Methicillin-resistant Staphylococcus aureus (MRSA) is a distinct and clinically significant strain of Staphylococcus aureus, responsible for a wide range of infections, from mild skin and soft tissue infections to severe conditions such as pneumonia, bacteraemia, and endocarditis. Managing MRSA infections presents as a therapeutic challenge due to the bacteria’s ability to develop and disseminate resistance mechanisms, either through intrinsic pathways or via horizontal gene transfer[7].

Among the antibiotics used for MRSA treatment, Clindamycin, a member of the Macrolide-Lincosamide-Streptogramin B (MLSB) group, is frequently preferred due to its excellent tissue penetration, ability to inhibit toxin production, and efficacy in both hospital- and community-acquired infections[5]. However, its widespread and often indiscriminate use has led to the emergence of resistance in MRSA strains, further complicating therapeutic decisions[6].

The primary mechanism of Clindamycin resistance in MRSA is through ribosomal target modification, which prevents the antibiotic from effectively binding to bacterial ribosomes, inhibiting protein synthesis [2]. This resistance is categorized into constitutive resistance and inducible resistance. Constitutive resistance occurs when MRSA remains resistant to Clindamycin at all times, regardless of the presence of inducers such as Erythromycin[8]. Inducible resistance is not immediately apparent but is activated upon exposure to macrolides, leading to treatment failure if not detected[4]. This phenomenon is mediated by the erm (erythromycin ribosomal methylase) gene, which modifies the bacterial ribosomal binding site, reducing Clindamycin's efficacy and creating resistance. Additionally, some strains demonstrate efflux-mediated resistance, in which bacteria actively pump Clindamycin out of their cells, further limiting its therapeutic efficacy[2].

Given the therapeutic implications of inducible resistance, performing the D-test is crucial in antimicrobial susceptibility testing. The D-test is a phenotypic method that helps to detect inducible Clindamycin resistance, ensuring accurate antibiotic selection and preventing therapeutic failures [5]. Failure to perform this test may lead to unrecognized inducible resistance, resulting in ineffective treatment and poor clinical outcomes.[6]

Determining the minimum inhibitory concentration (MIC) of Clindamycin provides a quantitative assessment of bacterial susceptibility, allowing clinicians to predict drug efficacy more accurately.  Several studies have demonstrated a strong correlation between D-test results and MIC values, reinforcing the clinical relevance of both methods in antimicrobial stewardship [4,2]

The study is intended to find out inducible resistance pattern of Clindamycin along with its correlation to minimum inhibitory concentration The results help to reinforce the importance of routine D-testing in clinical microbiology laboratories to ensure appropriate antimicrobial stewardship and prevent treatment failure.

Study Design and Setting

This prospective study was conducted over a duration of six months in the Department of Microbiology, Government Medical College, Ongole, Andhra Pradesh. Institutional Ethical Committee approval was obtained (IEC approval no: IEC/GMC-OGL/78/2023).

Study Population and Sample Collection

A total of 350 clinical exudate samples were collected and included in this study. Participants of all age groups and both genders who provided written informed consent were included. Patients who declined consent and samples from which organisms other than Staphylococcus aureus were isolated were excluded to maintain ethical integrity and specificity of the study.

Isolation and Identification of Staphylococcus aureus

The exudate samples collected were cultured using standard microbiological methods on appropriate media. Staphylococcus aureus isolates were initially identified by colony morphology, Gram staining, and biochemical testing, including catalase and coagulase tests.

Antibiotic Susceptibility Testing

Antimicrobial susceptibility testing of Staphylococcus aureus isolates was conducted using the Kirby-Bauer disc diffusion method[10].. Antibiotic discs were placed on Mueller-Hinton agar plates previously inoculated with the test organism. Plates were incubated aerobically at 35–37°C for 16–18 hours. Results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.

Detection of Methicillin Resistance (MRSA)

Methicillin resistance in Staphylococcus aureus isolates was determined using the Cefoxitin disc diffusion method. Briefly, a cefoxitin disc (30 µg) was placed onto a Mueller-Hinton agar plate inoculated with a standardized bacterial suspension. Plates were incubated at 35–37°C for 16–18 hours. Zones of inhibition were interpreted following CLSI guidelines: isolates with a zone diameter of ≤21 mm were identified as MRSA, while those with ≥22 mm were classified as Methicillin-sensitive Staphylococcus aureus (MSSA).

Detection of Inducible Clindamycin Resistance (D-Test)

The disk approximation test (D-test) was employed to screen for inducible Clindamycin resistance among MRSA isolates. Mueller-Hinton agar plates inoculated with a standardized suspension of the bacterial isolate (equivalent to 0.5 McFarland standard) were used. An erythromycin disc (15 µg) was placed approximately 15–20 mm away from a Clindamycin disc (2 µg) on the inoculated agar surface. After incubation at 35–37°C for 16–18 hours, the presence of a flattened inhibition zone around the Clindamycin disc adjacent to the erythromycin disc, forming a characteristic “D” shape, indicated inducible Clindamycin resistance.

Determination of Minimum Inhibitory Concentration (MIC) by Broth Microdilution Method

The MIC for Clindamycin was determined using the broth microdilution method following CLSI guidelines. A stock solution of Clindamycin (2560 µg/ml) was prepared by dissolving 582 mg of Clindamycin powder in 200 ml of distilled water, with a potency of 879 µg/mg as per manufacturer specifications. Serial two-fold dilutions ranging from 256 µg/ml to 0.25 µg/ml were prepared in cation-adjusted Mueller-Hinton broth (CAMHB).

A bacterial inoculum was prepared from 4–5 colonies suspended in trypticase soy broth incubated at 35–37°C for 2–6 hours until the turbidity matched a 0.5 McFarland standard. This suspension was diluted at a 1:20 ratio to achieve a final inoculum concentration of approximately 5×10⁵ CFU/ml.

Each well of the microdilution tray received 0.1 ml of Clindamycin dilution followed by the addition of 0.01 ml inoculum. Plates were sealed with a plastic cover and incubated aerobically at 35–37°C for 16–20 hours. The lowest concentration of antibiotic at which visible bacterial growth was inhibited was recorded as the MIC[9].

Out of 350 exudate samples, 220 were culture positive and 130 were culture sterile. 120 Staphylococcus aureus was isolated from culture positive samples of which 72 were MRSA. Our study analysed the resistance patterns of 72 MRSA isolates to Clindamycin and Erythromycin using the D-test as per CLSI guidelines. Among 72 MRSA isolates, 49 (68.05%) isolates exhibited zones of inhibition measuring ≥21 mm for Clindamycin and ≥23 mm for Erythromycin, indicating susceptibility to both antibiotics and a non-inducible pattern of resistance. In 14 isolates (19.44%) flattening of the zone of inhibition of Clindamycin at the side adjacent to Erythromycin was observed with a characteristic “D” shape indicating an inducible pattern of resistance. 7 isolates (9.72%) exhibited complete absence of inhibition zones for both Clindamycin and Erythromycin, consistent with constitutive resistance. Finally, 2 isolates (2.77%) showed inhibition zones of ≥21 mm for Clindamycin but ≤14 mm for Erythromycin, with the absence of a D-shaped zone of inhibition, suggesting Clindamycin susceptibility and Erythromycin resistance likely due to an efflux-mediated resistance mechanism.

Minimum inhibitory concentration to Clindamycin was tested by broth microdilution method. According to the CLSI breakpoint guidelines outlined in the M100 document, an MIC of ≤0.5 μg/mL is considered sensitive, while an MIC of ≥4 μg/mL is classified as resistant. A total of 49 strains (68.05%) were identified as non-inducible, with MIC values of  ≤0.5 µg/mL, indicating a high level of susceptibility to Clindamycin without any evidence of resistance. 14 (19.44%) strains were found to exhibit inducible resistance. Among these, 12 strains (17.0%) had MIC values of ≤2 µg/mL, suggesting partial susceptibility. However, 2 of these strains (3.0%) showed higher MIC values of ≥4 µg/mL, indicating resistance. 7(9.72%) strains displayed constitutive resistance, with very high MIC values of ≥128 µg/mL, demonstrating complete resistance to clindamycin. Lastly, 2 strains exhibited characteristics of an efflux-mediated resistance mechanism, with MIC values of ≥4 µg/mL.

TABLE 1.1- D TEST AND CLINDAMYCIN RESISTANCE PATTERNS

TABLE 1.2- MINIMUM INHIBITORY CONCENTRATIONS (MIC) FOR CLINDAMYCIN

The present study highlights the critical importance of detecting inducible Clindamycin resistance among methicillin-resistant Staphylococcus aureus (MRSA) isolates using the D-test. Macrolide-lincosamide-streptogramin B (MLSB) antibiotics, particularly Clindamycin, are commonly employed to treat various infections caused by Staphylococcus aureus, including those caused by MRSA. However, resistance to MLSB antibiotics is typically mediated by ribosomal target modifications attributed to the erm gene, leading to inducible or constitutive resistance patterns. Accurate identification of these resistance mechanisms is essential for guiding effective antimicrobial therapy and preventing therapeutic failures.

Inducible resistance arises from an initially inactive mRNA that is unable to produce functional methylases unless exposed to an effective inducer such as erythromycin. Upon induction, active mRNA encoding methylases is synthesized, conferring resistance to Clindamycin and related antibiotics [3]. Our study identified inducible Clindamycin resistance in 19% of isolates, which aligns closely with findings reported by Muluneh Assefa et al. (18.9%) [4] and P. Sireesha et al. [2]. These findings reinforce the significance of routine detection to ensure optimal antimicrobial treatment decisions.

In contrast, constitutive resistance is characterized by the continuous production of methylases, even without exposure to an inducer, due to persistently active methylated mRNA. This resistance mechanism confers a consistent and robust resistance profile. Our study reported that 10% of isolates exhibited constitutive resistance, and an additional 3% showed evidence suggestive of an efflux mechanism. These findings parallel the results observed by P. Sireesha et al. [2] and Leclercq et al. [3], who also documented similar percentages of constitutive resistance, thereby

emphasizing the importance of identifying these resistance profiles in clinical isolates.

The correlation observed between the D-test results and the Minimum Inhibitory Concentration (MIC) values further underscores the clinical relevance of our findings. Isolates exhibiting an "S" phenotype (sensitive to both Clindamycin and Erythromycin) demonstrated MIC values ≤0.5 µg/ml. In comparison, isolates with a positive D-test (inducible Clindamycin resistance, D phenotype) primarily exhibited MIC values ≤2 µg/ml, although notably, 2 isolates (3%) within this group showed higher MICs (≥4 µg/ml). This increased MIC may be attributable to resistance mechanisms not detected in the absence of the inducer during the broth microdilution assay.

Isolates showing potential efflux mechanisms consistently exhibited elevated MIC values (≥4 µg/ml), indicating variable resistance levels that could significantly impact clinical outcomes. Furthermore, isolates demonstrating constitutive resistance (R phenotype), resistant to both Clindamycin and Erythromycin, consistently displayed very high MIC values (≥128 µg/ml). This pattern mirrors observations by P. Sireesha et al. [2] and Patel et al. [7], highlighting the complete inefficacy of Clindamycin against these resistant strains, thus emphasizing the need for alternative therapeutic options.

According to the CLSI breakpoints defined in documents M100 and M07 [8,9], MIC values ≤0.5 µg/ml were considered sensitive, while those ≥4 µg/ml indicated resistance. These standardized guidelines provide critical benchmarks that facilitate reliable interpretation of susceptibility results in clinical microbiology laboratories[10][11].

Our results align well with previous research demonstrating the utility and necessity of the D-test as a routine diagnostic tool for antimicrobial susceptibility testing. Studies by Mokta et al. [5] and Gadepalli et al. [6] have similarly advocated for regular implementation of the D-test to ensure precise detection of inducible resistance patterns[12]. Implementing routine D-testing can significantly enhance antimicrobial stewardship, aiding in the effective management of MRSA infections and reducing the emergence and dissemination of resistant bacterial strains[13][14].

The present study concludes that the detection of inducible Clindamycin resistance among MRSA isolates using the D-test is crucial to avoid therapeutic failure. Determining the Minimum Inhibitory Concentration (MIC) values further enhances the prediction of Clindamycin efficacy, supporting informed clinical decisions and optimal antimicrobial therapy choices. Routine application of the D-test in clinical laboratories significantly contributes to effective antimicrobial stewardship by accurately identifying resistance mechanisms. This practice ensures the selection of appropriate antibiotic treatments, ultimately preventing therapeutic failures and controlling the emergence and dissemination of resistant bacterial strains. Therefore, integrating routine D-testing into susceptibility testing protocols is highly recommended for improving patient care outcomes

LIMITATIONS

The limitation of the study is mainly the absence of an inducer in broth microdilution method which would have preferably generated better understanding of minimum inhibitory concentrations in the efflux mechanism

CONFLICT OF INTEREST

The author declares that there are no conflicts of interest for publishing this study

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