Staphylococci are responsible for a wide spectrum of diseases. Currently, the organism is posing a global threat due to high rate of antibiotic resistance. Antibiotic susceptibility testing of 10 clinically relevant antibiotics was performed for 60 erythromycin resistant isolates. The isolates were found resistant to Fluoroquinolone group of antibiotics, 70% isolates showing resistant to ofloxacin and ciprofloxacin. However, most of the isolates (88.3%) were susceptible to linezolid. Methicillin resistance was observed among 56% Staphylococci with 42% S. aureus and 25% CNS. This result is in accordance with the findings disseminated by other studies done in various regions of Nepal [17, 18] and of the world [11, 19, 20, 21]. A marked variation has been observed in methicillin resistance isolated among different geographical condition as well as among hospitals of same country. In Nepal, a relatively lower rate of MRSA and MRCNS (18% & 9%) was reported by Thapa and Sapkota [18]. Another report, however, showed alarmingly high MRSA prevalence of 75.5% and 69% [22, 23]. Inappropriate use of antibiotics, improper infection control procedure in hospitals, increased use of medical implants may contribute to emerging methicillin resistant isolates.
Increasing frequency of MRSA and MRCNS infection and changing pattern in antibiotic resistance have sparked renewed interest in the use of MLSB antibiotic. Particularly, clindamycin has become an excellent drug for Staphylococcal infection as an alternative to patients allergic to β lactam antibiotics because of its low cost, low side effects and good tissue penetration [11, 16, 24]. Steward et al. [21] have described different phenotypes which include iMLSB, cMLSB, moderate sensitive (MS) and sensitive (S) among Staphylococcal isolates resistant to macrolide. Macrolide resistant Staphylococcal isolates may have constitutive or inducible resistant to clindamycin which is difficult to detect in routine laboratory test if they are not placed adjacent to one another. During clindamycin therapy, these inducible phenotypes can gradually develop constitutively resistant mutants both in vitro and in vivo. Hence, detection of such resistant phenotypes is important to minimize treatment failure [7]. Since the iMLSB resistance mechanism is unrecognized by using standard susceptibility test methods and its prevalence varies according to geographic location, D-test becomes an imperative part of routine antimicrobial susceptibility test for all clinical isolates [25, 26].
In this study, almost all isolates of Staphylococci presented MLSB resistant phenotypes. In fact, cMLSB resistant phenotype was the most common and highest (40%) followed by MSB (37%) and iMLSB (23%) phenotypes. Varying prevalence rates of MLSB resistance phenotype are reported by other studies [15, 17, 27, 28, 29]. iMLSB was found higher (44%) in MRSA whereas it was cMLSB (36%) in MSSA. Among 21 CNS isolates, iMLSB, cMLSB and MS phenotype was detected in 2 (3.3%), 16 (26.7%) and 3 (5%) respectively. Constitutive resistance among CNS was observed in various studies as well [20, 29, 30]. cMLSB was detected among 80% MRCNS and 67% MSCNS while iMLSB was observed only among MRCNS (13.3%) and not in MSCNS. Variations in these results depend on factors like sample size, patient’s age, geographical region, population studied, trends of antibiotic prescription, circulating clones and origin of isolates [31].
Studies on the prevalence of MLSB resistance in Staphylococci using phenotypic method is available to some extent but data on genetic determinants is limited in Nepal. Resistance to MLSB is mostly based on ribosomal target modification encoded by erm gene for enzyme methylase. The resistance mechanism is the methylation of 23S binding site to cause premature dissociation of the peptidyl tRNA from the ribosome halting further protein synthesis. In inducible resistance, the bacteria produce inactive mRNA that is unable to encode methylase. The mRNA becomes active only in the presence of a macrolide inducer. By contrast, in constitutive expression, active methylase mRNA is produced even in the absence of an inducer. The strains harbouring an inducible erm gene are resistant to the inducer but remain susceptible to non inducer macrolides and lincosamides. Mutations in the promoter region of erm allow production of methylase without an inducer [4, 32].
According our findings, the ermC gene was the most prevalent among Staphylococci isolates (22, 37%) followed by ermB among 6 (10%) isolates while ermA gene was not detected. The presence of erm genes varied in studies carried out by different researchers. The study carried out by Martineau et al. [33] in Canada, ermA gene was detected among 20.9% S. aureus and 66% CNS. Also, a multi-centre study in 24 European University hospitals, prevalence of ermA gene was higher than ermC and ermB genes among 851 S. aureus [19]. Lina et al. [29] showed 63.2% S. aureus positive for ermA gene and 44% CNS positive for ermC gene while ermB was positive only in 1% Staphylococci. As opposed to these studies, our study did not detect any ermA gene.
The strains with MS phenotype are resistant to macrolide and streptogramin but are susceptible to clindamycin. Such resistance is encoded by msr gene, either msrA or msrB [4]. Conferring active efflux of antibiotics such that intracellular concentration becomes low and ribosomes are free from the antibiotics. In this study, 6 (10%) isolates were detected with the presence of msrA and 7 (12%) with msrB genes. Export of macrolides is rarely seen in S. aureus but seems to be more frequent in CNS [29].
The erm genes were detected in 5 isolates showing cMLSB, 12 isolates showing iMLSB and 2 isolates with MS phenotype. Similarly, ermB gene was detected 3 isolates showing cMLSB, 2 isolates showing iMLSB and a single isolate with MS phenotype. None of the isolates with MLSB resistance were detected with ermA gene. This result is in accordance with the study carried out in Germany with 63% ermC showing constitutive resistance [34]. In contrast to situations reported by other studies [11, 29], in which constitutive resistance tends to be caused by ermA and the inducible phenotype is caused by ermC.
None of the erythromycin resistant isolates were encountered without any of the tested resistant mechanism. This is in contrast to other previous studies where unidentified resistance mechanism were observed among Staphylococcal isolates [11, 20]. Additionally, resistant genes were not detected among phenotypically erythromycin susceptible isolates. Our findings show a correlation between the presence of specific genes or sets of genes and the phenotypic MLSB resistance.