Inducible clindamycin resistance in staphylococcus isolates from a tertiary care hospital in Eastern India

Purpose: The increasing resistance to antimicrobial agents among Staphylococci has led to renewed interest in the usage of macrolide-lincosamide-streptogramin B (MLS _B ) antibiotics to treat Staphylococcus aureus (S. aureus) infections. The resistance to macrolide can be mediated by msr A gene coding for the efflux mechanism or via erm gene encoding for enzymes that confer inducible or constitutive resistance to MLS _B antibiotics. 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 a routine basis. Materials and Methods: One hundred and ninety-five S. aureus isolates were subjected to routine antibiotic susceptibility testing including cefoxitin (30 μg) by a modified Kirby Bauer disk diffusion method. Inducible resistance to clindamycin in S. aureus was tested by the “D test” as per CLSI guidelines. Results: Thirty-three (16.9%) isolates showed inducible clindamycin resistance, 45 (23%) showed a constitutive resistance while remaining 33 (16.9%) showed the MS phenotype. Inducible resistance and constitutive resistance were found to be higher in MRSA compared to MSSA (22.6%, 35.5%, and 11.8%, 11.8%, respectively). Conclusion: Clindamycin is kept as a reserve drug and is usually advocated in severe MRSA infections depending upon the antimicrobial susceptibility results. This study showed that a D test should be used as a mandatory method in routine disk diffusion testing to detect inducible clindamycin resistance in Staphylococci for the optimum treatment of patients.

Keywords: Clindamycin Resistance, Constitutive MLS _B Phenotype, Inducible MLS _B Phenotype, Methicillin-resistant S. aureus, MS Phenotype

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 to its excellent pharmacokinetic properties. ^[1]

However, widespread use of MLS _B antibiotics has led to an increase in the 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 the 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]

The present study was aimed to find out the percentage of S. aureus having inducible clindamycin resistance (iMLS _B ) in our geographic area using the D test as per CLSI guidelines. ^[4] Also, we tried to ascertain the relationship between methicillin-resistant S. aureus (MRSA) and inducible clindamycin resistance.

The study was conducted from May 2010 to February 2011. A total of 195 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 disk diffusion method on Mueller Hinton agar plates using erythromycin (15 μg), penicillin (10 U), ciprofloxacin (5 μg), and cefoxitin (30 μg) as per CLSI guidelines. Methicillin resistance was detected using oxacillin (1 μg) on Mueller Hinton agar supplemented with 2% NaCl followed by incubation at 35°C and cefoxitin (30 μg) disk. ^[4]

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

1. MS phenotype – Staphylococcal isolates exhibiting resistance to erythromycin (zone size ≤13 mm) while sensitive to clindamycin (zone size ≥21 mm) and giving circular zone of inhibition around clindamycin was labeled as having this phenotype.
2. Inducible MLS _B phenotype – Staphylococcal isolates showing resistance to erythromycin (zone size ≤13 mm) while being sensitive to clindamycin (zone size ≥21 mm) and giving a D-shaped zone of inhibition around clindamycin with flattening toward erythromycin disk were labeled as having this phenotype.
3. 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.
4. One hundred ninety five staphylococcal isolates were tested for susceptibility to erythromycin and other antibiotics of the panel by routine disk diffusion testing; 111 (56.9%) of them were erythromycin resistant. These isolates when subjected to the D test showed 33 (35.5%) isolates resistant to both erythromycin and clindamycin indicating constitutive MLS _B phenotype; 66 isolates showed clindamycin sensitivity. Out of these, 33 isolates showed a positive D test indicating inducible MLS _B phenotype while 33 gave a negative D test indicating the MS phenotype [Table 1]. The overall percentage resistance for all three phenotypes was as follows:Inducible clindamycin resistance – 16.9%, constitutive clindamycin resistance – 23.0%, MS phenotype – 16.9%. The percentage of both inducible and constitutive resistance was higher among MRSA isolates compared to MSSA [Table 1].
   

   Table 1: Distribution of isolates Click here to view

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 the institution of inappropriate clindamycin therapy. On the other hand a negative result for inducible clindamycin resistance confirms clindamycin susceptibility and provides a very good therapeutic option. ^[7]

In our study we found a high percentage of erythromycin resistant isolates [111 (56.9%)]. Among them 33 (16.9%) isolates tested positive for inducible clindamycin resistance by the D test while rest of the isolates were negative for the D test, out of which 45 (23.0%) were shown to have constitutive clindamycin resistance and 33 (16.9%) showed true sensitivity to clindamycin (MS phenotype). These observations suggest that had a 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 was higher among MRSA (22.6%) compared to MSSA (11.76%). In different studies the inducible clindamycin resistance ranged from 20% to 74% [Table 2]. On the contrary, Schreckenberger et al.^[12] and Levin et al.^[13] showed a higher percentage of inducible resistance in MSSA compared to MRSA, 7-12% in MRSA and 19-20% in MSSA; 12.5% MRSA and 68% MSSA, respectively.

Table 2: Various studies reporting the incidence of inducible clindamycin resistance Click here to view

Constitutive resistance in our study was seen in 35.5% of MRSA isolates and 11.8% in MSSA isolates which is similar to the study by Ciraj et al. In different studies the constitutive clindamycin resistance ranges from 0% to 43.7%. ^[10],[14]

Accurate susceptibility data are important for appropriate therapy decisions. The pattern of macrolide resistance in S. aureus varies in different regions. Depending upon this the prescription rate will not be uniform in different regions. There are no substantial data regarding clindamycin prescription from India. It is kept as a reserve drug and is usually advocated in severe in-patient MRSA infections depending upon the antimicrobial susceptibility results. Further, by using clindamycin, use of vancomycin can be avoided. ^[11] However, expression of inducible resistance to clindamycin could limit the effectiveness of this drug. ^[14] So, clinical microbiology laboratories should report inducible clindamycin resistance in S. aureus, and a D test can be used as a simple, auxiliary, and reliable method to delineate inducible and constitutive clindamycin resistance in routine clinical laboratories.

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1755-6783.105134

[Table 1], [Table 2]