INDUCIBLE CLINDAMYCIN RESISTANCE AND METHICILLIN RESISTANCE AMONGST STAPHYLOCOCCUS AUREUS ISOLATES: A PHENOTYPIC DETECTION

Abstract

Background: Increasing prevalence of Methicillin resistant staphylococcus aureus is global public health issue in 
both community and hospital settings. Management of MRSA infections is tough owing to its resistance to many 
antibiotics. Macrolide-lincosamide-streptogramins B (MLSB) antibiotics are commonly used for the management of 
MRSA. Clindamycin is being the preferred agent due to its excellent pharmacokinetic properties. However, use of 
clindamycin in erythromycin resistant Staphylococcus isolates could result in treatment failure as a result of 
inducible clindamycin resistance in spite of showing in vitro sensitivity.  
Aim: This study was aimed to find out the percentage of S. aureus having inducible clindamycin resistance 
(iMLSB) in our geographic  area using D-test and to ascertain the relationship between methicillin-resistant S. 
aureus (MRSA) and inducible clindamycin resistance. 
Methods: A total of 822 Staphylococcus aureus isolated from different clinical samples were subjected to routine 
antibiotic sensitivity testing by Kirby Bauer disc diffusion method. All isolates were tested for Methicillin resistance 
by using cefoxitin 30 µg discs. Inducible clindamycin resistance was detected by ‘D’ test as per CLSI guidelines. 
Results: Out of the 822 Staphylococcus aureus isolates, 395 (48.05%) were MRSA and 427 (51.94%) were MSSA. 
482 (58.63%) isolates were erythromycin resistant. These erythromycin resistant isolates when subjected to ‘D’ test, 
89 isolates showed MS phenotype, 148 showed inducible MLSB phenotype and 245 showed Constitutive MLSB 
phenotype. Out of 395 MRSA isolates 116 (29.36%) showed Inducible MLSB phenotype and 190 (48.10%) showed 
Constitutive MLSB phenotype, while in 427 methicillin sensitive Staphylococcal isolates 32(7.49%) showed 
Inducible MLSB phenotype and 55 (12.88%) showed Constitutive MLSB phenotype. The percentage of inducible 
and constitutive resistance was higher amongst MRSA isolates as compared to MSSA isolates. 
Conclusion: Considering the higher   prevalence of clindamycin resistance in MRSA isolates as compared MSSA 
isolates, routine D- test of S.aureus isolates is strongly recommended to prevent treatment failure. Therefore 
inducible clindamycin resistance detection should be the part of S.aureus sensitivity testing in all the microbiology 
11 
Jadhav AG et al. International Research Journal Of Pharmacy, 2023,14:7:11-16. 
laboratories. 
Keywords: Clindamycin, MRSA, D- Test, constitutive MLSB phenotype, Erythromycin, Inducible MLSB phenotype 
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INTRODUCTION: 
Staphylococcus aureus, one of the most common nosocomial and community acquired pathogens has now 
emerged as an ever increasing problem due to its increasing resistance to several antibiotics.1Emergence of 
methicillin resistance in S. aureus has left very few therapeutic alternatives. The macrolide-lincosamide- 
streptogramin B (MLSB) family of antibiotics serves as one such alternative, with clindamycin being the preferred 
agent due to its excellent pharmacokinetic properties.2 The advantages of choosing clindamycin are availability of 
both parenteral and oral formulations, high bioavailability, soft tissue permeability, inhibits toxin production ,no 
dosage adjustments are required in the presence of renal disease, can be given in penicillin allergic patients and is 
relatively cheap. However its increased use has resulted in widespread resistance against clindamycin.3,4 However, 
resistance to this drug is again a problem. Resistance to MLSB can occur by two mechanisms: an active efflux 
mechanism encoded by the msrA gene and target site modification mediated by erm genes, which can be expressed 
either constitutively (constitutive MLSB  Phenotype) or inducible (inducible MLSB Phenotype).5 It is very difficult 
to detect the inducible clindamycin resistance 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 case of another 
mechanism of resistance mediated through msrA genes i.e. efflux of antibiotic, staphylococcal isolates ap- pear 
erythromycin-resistant and clindamycin-sensitive both in vivo and in vitro and the strain do not typically be- 
come clindamycin resistant during therapy.6 Thus to avoid clinical therapeutic failure in the resistance case me- 
diated by erm gene, it is very important to detect inducible clindamycin resistance phenotypes in vitro which 
can be made by erythromycin-clindamycin disc approximation test (D-test) as its sensitivity was found 100% in 
different studies when compared with erm and msr gene detection by polymerase chain reaction. There is a 
wide variation in the rate of inducible clindamycin resistance in different places.7 This study was conducted to 
determine the prevalence of inducible clindamycin resistance among clinical S. aureus isolates and also to study 
their association with MRSA in our set up. 
MATERIALS AND METHODS 
This observational study was conducted in the Department of Microbiology, Swami Ramanand Teerth Rural 
Medical College, Ambajogai, Beed, Maharashtra for a period of 1 year and 6 months from January 2022 to June 
2023 .A total of 822 isolates of Staphylococcus aureus were isolated from various clinical samples e.g. pus, blood, 
urine, sputum, body fluids, throat swabs, swabs from surgical and non-surgical wounds  sent  for bacteriological 
cultures from patients of all age groups and both sexes from various departments. Repeated samples and samples 
showing the possible signs of contaminations were excluded. Isolates were identified on the basis of colony 
characteristics, Gram staining, catalase test, slide coagulase test, tube coagulase test, growth on mannitol salt agar 
and DNase test.8 Antibiotic susceptibility pattern of S. aureus was carried out by modified Kirby Bauer disc 
diffusion method on Mueller Hinton agar. Methicillin-resistance was detected using a 30 mg cefoxitin disc and 
inducible resistance to clindamycin was tested by D-test as per Clinical and Laboratory Standards Institute (CLSI) 
guidelines.9 A lawn culture of the isolate which was adjusted to 0.5 McFarland’s concentration was made on a 
Mueller-Hinton agar plate and discs of  clindamycin (2 mg) and erythromycin (15mg) (Hi-Media, Mumbai, 
India) were placed at a distance of 15 mm (edge to edge) as per the CLSI recommendations, along with routine 
antibiotic susceptibility testing. Three different phenotypes were appreciated and interpreted. This interpretation 
was done only for erythromycin resistant S. aureus strains. 
MS phenotypes: MS phenotypes were the staphylococcal isolates exhibiting resistance to erythromycin (zone size 
≤13 mm) while sensitive to clindamycin (zone size ≥21 mm) and giving a circular zone of inhibition around 
clindamycin. 
Inducible MLSB (iMLSB) phenotype: Inducible MLSB ( iMLSB) phenotypes were the staphylococcal isolates showing 
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 toward erythromycin disc.  
Constitutive MLSB (cMLSB) phenotype: Constitutive MLSB (cMLSB) phenotypes were labeled for 
erythromycin zone size ≤13 mm and clindamycin zone size ≤14 mm with the circular shape of the zone of 
inhibition (if any) around clindamycin or with Staphylococcus isolates showing no zones of inhibition around both 
erythromycin and clindamycin. Quality control of the erythromycin and clindamycin discs was performed with S. 
aureus ATCC  25923, S. aureus.