Inducible Clindamycin Resistant Staphylococcus aureus among Patients Attending Tertiary Care Centre: A Descriptive Cross-sectional Study

ABSTRACT Introduction: Staphylococcus aureus, a superbug, resistant to multiple antibiotics led to growing interest in the usage of macrolide-lincosamide-streptogramin B antibiotics, which are now rapidly developing resistance. This study aims to find the prevalence of inducible clindamycin-resistant Staphylococcus aureus among obtained clinical samples from in-patient and out-patient departments of a tertiary care center. Methods: This is a descriptive cross-sectional study done in clinical samples from the in-patient and out-patient departments of a tertiary care center from September 2020-May 2021. Ethical clearance was taken from the Institutional Review Committee (Ref: 068/2077/2078). Staphylococcus aureus were isolated and antibiotic susceptibility tests were performed by disc diffusion method. Inducible clindamycin and methicillin resistance Staphylococcus aureus were detected using D-test and cefoxitin disc according to Clinical and Laboratory Standards Institute guidelines. Convenient sampling was done and the data was analyzed using Statistical Package for Social Sciences version 20. Point estimate at 95% confidence interval was calculated along with frequency and proportion for binary data. Results: Among a total of 141 Staphylococcus aureus isolated, the prevalence of inducible clindamycin resistant phenotype was 41 (29.1%) (21.6-36.59 at 95% Confidence Interval). Whereas, 30 (21.3%) were constitutive clindamycin resistant. The inducible 28 (47.5%) and 19 (32.2%) constitutive clindamycin resistance was higher among methicillin-resistant Staphylococcus aureus. Conclusions: The frequency of inducible clindamycin resistance among methicillin resistant Staphylococcus aureus was high, which alarms the use of macrolide-lincosamide-streptogramin B antibiotics in Staphylococcus aureus infections. Hence, D-test should be performed to detect inducible clindamycin resistance in routine testing to prevent treatment failure.


INTRODUCTION
Staphylococcus aureus is an aggressive pathogen, resistant to several antibiotics that cause nosocomial as well as community-acquired infections. 1,2 With the rise of methicillin-resistant S. aureus (MRSA), therapeutic options for S. aureus infection have narrowed. 3,4,5 Clindamycin (CL), a macrolide, lincosamide, streptogramin B (MLS B ) group of antibiotics is now preferred for treating methicillin-resistant S. aureus infections. 3 However, its extensive use has led to increasing resistance among Staphylococcal strains. 6 MLS B resistance occurs frequently due to erm genes, which can be expressed inducibly (iMLS B , inducible Macrolide-Lincosamide-Streptogramin B phenotypes) or constitutively (cMLS B , constitutive Macrolide-Lincosamide-Streptogramin B phenotype). 7 In vitro, inducible clindamycin resistant strains show clindamycin sensitive which results in treatment Free Full Text Articles are Available at www.jnma.com.np failure. 8,9 Detection of iMLS B can be done by D-test, which has high sensitivity and specificity. 3 There are limited studies of the prevalence of iMLS B S. aureus from Nepal. 4 Thus, this study aims to find out the prevalence of inducible clindamycin resistant S. aureus using D-test among obtained clinical samples of a tertiary care center. Overall 3428 samples were processed using standard microbiological techniques without delay. A total of 141 S. aureus isolates were identified by their colony characteristics, Gram staining, catalase test, slide and tube coagulase test, and growth on mannitol salt agar. 10 Furthermore, an antibiotic susceptibility test was performed by modified Kirby Bauer's disc diffusion method on Mueller Hinton Agar (MHA) plates as per Clinical and Laboratory Standards Institute (CLSI) guidelines. 11 S. aureus isolates with cefoxitin zone size ≥22 mm were considered methicillin-susceptible and those with ≤ 21mm were considered resistant. Isolates that showed erythromycin (E) resistant (zone size ≤ 13mm) and clindamycin sensitive (zone size ≥21mm) were subjected to D-test by keeping the erythromycin and clindamycin discs 15mm apart on MHA plate. 11 Isolates with flattening of the zone of inhibition (ZOI) of clindamycin at the side adjacent to erythromycin were considered D-test positive (iMLS B phenotype) whereas isolates with no flattening of ZOI around CL were D-test negative (MS phenotype). The cMLS B phenotypes were resistant to both CL and E and susceptible phenotypes were sensitive to both discs. S. aureus ATCC 25923 was used to carry out quality control.

METHODS
All the data collected were entered and statistical analysis was done using Microsoft Excel 2016 and Statistical Package for the Social Sciences (SPSS) version 20. Point estimate at 95% CI was calculated along with frequency and proportion for binary data.

RESULTS
From a total of 141 clinical isolates of S. aureus tested for antibiotic susceptibility to erythromycin (E), clindamycin (CL), and other antibiotics by routine disc diffusion method, 86 (61.0%) isolates were resistant to erythromycin and 109 (77.3%) isolates were sensitive to clindamycin. The D-test performed on them showed the overall prevalence of inducible clindamycin resistance (iMLS B ) to be 41 (29.1%) (21.6-36.59) at 95% Confidence Interval. Different types of susceptibility patterns of S. aureus isolate to clindamycin were observed. There were 30 (21.3%) isolates of constitutive resistant (cMLS B) phenotype whereas there was 2 (1.4%) uncommon variant of S. aureus isolates which showed erythromycin sensitive and clindamycin resistance (E-S, CL-R) ( Table 1).

DISCUSSION
S. aureus is of major concern, particularly due to its increased resistance to various antimicrobials, which has narrowed the therapeutic options for clinicians. Globally, Asia is among the region with the highest burden of MRSA. 12 2,15 However, the lower incidence has also been reported by Adhikari et al (11.48%) and Sah et al (12%). 4,16 As the inducible resistance was noted, it should be reported as clindamycin resistant to prevent therapeutic failure. Hence, it is necessary to perform D-test on a routine basis as false interpretation can lead to constitutive resistance among them causing therapeutic failure. There was 21.3% of cMLS B S. aureus strain observed in our study which was consistent with the other studies. 2,7,17 Therefore, the overall resistance to clindamycin in this study was73 (51.8%) which was alarmingly high. Thus, this data reflects the overuse of MLS antibiotics in our institution, which is of major concern.
The present study identified 41.8% MRSA which was in accordance with the other studies from Nepal, 3 S. aureus is a skin flora that can penetrate through trivial injuries, cuts, surgical incisions, etc. In the current study, the majority of the S. aureus was isolated from swabs and pus samples. Reports from other authors have also shown similar findings. 5,25 Thus, these evidence proves the fact that S. aureus is an important pathogen to cause pyogenic infections.
The limitation of this study was that the molecular method was not possible because of limited resources. Moreover, the molecular method could have explored the genetic characteristic of the two uncommon variants. A multicentric study within and out of this region would have strengthened the data.

CONCLUSIONS
This study demonstrates that S. aureus is the most common pathogen causing soft tissue and wound infections. With the increase in the frequency of MRSA in our hospital setup, the treatment options for MRSA are becoming limited, as most of them are resistant to erythromycin and clindamycin in the form of iMLS B and cMLS B . This indeed calls for the injudicious use of drugs like vancomycin and linezolid which are kept as a last resort for serious Staphylococcal infections. Therefore, there should be regular monitoring of the antimicrobial susceptibility pattern and proper enforcement of the empirical treatment protocols, to prevent antibiotic-resistant S. aureus. D-test should be mandatory on routine antibiogram testing as this will check the false clindamycin sensitive strains.