Introduction

Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are important causes of nosocomial and community-acquired infections. Treatment of these infections is a growing problem because of the increasing methicillin resistance among staphylococci [1,2]. ^{The macrolide-lincosamide-streptogramin B (MLS_B) family of antibiotics serve as an alternative, with clindamycin being the preferred agent due to its excellent pharmacokinetic properties [3]. However, widespread use of MLS¬ _B antibiotics has led to an increase in number of staphylococcal strains acquiring resistance to MLS _B antibiotics [4].}

Although erythromycin and clindamycin are in separate antimicrobial agent classes, macrolides and lincosamides, respectively, their mechanisms of action (inhibition of protein synthesis) and mechanisms of resistance are similar [5]. The cross-resistance for 3 antibiotic families (macrolides e.g., erythromycin, clarithromycin, azithromcyin; lincosamides e.g., clindamycin; and group B streptogrammins e.g., quinupristin) that share a common binding site is called as the MLS_B phenotype [6]. The two main mechanisms of resistance are production of methylase enzyme encoded by a multiallele plasmid-borne gene erm that alters the ribosomal binding site of the antimicrobial agents and efflux pumps. In staphylococci, the MLS _B resistance can be either constitutive (cMLS_B) or inducible (iMLS_B) [6]. If it is constitutive, in vitro susceptibility tests will show resistance to all 3 antibiotic classes, while if it is inducible, in vitro tests will show resistance to macrolides, but susceptibility to clindamycin will be retained, unless induced by a macrolide (i.e. erythromycin). Isolates that are erythromycin resistant but clindamycin susceptible may either possess inducible clindamycin resistance (iMLS_B) or have efflux pumps that remove macrolides but not clindamycin from the microbe [6].

It is important to determine if resistance (whether inducible or constitutive) to clindamycin exists when it is being considered for therapy. Antimicrobial susceptibility data are important for the management of infections, but false susceptibility results may be obtained if staphylococci are not tested for inducible CL resistance by the disk diffusion induction test (D-test). We performed this study to determine the prevalence of inducible clindamycin resistance in clinical isolates of Staphylococcus spp. and the susceptibility pattern of the isolates.

Material and Methods

Study design and setting

Clinical samples and bacterial isolates

Laboratory procedures

D-test

Results

The demographic details of the patients included in the study are summarized in Table 1. A total of 300 staphylococci were isolated from various types of clinical samples obtained from these patients. Of these 300 isolates, 176 were identified as S. aureus, while 124 were CoNS. Of the 176 S. aureus, 35 (19.89%) were methicillin resistant S. aureus (MRSA), while 25 of the 124 (20.2%) CoNS were methicillin resistant. The erythromycin and clindamycin resistance patterns of the isolates based on disc diffusion method are shown in Table 2 & Fig1. Majority of the MRSA (80.0%) were erythromycin resistant and clindamycin sensitive, while most (63.8%) of the MSSA were sensitive to both erythromycin and clindamycin.

Of the 300 staphylococcal isolates, 121 (40.33%) were erythromycin resistant and clindamycin sensitive (Table 2). These were subjected to D- test for detecting inducible clindamycin resistance. The rates of inducible clindamycin resistance of the different staphylococcal isolates are shown in Table 3. The inducible clindamycin resistance was significantly more among MRSA compared to methicillin sensitive S. aureus (MSSA) (P value < 0.0001).

The antibiotic susceptibility patterns of the different staphylococcal isolates are summarized in Table 4. Majority of the MRSA isolates were susceptible to clindamycin, vancomycin and linezolid, while most of them were resistant to erythromycin, gentamicin, ciprofloxacin, tetracycline and sulfamethoxazole-trimethoprim.

Figure 1

Table 1. Demographic data of the patients included in the study

Figure 2

Table 2. Macrolide resistance of the isolates based on disc diffusion method

Figure 3

Table 3. Inducible clindamycin resistance among the isolates based on D test

Figure 4

Table 4. Antibiotic susceptibility pattern of the isolates

Figure 5

Figure 1: Positive D Test

Discussion

The performance of antimicrobial susceptibility testing remains a crucial component of the microbiology laboratory. Due to the emergence of methicillin resistance in Staphylococcus spp., only a few therapeutic alternatives are 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. Clindamycin has excellent tissue penetration, accumulates in abscesses, and no renal dosing adjustments are needed [9]. Also, it has good oral bioavailability making it a good option for outpatient therapy and changeover after intravenous antibiotics [10]. ^{However, one of the major concerns regarding the use of clindamycin to treat staphylococcal infections is the possible presence of inducible resistance to clindamycin [11,12].}

In S. aureus and CoNS, resistance to macrolides (e.g. erythromycin), lincosamides (e.g. clindamycin) and type B streptogramins (MLS_B ) can be the result of ribosomal target modification in which enzymes encoded by erm genes confer constitutive or inducible resistance to MLS drugs through methylation of the 23S rRNA [2]. Also, staphylococci can have an active efflux mechanism (encoded by msrS genes) that confers resistance to MLS _B only, but not to lincosamides [7,13]. Isolates with constitutive resistance can be detected readily by standard susceptibility testing methods [14].When tested by standard methods , clindamycin may appear active against staphylococci with inducible clindamycin resistance, and so this mode of resistance is identified by the disk- diffusion induction test (D- test) [2,13,15].

Among the 176 Staphylococcus aureus strains, we found 35 (19.9%) to be MRSA, which is lower than that reported by Gupta et al in North India ^{[16]. In our study, 33 (42.3%) S. aureus were of the iMLS_B phenotype, whereas in other studies only about 20 – 30% of the S. aureus showed iMLS_B phenotype [3,17,18]. In our study, 24.0% MSSA isolates were of the iMLS_B phenotype, which is higher than that reported by other workers who have found that 4 – 15% of their MSSA isolates were of the iMLS_B phenotype [19-21]. But in study from Thailand, 22% of MSSA were noted to be of the iMLS_B phenotype similar to our study [22]. Several studies from different parts of India have reported that 30- 64% of their MRSA isolates were of iMLSB phenotype [4,17,19,21,23]. Our study showed a higher value with 75% MRSA isolates found to be of iMLS_B phenotype.}

In the present study the constitutive clindamycin resistance was present in 2.9% of MRSA and 4.7% of MSSA isolates. This trend is in contrast with other studies from Korea where the majority of MRSA had constitutive resistance (cMLSB) [24]. This indicates that the incidence of constitutive and inducible resistance in staphylococcal isolates varies widely by hospital and geographic region. The low constitutive clindamycin resistance in our study may also be attributed to the fact that drug is not commonly used and hence there is less selection of resistant strains.

In our study, majority of the MRSA isolates were susceptible to clindamycin, vancomycin and linezolid, while most of them were resistant to erythromycin, gentamicin, ciprofloxacin, tetracycline and sulfamethoxazole-trimethoprim, similar to the study by Mallick et al [18]. In our study, S. aureus was commonly isolated from hospitalized patients with surgical site infections and orthopedic patients with fracture and bone infection. Similar observations have been reported elsewhere that S. aureus is the cause of most wound infections among hospitalized patients [25].

In conclusion, resistance to antimicrobials such as macrolides might not be readily apparent by routine testing. The D-test is easy to perform and inexpensive for practical work. We feel that this test should be made mandatory as a routine work in clinical microbiology laboratories. Therapeutic failures can be prevented if clindamycin is not used for treatment of patients with infections caused by staphylococci with inducible clindamycin resistance.