CHARACTERIZATION AND PREVALENCE OF CLINDAMYCIN RESISTANCE STAPHYLOCOCCUS AUREUS FROM CLINICAL SAMPLES OF NATIONAL MEDICAL COLLEGE AND TEACHING HOSPITAL, NEPAL

Objective: Clindamycin is the drug of choice for the treatment of severe form of skin, soft tissue, and blood infections caused by resistant Staphylococcus aureus in the form of methicillin-resistant S. aureus (MRSA) and erythromycin-resistant S. aureus. In this research, we determine the susceptibility pattern of isolated S. aureus strains against antibiotics and the prevalence of resistant S. aureus in the form of MRSA, inducible clindamycin-resistant S. aureus (inducible macrolide-lincosamide-streptogramin B [iMLSB]) and constitutive clindamycin-resistant S. aureus (cMLSB). Methods: A total of 310 isolated S. aureus among 2000 different clinical samples were subjected to oxacillin (1 μg) as per the Kirby-Bauer disk diffusion method for MRSA. Clindamycin-resistant either in the form of iMLSB or cMLSB was determined through double disk diffusion method or D-test by use erythromycin (2 μg) and clindamycin (15 μg) as per the CLSI guidelines. Results: Out of total S. aureus, MRSA and methicillin-sensitive S. aureus (MSSA) were 78.06% and 20.64%, respectively. This study showed that iMLSB and cMLSB were 34.19% and 23.22%. Both iMLSB and cMLSB were found more among MRSA than MSSA (43.80%, 26.85% and 40.62%, 10.93%), respectively. Conclusion: This study helps for the characterization of different resistant strains of S. aureus along with the determination of the prevalence rate of these mutant forms causing nosocomial infections.


INTRODUCTION
Staphylococcus aureus is recognized as the causative agent of skin, soft tissue, and systemic infection. It frequently associated with the pus formation in different body sites infections. It is also more common nosocomial as well as opportunistic pathogens [1]. It causes different types of diseases such as skin infections, rhinitis, otitis media infection, mastitis, suppurative wounds osteomyelitis, urinary tract infections, and septic arthritis with life-threatening invasion diseases such as pneumonia, septicemia, endocarditis, bacteremia, and toxic shock syndrome [2].
Origin of methicillin-resistant S. aureus (MRSA) made serious problems for the treatment and selection of therapeutics available alternatives for staphylococcal infections [3]. Earlier staphylococcal infections have been treated by the use of erythromycin since three-four decades and become resistant against it has been reported since long [4]. The increase of resistant staphylococcal infection with MRSA has led to renewed interest in the use of macrolide-lincosamide-streptogramin B (MLSB) antibiotics for the treatment of such infections [5]. These family MLSB antibiotics with clindamycin are the preferred antibiotic due to its best pharmacokinetics property [6].
Macrolides (erythromycin, clarithromycin, and roxithromycin) resistance may be constitutive and inducible in the presence of a macrolide inducer being the most common erythromycin [7]. It is the best inducer for the synthesis of erythromycin ribosome methylase (erm) enzyme. In inducible clindamycin resistance S. aureus (iMLSB), erm enzyme only produced in the presence of erythromycin whereas in constitutive phenotype (constitutive clindamycin-resistant S. aureus The double disk diffusion test (D-test) is used to distinguished inducible clindamycin resistance from constitutive clindamycin-resistant S. aureus among erythromycin-resistant isolates to determine therapeutic for clindamycin to be used as a therapeutic option [9].
In this present research, we aimed to determine the prevalence of MLSBi and MLSBc among hospital-based isolates of S. aureus in the form of MRSA and methicillin-sensitive S. aureus (MSSA).

METHODS
This research included 2000 different clinical samples; pus, blood, urine, sputum, throat swab, pleural fluid, synovial fluid, ascitic fluid, ear swab, cerebrospinal fluid, stool, and high vaginal swab and processed in National Medical College and Teaching Hospital, Birgunj, Nepal from February 2017 to December 2017. Among these, samples 310 (15.5%) S. aureus was isolated by growth on blood agar, nutrient agar, and mannitol salt agar. These isolates were confirmed by use catalase and coagulase test as per the standard operative procedure CLSI [10].
MRSA was identified by the oxacillin disk diffusion method. In this method, 0.5 MacFarland standard inoculums of S. aureus was inoculated on Muller-Hinton agar (MHA) media through swabbing and left for 1 h. The oxacillin disc (1 µg) was now put on inoculated MHA media and incubated at 37°C for 24 h. By the measurement of the zone of diameter,
iMLSB and cMLSB were identified by the use of erythromycin (15 μg) and clindamycin (2 μg) disks through D-test. In this test 0.5, MacFarland standard inoculums of S. aureus were swabbed on the surface of MHA media. The erythromycin and clindamycin disks were placed at a distance of 15-20 mm distant edge to edge from each other incubated at 37°C for 24 h. Through the observation as flattening of the zone of inhibition around the clindamycin proximal to erythromycin disk (D shaped zone of inhibition) indicated as iMLSB phenotypes. There no formation of any zone of inhibition around both antibiotic disks that was defined as cMLSB phenotypes [11].

DISCUSSION
Empirical therapy for staphylococcal infection has become more limited due to the emergence and the prevalence of MRSA. Among options available for the treatment of MRSA and MSSA, clindamycin is one of the good alternatives [6]. However, therapeutic failure caused by iMLSB strains is now being reported commonly.
In one study, all iMLSB shown resistance against macrolides and made either low-level resistance or susceptibility to clindamycin without induction by the use of erythromycin but with induction MICs of clindamycin were noted as an increase from 16 to 256 g/ml in iMLSB indicating inducible resistant [18]. Staphylococci making inducible    [15].

Shidiki et al.
resistance to MLS antibiotics are now common to clinical practice, especially in developing countries. There should be cautious for using the clindamycin in a severe case of infections due to making constitutive mutants during the course of clindamycin therapy in patients with iMLSB [19].
At present, some authors suggested less or no use of clindamycin for serious infections with high burden bacteria such as abscess or osteomyelitis [20]. Conversely, all erythromycin-resistant S. aureus was labeled as clindamycin resistant would prevent the use of clindamycin in infections caused by clindamycin-susceptible isolates. Clindamycin is now mostly used in non iMLSB or less severe form of infections caused by S. aureus.