Recent Indian studies have established inducible macrolide-Lincosamide-streptogramin B (MLSB) resistance as a clinically relevant resistance mechanism in Staphylococcus aureus, particularly in tertiary-care hospital settings. Majhi et al. demonstrated both inducible and constitutive MLSB resistance among clinical isolates in Eastern India, highlighting the inadequacy of routine susceptibility testing without D-test confirmation.⁴

Earlier investigations from South India reported that erythromycin-resistant, clindamycin-susceptible isolates frequently harbour inducible resistance detectable only by the D-test, emphasizing the risk of false clindamycin susceptibility reporting.⁵ Similar findings were subsequently documented from Maharashtra, where inducible MLSB resistance was identified in isolates that would otherwise be misclassified using routine disc diffusion alone.⁶

Independent studies from Karnataka, North India, Kashmir, and Eastern India have consistently confirmed the presence of inducible⁸ MLSB resistance across diverse geographic regions, underscoring the need for routine D-test implementation in clinical microbiology laboratories.⁷

The present study was undertaken to evaluate the diagnostic utility of the D-test for detecting MLSB resistance among Staphylococcus aureus isolates in a tertiary care hospital in Central India.

AIM

To assess the diagnostic efficacy of the D-test in identifying MLSB resistance patterns among Staphylococcus aureus isolates.

OBJECTIVES

1. To isolate and identify Staphylococcus aureus from various clinical specimens using standard microbiological techniques.
2. To determine the prevalence of MLSB resistance phenotypes among Staphylococcus aureus isolates.
3. To detect inducible clindamycin resistance using the D-test in erythromycin-resistant isolates.

MATERIALS AND METHODS

This prospective laboratory-based observational study was conducted in the Department of Microbiology of a tertiary care hospital in Central India.

Study Duration:

This prospective laboratory-based observational study was conducted over a period of 3 months, in the Department of Microbiology of a tertiary care hospital in Central India.

Sample Size Calculation:

The sample size was calculated based on the expected prevalence of inducible clindamycin resistance (iMLSB phenotype) among Staphylococcus aureus isolates, as reported in Indian studies.

Previous Indian studies have reported the prevalence of inducible MLSB resistance ranging from 10% to 20%. Considering an expected prevalence (p) of 15%, a confidence level of 95%, and an absolute allowable error (d) of 10%, the sample size was calculated using the standard formula for estimating a single proportion:

Where:

• = required sample size
• = standard normal variate at 95% confidence level (= 1.96)
• = expected prevalence = 0.15
• = = 0.85
• = allowable error = 0.10

Substituting the values:

Thus, the minimum required sample size was calculated to be 49 isolates.

In the present study, a total of 51 non-duplicate Staphylococcus aureus isolates were included over the 18-month study period, which satisfies the calculated sample size requirement.

Clinical specimens including pus, wound swabs, blood, ear discharge, and other sterile body fluids were processed according to standard microbiological protocols. Identification of Staphylococcus aureus was performed using colony morphology, Gram staining, catalase test, and slide and tube coagulase tests.

Antimicrobial susceptibility testing was carried out on Mueller–Hinton agar using the Kirby–Bauer disc diffusion method. Erythromycin and clindamycin discs were placed 15–20 mm apart for performance of the D-test. Flattening of the clindamycin inhibition zone adjacent to the erythromycin disc was interpreted as inducible MLSB resistance.

RESULTS

Distribution of Isolates

A total of 51 non-duplicate clinical isolates of Staphylococcus aureus were included in the study. All isolates fulfilled the inclusion criteria and were subjected to antimicrobial susceptibility testing followed by D-test where indicated.

Detection of Inducible Clindamycin Resistance

Out of 51 isolates, 7 (13.7%) demonstrated a positive D-test, indicating inducible MLSB resistance. The remaining 44 isolates (86.3%) were D-test negative. All D-test positive isolates appeared clindamycin susceptible on routine disc diffusion testing but showed characteristic flattening of the clindamycin inhibition zone adjacent to the erythromycin disc on D-test.

Gender-wise Distribution

All 7 D-test positive isolates (100%) were recovered from male patients. No inducible MLSB resistance was detected among female patients. Statistical analysis did not demonstrate a significant association between gender and D-test positivity (Chi-square test, p = 0.18).

Age Distribution

Patients yielding D-test positive isolates had a higher median age compared to those with D-test negative isolates. Non-parametric analysis using the Mann–Whitney U test demonstrated a statistically significant association between increasing age and D-test positivity (p = 0.033).

Summary of Observations

The D-test enabled identification of inducible clindamycin resistance in isolates that would have been misclassified as clindamycin susceptible on routine testing, thereby improving the accuracy of suscept

DISCUSSION

The prevalence of inducible MLSB resistance observed in the present study (13.7%) is comparable with findings reported from several Indian tertiary-care centres.

Majhi et al. reported inducible clindamycin resistance among Staphylococcus aureus isolates in Eastern India, emphasizing the diagnostic gap associated with routine susceptibility testing without D-test confirmation.⁴ Gade and Qazi similarly documented inducible resistance among erythromycin-resistant isolates in South India.⁵

Studies from Maharashtra and Karnataka have reported comparable prevalence of inducible MLSB resistance, reinforcing the reproducibility and reliability of the D-test across laboratories.^4,5,8 . Reports from North India, Kashmir, and Eastern India have further confirmed the widespread distribution and clinical relevance of inducible MLSB resistance.^6, ⁹⁻¹¹

The findings of the present study are consistent with these reports and support the routine incorporation of the D-test into antimicrobial susceptibility testing. Accurate detection of inducible clindamycin resistance is essential to prevent inappropriate clindamycin therapy and potential treatment failure, particularly in settings with a high burden of MRSA infections.

CONCLUSION

The D-test is a simple, reliable, and essential phenotypic method for detecting inducible clindamycin resistance in Staphylococcus aureus. Routine implementation of the D-test is recommended in clinical microbiology laboratories to ensure accurate susceptibility reporting and rational antimicrobial therapy ability reporting.

Table 1. Distribution of D-test Results among Staphylococcus aureus Isolates (n = 51)

Table 2. Association of D-test Positivity with Gender

Figure 1. Distribution of D-test results among Staphylococcus aureus isolates

Bar diagram showing the proportion of D-test positive and D-test negative Staphylococcus aureus isolates.

Figure 2. Percentage Distribution of MLSB resistance phenotypes among Staphylococcus aureus isolates

Bar diagram showing the percentage distribution of inducible MLSB resistance and other resistance phenotypes among Staphylococcus aureus isolates studied (n = 51).

REFERENCES

1. Majhi S, Dash M, Mohapatra D, Mohapatra A, Chayani N. Detection of inducible and constitutive clindamycin resistance among Staphylococcus aureus isolates in a tertiary care hospital, Eastern India. Avicenna J Med. 2021;11(3):147-152.
2. Gade ND, Qazi MS. Inducible clindamycin resistance in Staphylococcus aureus isolates from clinical samples. Med J Armed Forces India. 2013;69(1):53-57.
3. Drinkovic D, Fuller ER, Shore KP, et al. Clindamycin treatment of Staphylococcus aureus expressing inducible clindamycin resistance. J Antimicrob Chemother. 2001;48(2):315-316.
4. Deotale V, Mendiratta DK, Raut U, Narang P. Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples. Indian J Med Microbiol. 2010;28(2):124-126.
5. Prabhu K, Rao S, Rao V. Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples. J Lab Physicians. 2011;3(1):25-27.
6. Gupta V, Datta P, Rani H, Chander J. Inducible clindamycin resistance in Staphylococcus aureus: a study from North India. J Postgrad Med. 2009;55(3):176-179.
7. Sasirekha B. Prevalence of inducible clindamycin resistance among community- and hospital-associated Staphylococcus aureus isolates. J Clin Diagn Res. 2014;8(8):DC19-DC22.
8. Shantala GB, Prakash R, Rani MS, Vijaya D. Inducible clindamycin resistance in Staphylococcus aureus isolates. J Lab Physicians. 2011;3(1):25-27.
9. Fomda BA, Thokar MA, Khan A, et al. Prevalence of inducible clindamycin resistance in Staphylococcus aureus in a tertiary care hospital in Kashmir. Indian J Pathol Microbiol. 2013;56(3):413-414.
10. Pal N, Sharma S, Sharma P, et al. Inducible clindamycin resistance in Staphylococcus aureus isolates from Eastern India. Indian J Med Microbiol. 2010;28(4):409-410.
11. Ghosh A, Banerjee T, Anupurba S. Prevalence of inducible clindamycin resistance in Staphylococcus aureus in a tertiary care hospital in Eastern India. Indian J Med Microbiol. 2011;29(3):345-346.