INCIDENCE OF INDUCIBLE CLINDAMYCIN RESISTANCE IN CLINICAL ISOLATES OF STAPHYLOCOCCI AND THEIR ANTIBIOTIC SENSITIVITY PATTERN.

The grassland resource could be better managed if the effect of different defoliation regimes on the amount of the dry matter and nutritive value was known. Consequently, 9 accessions of Andropogon gayanus Kunth were evaluated in south region of Benin with an average 1100 mm annual rainfall during 3 years for ley pasture without any fertiliser input. Three cutting regimes (3-10-3, 5-6-5 and 6-4-6-week) were tested for dry matter production (DM), crude protein (CP) content, CP production and mineral (Ca,

Methicillin resistance in Staphylococcus aureus and MRCoNS is due to the expression of the penicillin binding protein 2a (PBP2a), which is a transpeptidase with a low affinity for β-lactams, encoded by the mecA gene. The mobile genetic element, the staphylococcal cassette chromosome (SCCmec), is responsible for transmission of the mec genes determining the resistance to methicillin and almost all β-lactam antibiotics. The horizontal transmission of the resistance genes from MRCoNS onto Staphylococcus aureus has been confirmed, contributing significantly to the development of methicillin-resistant Staphylococcus aureus. 2 The increasing prevalence of methicillin resistance among Staphylococci is an increasing problem. This had led to renewed interest in the usage of Macrolide-Lincosamide-Streptogramin B (MLS B ) antibiotic to treat S. aureus infections 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 the number of Staphylococcal strains acquiring resistance to MLS B antibiotics. 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 mutants leading to clinical therapeutic failure. 3 Most common mechanism for such resistance is target site modification mediated by ern gene which can be expressed either constitutively ( The presence study was undertaken to find the incidence of inducible clindamycin resistance using D-test amongst clinical isolates of Staphylococcus aureus and Coagulase-negative staphylococcus and to know there antibiotic sensitivity pattern.

Material and methods:-
After the permission from institutional ethical committee, present study was conducted at Department of Microbiology. A total of 368 non repetitive Staphylococcus aureus isolates form various clinical specimens like pus, blood, body fluids, sputum, and urine were included in the study.
Isolation and Identification of Staphylococcus 9 : Culture and microscopy : All samples were inoculated on Blood agar and MacConkey's agar. The plates were incubated at 37 o C in for 18 hours and staphylococcus was identified by standard bacteriological procedures like grams staining, colony characters and biochemical testing. 9 All staphylococci were subjected first to slide and tube coagulase test and manitol fermentation on manitol salt agar (MSA). A isolate was labeled as S. aureus if positive for slide and/or tube coagulase positive and manitol fermenting (yellow colonies on MSA). Isolate with negative coagulase test and maintol non fermenting (Pink colonies on MSA) was labeled as coagulase negative staphylococci.

Detection of Methicillin resistance in S. aureus and Coagulase Negative Staphylococci.
Methicillin resistance was detected by performing cefoxitin disc diffusion test as per CLSI 2016 guidelines. For this a 30 µg cefoxitin disc was placed on Muller Hinton agar (MHA) inoculated with the staphylococcus isolate. A isolate was considered as MRSA if it was resistant to cefoxitin i.e. having a zone of inhibition of ≤ 21 mm diameter and MRCoNS if having a zone of inhibition of ≤ 24 mm diameter. 10 Detection of Inducible Clindamycin resistance: D-test 11 All MRSA and MRCoNS isolates exhibiting erythromycin resistance and clindamycin susceptibility on primary antibiotic susceptibility testing were further subjected to D-test, to detect inducible clindamycin resistance.

D-test:
For this Erythromycin (15µg) disc was placed at a distance of 15 mm (edge to edge) from Clindamycin (2 µg) disc on Mueller Hinton agar plate previously inoculated with 0.5 McFarland suspension of the isolate and incubated at 37 o C for 16 to18 hrs.
Three different phenotypes were appreciated. 1. MS Phenotype-Staphylococci isolates exhibiting resistance to erythromycin (Zone size <13mm) while susceptible to clindamycin (zone size > 21mm) and giving a circular zone of inhibition around Clindamycin were labelled as having this phenotype. 2. Inducible MLSB(iMLSB) Phenotype -Staphylococcal isolates showing resistance to erythromycin (zone size ≤13 mm) while being susceptible to clindamycin (zone size ≥21 mm) and giving a D shaped zone of inhibition around clindamycin with flattening towards erythromycin disc were labelled as having this phenotype. 3. Constitutive MLSB (cMLSB) Phenotype -this phenotype was labelled for those staphylococcal isolates which showed resistance to both erythromycin (zone size ≤13 mm) and clindamycin (zone size ≤14 mm) with a circular shape of zone of inhibition if any around clindamycin.

Quality control:
For AST testing Staphylococcus aureus ATCC-25923was used for quality control. For D test ATCC BAA-977 was used as the positive control for iMLSB.

Results:
A total of 368 isolates of Staphylococci from various clinical samples were included in the present study. The clinical samples were Pus 117, Blood 158, Urine 30, Sputum 20, ET secretion 20, CSF 5, Body Fluid 18.      For MS phenotype in MRSA isolates the best sensitivity was for tetracycline and norfloxacin of 75 % followed by gentamycin (50%). Linezolid, teicoplanin and co-trimoxazole had a similar sensitivity of 25 %. For MRCoNS with MS phenotype linezolid was with highest sensitivity of 89.28 % followed by 64.28 % for Gentamycin, tetracycline and teicoplanin had a showed a decent sensitivity of 57.14 % and 53.57 % respectively. Co-trimoxazole was least sensitive with 14.28%.
The AST pattern of MRSA isolates with cMLSB phenotype showed 100 % sensitive to teicoplanin. The next best sensitivity was of 66.66 % for gentamycin, linezolid and co-trimoxazole not isolates were sensitive to norfloxacin. For MRCoNS having cMLSB phenotype the highest sensitive of 73.33 % was for linezolid followed by gentamycin 60 % other antibiotics had a decent sensitivity from 46 % to 53 %.

Discussion:-
The increasing prevalence of methicillin resistance among staphylococci is an increasing problem 13 Macrolide lincosamide-streptogramin B (MLSB) family of antibiotics is a choice of treatment of staphylococcal infection strains. 14 Clindamycin is frequently used to treat skin & soft tissue infections because of good oral bioavailability, low cost, excellent penetration and the fact that it accumulates in abscesses. 15 There is concern about the use of these antibiotics in the presence of erythromycin resistance because of the possibility of induction of cross resistance among members of the MLSB phenotype. 16 Inducible MLSB resistance is not recognized by using standard susceptibility test methods. The strains appear resistance to erythromycin and sensitive to clindamycin by routine susceptibility testing. In such cases in vivo therapy with clindamycin will lead to clinical therapeutic failure. 17 In the present study for MRSA incidence of iMLSB was more than the incidence of cMLSB and MS phenotypes.
For MRSA the incidence of iMLSB being more than cMLSB is similar to the finding of study by Lall M et al al. 15 However Yilmaz etal have reported a higher incidence of cMLSB than iMLSB. 13 For MRCoNS in the present study the incidence of cMLSB was more as compared iMLSB. If we analyze the susceptibility of the isolates as per the phenotypes, linezolid and gentamycin were uniformly having the best sensitivity for all phenotypes except for MRSA isolates having the MS phenotype which showed lower susceptibility rates. Teicoplanin resistance is 100% only for MRSA isolates having constitutive phenotype. For other phenotypes in MRSA and MRCoNS the teicoplanin susceptibility ranges from 25% to 53.57%. For norfloxacin, tetracycline, teicoplanin, co-trimoxazole there is a mixed susceptibility pattern with no single drug having uniformly good susceptibility in all the phenotypes. These drugs have shown moderate sensitivity and thus can be used for empirical treatment in patents not having serious infections. Knowing the antibiotic sensitivity pattern of these antibiotics will play an important role in treatment of infections with MRSA and MRCoNS.
The D test is usually done on erythromycin resistant and clindamycin sensitive isolates, as is the case in the present study. This would take another day or 18 hours for the result of the D test to be available and thus delay in reporting to the clinician. For this studies should be correct out to see whether the D test can be performed on the primary