Phenotypic Characterization of Macrolide- Lincosamide-Streptogramin B Resistance in Staphylococcus aureus

Staphylococcus aureus (S.aureus) is a prevalent organism causing infections in the community and hospital. A variety of antibiotics are used, including the Macrolide-Lincosamide-StreptograminB (MLSB) family of antibiotics in which clindamycin is the preferred agent. Widespread use of these antibiotics leads to resistance to these MLSB antibiotics; a D-test can characterize the different MLSB phenotypes. This study was taken up with an objective to perform a double disc diffusion test for detecting different phenotypes in S.aureus with particular reference to inducible clindamycin resistance. Out of a total of 174(100%) strains of S.aureus, 98(56.32%) were MRSA, and 76(43.68%) were MSSA. All isolates were tested by D-test. A total of 47(27.01%) were of cMLSB phenotype, 31(17.82%) were of iMLSB phenotype, and 96(55.17%) were of MS phenotype. The majority of MRSA strains were cMLSB phenotype(76.60%) and iMLSB phenotype (64.52%) in comparison to MSSA isolates. Although iMLSB phenotypes are present in both MRSA and MSSA, iMLSB was more in MRSA isolates. Appropriate susceptibility data is essential for a clinician to start clindamycin therapy to prevent therapeutic failures with inducible MLSB resistance in S.aureus isolates. It will be appropriate for all the clinical laboratories to report inducible Clindamycin resistance in S.aureus strains (both MRSA and MSSA), for which D-test is a reliable testing method.


InTRODuCTIOn
Staphylococcus aureus (S.aureus) is a commonly encountered organism causing infections both in hospital and community settings 1 .The genus Staphylococcus contains 32 species, of which 16 species are found in humans. S.aureus is one of the most virulent species having many virulence factors like surface proteins, capsular polysaccharides, cytotoxins, superantigens, enzymes responsible for producing an array of ailments from superficial infections to deep-seated and life-threatening infections 2 .
Treatment of S.aureus infections is usually with antibiotics like β lactams, glycopeptides, quinolones, oxazolidinone, etc. S.aureus has developed resistance to multiple antibiotics by various mechanisms like efflux of the drug, drug inactivation, target alteration, production of β lactamase, etc. 3 Emergence of Methicillin-Resistant S.aureus(MRSA) strains which is a typical hospital acquired organism and acquiring multidrug resistance has still complicated the treatment . The Macrolide-Lincosamide-StreptograminB(MLS B ) family of antibiotics are the agents used against such strains. MLS B includes Macrolide (Eg: Erythromycin, Azithromycin, Spiramycin), Lincosamides (Eg: Clindamycin, Lincomycin), and StreptograminB (Eg: Quinupristin, Dalfopristin). These agents are different chemically, but all of them act by inhibition of protein synthesis, among which clindamycin is the recommended agent due to its pharmacokinetics, and its ability to reach various tissues, including bones 4 .
Widespread use of the MLS B group of antibiotics leads to an increase in S.aureus strains becoming resistant to these drugs, which can be due to any of the following mechanisms: 1. erm, a gene of S.aureus produces rRNA methylase, which brings about changes in the antibiotic binding site. The production of the erm gene can be either constitutive or inducible, leading to cMLS B or iMLS B phenotypes, respectively. 2. Efflux of antibiotics by msrA gene, which is called MS phenotype. 3. Inactivation of lincosamide by chemical alteration by the inuA gene 5 .
Organisms develop resistance to these groups of antibiotics by acquiring genes called erm genes responsible for producing methylases.
S.aureus strains harbor the genes like erm A, B, C & Y in their plasmids, conferring resistance to MLS B antibiotics. The resistance can be inducible resistance where the strains exhibiting this type of resistance don't encode for methylases but become active only in the presence of antimicrobial agents like erythromycin, which is an inducer of erm genes. Another type of resistance exhibited by organisms is called constitutive resistance, in which methylases are produced even in the absence of inducer like erythromycin 6 .
The isolates having the inducible erm gene exhibit resistance to agents like erythromycin, which are the inducer but will appear to be susceptible to the lincosamide and the noninducer macrolides. Hence, using antibiotics like clindamycin will lead to the selection of constitutive mutants leading to treatment failures 7 .
So while testing in vitro, interpretation of different phenotypes has to be done. cMLS B phenotypes are resistant to macrolides like erythromycin and lincosamides like clindamycin. iMLS B phenotypes are resistant to erythromycin and appear sensitive to clindamycin when tested without an inducer. But, in the presence of inducer of erm gene like erythromycin, they are resistant to clindamycin with a D-shaped zone of inhibition. MS phenotypes are sensitive to clindamycin without a D zone and resistant to erythromycin due to drug efflux mechanisms 4 .
Determination of inducible clindamycin resistance by double disc diffusion test is advisable to avoid false sensitive reporting of clindamycin. The use of clindamycin in iMLS B phenotypes can lead to treatment failure because of the selection of cMLS B phenotypic strains. D-test which is an induction test useful in distinguishing S.aureus isolates which have inducible erm mediated resistance, i.e., iMLS B phenotypes from those with resistance due to drug efflux mechanism, i.e., MS phenotypes, and it is essential to test in vitro to differentiate iMLS B and MS phenotype strains to avoid clinical therapeutic failure 8,9 .
Inducible Clindamycin resistance can be tested phenotypically by double disc diffusion test (D-test) or genotypically by molecular methods like Polymerase Chain Reaction(PCR) for detecting erm gene. 10 Though molecular techniques like PCR are more sensitive, its cost, requirement for technical expertise, and non-availability at all testing facilities make it less preferable than simple, easy to perform D-test.

MATeRIALS AnD MeThODS
This is a descriptive cross-sectional study conducted in the microbiology department at Dr. Pinnamaneni Siddhartha Institute of Medical Sciences and Research Foundation, Andhra Pradesh, India, after the Institutional Ethics Committee approval for a period of two years, i.e., from January 2018 to December 2019.
A total of 339 Staphylococcus aureus isolates obtained from various clinical samples were incorporated in the study and were characterized by conventional tests, including Gram's staining, culture, and standard biochemical tests. Antibiotic sensitivity testing of all the isolates was done by Kirby Bauer disc diffusion method on Mueller Hinton agar (MHA) by using antibiotic discs (obtained from HIMEDIA lab Mumbai)of Penicillin (10units), Cefoxitin (30mcg), Ciprofloxacin (5mcg), Linezolid (30mcg), Erythromycin (15mcg) and Clindamycin (2mcg); interpreted as sensitive, intermediate and resistant as per CLSI guidelines. Vancomycin was reported by performing E-test. Identification of methicillin sensitive S.aureus (MSSA) and MRSA strains were according to CLSI guidelines 10 . Double disc diffusion test was done for all the isolates by placing Clindamycin(2mcg) and Erythromycin(15mcg) discs 15mm apart.
Flattening of the zone of inhibition around the clindamycin disc facing the Erythromycin disc was considered D-test positive, indicating inducible clindamycin resistance (Fig. 1). All such isolates were reported as clindamycin resistant.
The strains were interpreted as constitutive MLS B phenotype if resistant to erythromycin with zone size ≤13mm and clindamycin with zone size ≤14mm, and those strains that were resistant to erythromycin with zone size ≤13mm and sensitive to clindamycin with zone size ≥21mm without D-zone was interpreted as MS phenotype 11 . (Table 1) S.aureus ATCC 25923 was used as a control strain. Results tabulated and analyzed statistically.

ReSuLTS
Of the total 339 S.aureus isolates,165 were sensitive to both erythromycin and clindamycin. D-test further characterized the remaining 174 isolates resistant to either erythromycin or clindamycin, or both.  Out of the 174 isolates, 98(56.32%) were MRSA, and 76 isolates were MSSA(43.68%), as shown in Fig. 2.
All the 174 isolates of S.aureus were subjected to D-test to characterize as cMLS B , iMLS B, or MS phenotype. Among the 174 isolates tested, 47(27.01%) strains were of cMLS B phenotype, 31(17.82%) strains were of iMLS B phenotype, and 96(55.17%) strains were of MS phenotype, as shown in Table 2.
In the present study, the iMLS B phenotype was more in MRSA isolates (64.52%) than in MSSA isolates(35.48%).

DISCuSSIOn
Clindamycin is an excellent and preferred agent to treat superficial infections with S.aureus and a preferred antibiotic in patients allergic to penicillin 12 . Resistance to clindamycin in S.aureus strains with inducible phenotype may be reported as sensitive if not tested by D-test giving a false sensitive report which could result in treatment failure and also the emergence of constitutive erm mutants 13 .
The incidence of iMLS B in our study was 17.82% which was comparable with Toleti et al. 14 (18%), Lall et al. 9 (20.3%), and Adaleti et al. 15 (22%). Bingo et al. 16 had reported an incidence of iMLS B to be 28.5% which is higher than in our study. Prabhu K et al. 17 17 iMLS B phenotypes in MRSA was 20% and MSSA was 6.5%, and these results were similar to our study showing iMLS B phenotypes more in MRSA isolates than in MSSA isolates.

COnCLuSIOn
Clindamycin is a preferred antibiotic in superficial Staphylococcal infections and an alternative in penicillin-allergic patients.
False sensitive reports can lead to Clindamycin therapy failures and the selection of a constitutive resistant mutant in an iMLS B strain. So it will be appropriate that all clinical laboratories test and report inducible clindamycin resistance in both MRSA & MSSA by double disc diffusion test, which is a straight forward method to identify iMLS B phenotypes.