)] and 472 CNS [37.60% methicillin-resistant CNS (MRCNS), 17.86% methicillin-sensitive CNS (MSCNS)]. Categorization of the isolates along with sources is depicted in [Table 2].
Four hundred and thirty (50.52%) clinical isolates which showed erythromycin resistance were tested for inducible resistance by double disk approximation test. Out of 430 erythromycin-resistant strains 101 (23.48%) were iMLS B phenotypes [43.56% MRSA (44/101), 6.93% MSSA (7/101), 43.56% (44/101) MRCNS and 6.0% (6/101) MSCNS].
Eighty-eight (87.12%) iMLS B phenotypes were observed to be methicillin-resistant.
D-test yielded two distinct induction phenotypes, D-zone phenotype [Figure 1] was observed in 78 (18.13%) and D + phenotype [Figure 2] in 22 (5.34%) isolates. Both D and D + results were considered positive for CLI induction- iMLS B phenotypes. One hundred and twenty-seven (29.53%) isolates showed ERresistant and CLI-susceptible zone diameters with no blunting of the zones (MS phenotype). Two hundred and two (46.97%) isolates showed ER and CLI resistance (cMLS B phenotype), of which 98 were MRCNS, 85 MRSA and the rest were methicillin-sensitive [Table 3]. No hazy D zone (HD) phenotype was observed.
Susceptibility of iMLS B phenotypes isolated were ampicillin 37.5%, amoxiclav 39.13%, cefepime 12.5%, ceftriaxone 50.0%, cephotaxime 62.5%, cefoperazone-sulbactam 60.86%, cephalexin 23.07%, ciprofloxacin 78.78%, doxycycline 69.56%, linezolid 100%, netilmicin 46.60%, piperacillin-tazobactum 69.56% and vancomycin 100%.
In our study, 851 Staphyloccocal isolates were obtained over a period of nine months (2008-09) in which 50.52% were ER-resistant. Among the ER-resistant S. aureus iMLS B resistance was observed in 24.63% (51/207) similar to that reported by Gadepalli et al., Fiebelkorn et al., and Jorgensen et al. ,, Some investigators have reported a higher incidence ,,,,, (iMLS B resistance) while others have indicated lower incidence. ,,,, We observed almost a similar rate of iMLS B resistance among S. aureus (24.63%) and CNS (22.42%), while others have reported variable results. The different patterns of resistance observed in various studies are because iMLS B resistance varies by geographical region, age group, methicillin susceptibility and even from hospital to hospital.
As observed in studies by Steward et al., and Schreckenberger et al., our CLI induction results also showed two phenotypes, D (18.13%) and D + (5.34%) phenotypes and both are considered to be positive D-zone test. ,
All our iMLS B isolates were susceptible to linezolid and vancomycin while 78.78% to ciprofloxacin. Rahbar and Hajia also found all iMLS B isolates susceptible to linezolid and vancomycin. 
Due to the emergence of resistance to antimicrobial agents accurate drug susceptibility data of the infecting microbe is an essential factor in making appropriate therapeutic decisions. MLS B resistance is the most widespread and clinically important mechanism of resistance encountered with Gram-positive organisms due to the production of methylases and efflux proteins. In vitro susceptibility testing for clindamycin may indicate false susceptibility by the broth microdilution method and by disk diffusion testing with erythromycin and clindamycin disks in nonadjacent positions. Erythromycin-clindamycin disc approximation test or D-test is a simple, reliable method to detect inducible resistance to clindamycin in erythromycin-resistant isolates of Staphylococci. [2, 6, 14] Sensitivity of D-test performed at 15-20 mm disk spacing was 100% when correlated with detection of erm and msr genes by polymerase chain reaction(PCR). ,,,,
Clinically, bacterial strains exhibiting iMLS B have a high rate of spontaneous mutation to constitutive resistance and use of non-inducer antibiotics such as clindamycin can lead to selection of constitutive mutants at frequencies of 10 -7 cfu. ,,, McGehee and other investigators have confirmed this rapid in vitro conversion of inducible to constitutive MLS B resistance in Staphylococci. , There have also been a number of reported clindamycin or lincomycin therapy failures in serious infections due to Staphylococci with iMLS B resistance, indicating that it is not uncommon. , Clindamycin has long been an attractive option in the treatment of skin and soft tissue infections (SSTI) and serious infections because of its efficacy against MRSA and MSSA, as well as anaerobes. This has led to questioning the efficacy of clindamycin use against any erythromycin-resistant Staphylococci spp. However, if inducible resistance can be reliably detected on a routine basis in clinically significant isolates, clindamycin can be safely and effectively used in patients with true clindamycin-susceptible strains.
In the present study, 29.53% of erythromycin-resistant Staphylococcal isolates showed true clindamycin susceptibility (MS phenotype). Patients with infections caused by such isolates can be treated with clindamycin without emergence of resistance during therapy.
The high frequency of methicillin-resistance isolates (87.12%) with in vitro inducible clindamycin resistance at our institute raises concern of clindamycin treatment failures with methicillin-resistant infections.
We conclude that it is important for laboratories to be aware of the local prevalence of iMLS B isolates. On the basis of their data they can choose whether or not to perform the D-test routinely. The D-test is an easy, sensitive, and reliable means for detection of iMLS B strains in a clinical laboratory setting without specialized testing facilities.  This prevalence of iMLS B may change over time with the emergence of strains with different sensitivity patterns, so periodic surveys should be performed if testing is not routine.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]