Original article / research
| Year :
2025 |
Month :
April
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Volume :
14 |
Issue :
2 |
Page :
MO01 - MO05 |
Full Version
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Prevalence of Staphylococcus aureus Isolated from Clinical Samples that Exhibits Inducible Clindamycin Resistance: A Cross-sectional Study
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Akansha Goyal, Akansha Arya, Kajal Rajput, Sapna Chauhan
1. Assistant Professor, Department of Microbiology, Muzaffarnagar Medical College, Muzaffarnagar, Uttar Pradesh, India.
2. Postgraduate Student, Department of Microbiology, Muzaffarnagar Medical College, Muzaffarnagar, Uttar Pradesh, India.
3. Postgraduate Student, Department of Microbiology, Muzaffarnagar Medical College, Muzaffarnagar, Uttar Pradesh, India.
4. Professor and Head, Department of Microbiology, Muzaffarnagar Medical College, Muzaffarnagar, Uttar Pradesh, India.
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Correspondence
Address :
Dr. Sapna Chauhan,
Professor and Head, Department of Microbiology, Muzaffarnagar Medical College, Opp. Begrajpur Industrial Area, Muzaffarnagar-251001, Uttar Pradesh, India.
E-mail: connect.akansha@gmail.com; drsapnachauhan@gmail.com
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| | | ABSTRACT |  | : Introduction: The resistance to Macrolide-Lincosamide-Streptogramin B (MLSB) in Staphylococcus aureus is often caused by erm genes, which can either be constitutive MLSB (cMLSB) or inducible MLSB (iMLSB). Clindamycin (CLI), a lincosamide, and erythromycin (ERY), a macrolide, both act by binding to the 50S ribosomal subunits of bacterial cells, inhibiting protein synthesis. Methylation of the ribosomal target site in iMLSB-resistant isolates displays resistance to ERY but allows susceptibility to CLI. iMLSB resistance emerges when a strong inducer of the methylase enzyme, such as ERY, is present. Identifying iMLSB resistance is crucial for effectively managing S. aureus.
Aim: To detect the prevalence of inducible CLI resistance in S. aureus isolates from various clinical samples and to determine the association between methicillin resistance and inducible CLI resistance in S. aureus isolates.
Materials and Methods: The study was a cross-sectional study conducted at Muzaffarnagar Medical College and Hospital, Muzaffarnagar, Uttar Pradesh, India over a period of six months, from January 2024 to June 2024. A total of 100 isolates of S. aureus obtained from various clinical samples were included. Simple random sampling was employed to ensure an unbiased selection of isolates. Only culture-confirmed S. aureus isolates were considered, while duplicate isolates and samples showing polymicrobial growth were excluded from the study. The sample population comprised individuals of varying ages and genders who sought treatment at the hospital during the study period due to symptoms indicating potential infections. The S. aureus isolates were initially identified using standard biochemical techniques and were then tested for susceptibility using the modified Kirby-Bauer disc diffusion method on Mueller-Hinton agar plates, following Clinical and Laboratory Standards Institute (CLSI) guidelines. The CLSI guidelines were also followed to test for inducible resistance to CLI using the ‘D test’ method. Data were entered and analysed using the Statistical Package for the Social Sciences (SPSS) software, version 24.0, with statistical significance considered at a p-value <0.05.
Results: Routine disc diffusion testing was employed to examine antibiotic susceptibility in 100 S. aureus strains, revealing that 44 (44%) displayed resistance to ERY. Out of the 100 strains, 22 (22%) showed iMLSB resistance, 27 (27%) were cMLSB-positive, and 15 (15%) had the Macrolide-Streptogramin B (MS) phenotype. In this study, it was discovered that 18 (82%) of the S. aureus isolates that were positive for the D test were Methicillin-Resistant S. aureus (MRSA), while 4 (18%) were sensitive to cefoxitin (MSSA).
Conclusion: This study revealed a substantial prevalence of inducible CLI resistance among S. aureus isolates, with a pronounced association in MRSA strains. The strong correlation between methicillin and iMLSB resistance underscores the critical need for routine D-test screening to guide precise antimicrobial therapy, mitigate treatment failures and support effective antimicrobial stewardship practices. |
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| Keywords
: D-test, Methylation, Methicillin resistant, Staphylococcus aureus |
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| DOI and Others
: DOI: 10.7860/NJLM/2025/77170.2911
Date of Submission: Dec 06, 2024
Date of Peer Review: Feb 17, 2025
Date of Acceptance: Mar 26, 2025
Date of Publishing: Apr 01, 2025
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA
PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Dec 06, 2024
• Manual Googling: Mar 22, 2025
• iThenticate Software: Mar 24, 2025 (15%)
ETYMOLOGY: Author Origin
EMENDATIONS: 7 |
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INTRODUCTION |
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Staphylococcus aureus is a Gram-positive coccus from the Micrococcaceae family. Its cells can be found individually or grouped in pairs, tetrads, and grape-like structures. Staphylococcus aureus commonly resides in the human body without causing symptoms and is a prevalent opportunistic pathogen (1). However, in specific circumstances, this bacterium can cause a variety of more serious infections that impact soft-tissue and the outer layers of the skin. Certain strains can form enterotoxins, which lead to cases of food poisoning. The frequency of invasive infections, such as pneumonia, bloodstream infections and infections linked to catheters or artificial devices, has been increasing due to the presence of Community-Acquired (CA) and Hospital-Acquired (HA) MRSA [2-4]. S. aureus Bacteremia (SAB) often leads to metastatic conditions, such as septic arthritis, osteomyelitis and Infective Endocarditis (IE) (5).
The prevalence of MRSA infections is increasing, leading to a higher demand for antibiotics like macrolides, lincosamides, and streptogramin B (MLSB) to address S. aureus infections (6). MLSB resistance in S. aureus is usually caused by erm genes, which can be either cMLSB or iMLSB. Both CLI and ERY, belonging to different drug classes-lincosamides and macrolides, respectively-inhibit protein synthesis in bacterial cells by binding to their 50S ribosomal subunits. Methylation of the ribosomal target site of staphylococci may lead to resistance against both of these antibiotics (7). Through the constitutive resistance mechanism controlled by msrA genes, S. aureus strains exhibit resistance to ERY and susceptibility to CLI in both in-vivo and in-vitro settings. While being treated, the strains that are always resistant do not develop resistance to CLI. The isolates resistant to iMLSB display resistance to ERY but are sensitive to CLI. In the presence of ERY, a potent methylase enzyme inducer, iMLSB resistance emerges. Contrary to cMLSB resistance, standard susceptibility testing does not detect iMLSB resistance. The D-zone test can detect inducible CLI resistance, which is shown by a D-shaped inhibition zone around CLI when exposed to ERY in-vitro. Identifying iMLSB resistance is crucial for managing S. aureus effectively.
Alternatively, CLI usage may result in treatment ineffectiveness due to the emergence of inherent resistance [6,8]. However, the underreporting of inducible CLI resistance in S. aureus is primarily due to the lack of research on its identification. In hospital settings, traditional susceptibility tests for S. aureus paradoxically do not include detecting inducible CLI resistance. Local resistance data are crucial for effectively managing infections, maximising antibiotic use, and guiding empirical treatment.
The current study aimed to utilise the D-test to establish the prevalence of S. aureus strains exhibiting inducible CLI resistance iMLSB in our specific location. Present study also sought to establish the relationship between inducible CLI resistance and MRSA. The increasing worldwide problem of antibiotic resistance indicates that we may soon see the end of successful antibiotic treatment. Resistance impacts all aspects of medicine and makes it more challenging to carry out effective empirical therapy. Paradoxically, typical susceptibility tests for S. aureus in hospital settings still do not include the detection of methicillin and inducible CLI resistance.
Effective infection management, empirical treatment guidance and antibiotic usage optimisation all depend on local geographical data. The aim and purpose of the study were to investigate the prevalence of methicillin and inducible CLI resistance in S. aureus at a tertiary care hospital and to determine the association between them. The results of this surveillance study will contribute recent data to the existing literature and assist clinicians in the area in choosing the right medications, thereby enhancing the clinical management of infections.
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Material and Methods |
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This cross-sectional study was conducted at Muzaffarnagar Medical College and Hospital, Muzaffarnagar, Uttar Pradesh, India from January 2024 to June 2024. Clinical samples were collected in real-time during the study period, ensuring that newly acquired data were available for analysis. The procedures followed in this study were in accordance with the ethical standards set by the committee on human experimentation (both institutional and national). The study was approved by the Institutional Ethics Committee of Muzaffarnagar Medical College, UP, India (MMC/IEC/2024/166). Informed consent was obtained from patients prior to sample collection.
Sample size calculation: The sample size was determined using the Cochran’s formula (9).
n=Z2×(p×q)/e2
=1.962×(0.086×0.914)/0.062
=83.87
Where,
n=sample size,
Z=1.96 for 95% Confidence Interval (CI),
p=D-test positive prevalence was taken from the previous study, 08.64% (10)
q=1-p,
e=margin of error, 6%
The sample size calculation yielded 83.87; however, 100 samples were included to enhance statistical power, improve reliability, and account for potential data loss, ensuring more robust and optimisation findings.
One hundred S. aureus isolates were obtained from various clinical samples, including pus, wound swabs, aspirates, blood and sterile fluids, and were subsequently examined.
Inclusion criteria: All inpatients and outpatients from all age and sex groups who were asked by doctors to undergo standard microbiological culture and antibiotic susceptibility tests were included in the study.
Exclusion criteria: Individuals who had taken antibiotics in the previous four weeks and patients who refused to give consent were excluded from the study. Duplicate samples from the same patient were also omitted from the study.
Study Procedure
The S. aureus isolates were initially identified using standard biochemical techniques (catalase test, slide and tube coagulase test). They were then tested for susceptibility using the modified Kirby-Bauer disc diffusion method on Mueller-Hinton agar plates, following CLSI guidelines (11). The antibiotics tested included trimethoprim-sulfamethoxazole (SXT) (25 μg), ERY (15 μg), ciprofloxacin (5 μg), CLI (2 μg), linezolid (30 μg), tetracycline (30 μg), and cefoxitin (30 μg). A zone of inhibition of 19 mm or smaller around a cefoxitin disc signifies the presence of MRSA.
The CLSI guidelines were followed to test for inducible resistance to CLI using the ‘D test’ method (11). An agar plate with Mueller-Hinton agar was prepared using 0.5 McFarland standard bacterial suspensions. ERY (15 μg) and CLI (2 μg) discs were placed 15 mm apart on the plate. Inducible CLI resistance was indicated by the flattening of the D-shaped zone around the CLI disc between the two discs following overnight incubation at 37°C. After the testing process, three separate characteristics were identified and examined. This interpretation applied only to strains of S. aureus that showed resistance to ERY. All strains that were susceptible to ERY were eliminated from consideration.
MS phenotype: This characteristic was observed in a staphylococcal strain that displayed resistance to ERY (zone size ≤13 mm) but susceptibility to CLI (zone size ≥21 mm), resulting in a circular zone of inhibition around CLI (12).
Inducible MLSB (iMLSB) phenotype: This phenotype was found in a staphylococcal strain that was resistant to ERY (zone size ≤13 mm) but susceptible to CLI (zone size ≥21 mm), exhibiting a D-shaped area of inhibition around CLI that tapered toward the ERY disc (12).
Constitutive MLSB phenotype: Staphylococcal isolates showing resistance to CLI (zone size ≤14 mm) and ERY (zone size ≤13 mm), with a circular zone of inhibition around CLI, were categorised as having this characteristic (12).
Following the established Standard Operating Procedure (SOP) ensured data quality from data collection to laboratory identification. The culture media were prepared in accordance with the manufacturer’s guidelines and sterility was assessed by incubating a representative portion (5% of the total prepared media) at 37°C overnight, followed by evaluation for bacterial growth [11,13]. Media batches exhibiting growth were discarded and replaced with freshly prepared sterile media.
A standard disc diffusion procedure was used to perform Quality Control (QC) on the ERY and CLI discs with S. aureus ATCC 25923. Additional QC measures were implemented using in-house selected S. aureus strains, representing both positive and negative D-test results, to ensure the reliability of the testing process. All HiMedia discs were utilised for both the media and the antibiotics.
Statistical Analysis
Data were entered and analysed using SPSS software version 24.0, Chi-square test was used as a significance test with statistical significance established when the p-value was below 0.05.
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Results |
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Standard disc diffusion testing was employed to evaluate the antibiotic susceptibility of 100 S. aureus strains, revealing that 44 (44%) of them exhibited resistance to erythromycin (ERY). A total of 27 (27%) of the S. aureus isolates were resistant to both ERY and CLI, indicating a constitutive MLSB phenotype; 36 (36%) were sensitive to both drugs, 22 (22%) showed an inducible iMLSB phenotype, and 15 (15%) were negative for the D-test, indicating an MS phenotype. These findings have been illustrated in (Table/Fig 1).
The association between methicillin resistance and CLI resistance is demonstrated in (Table/Fig 2). A total of 18 (82%) of the S. aureus isolates that tested positive for the D-test were identified as MRSA, while 4 (18%) were sensitive to cefoxitin (MSSA) in this research.
(Table/Fig 3) shows that MRSA isolates exhibited a greater proportion of constitutive and inducible resistance compared to MSSA isolates. (Table/Fig 4) illustrates the D-test showing inducible CLI resistance, with the flattening of the zone of inhibition around the CLI disc clearly observed.
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Discussion |
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Clindamycin (CLI) has recently emerged as an effective treatment for certain Staphylococcal infections, especially those affecting the skin and soft tissues, and as a substitute for penicillin in individuals who are allergic to it (14). If S. aureus is reported to be susceptible to CLI without first undergoing testing for inducible resistance, CLI treatment may be administered inappropriately. Inducible resistance to CLI is indicated by a positive D-test.
In this current research, 44 out of 100 S. aureus isolates were found to be resistant to ERY, accounting for 44 (44%) of the total isolates. Every strain resistant to ERY underwent testing with the D-test. This research showed that D-test positivity was found in 22 (22%) of the S. aureus isolates. Numerous studies from India have documented a high prevalence of cMLSB and iMLSB phenotypes of S. aureus. Sefi N et al., reported 56 (26.54%) cMLSB isolates and 24 (11.37%) iMLSB isolates (15). Another study from eastern India by Dash M et al., documented 37 (17.7%) cMLSB and 46 (22%) iMLSB isolates (16). A study by Prabhu K et al., reported relatively lower prevalence of cMLSB and iMLSB phenotypes (18 (9.47%), 20 (10.52%)) (14). A comparison of CLI resistance prevalence from various studies is presented in (Table/Fig 5) (10),(14),(15),(16),(17),(18).
Most of the S. aureus isolates that tested positive in the D-test (iMLSB phenotype), specifically 18 (82%), were found to be MRSA. This result was similar to studies by Mansouri S and Sadeghi J, Prabhu K et al., Sefi N et al., Dash M et al., as well as Shetty J and Afroz Z (10),(14),(15),(16),(17). In contrast, the study by Thapa D et al., reported that 8 (57.2%) of D-test positive association were MSSA and 6 (42.8%) were MRSA (18). The association of iMLSB isolates and methicillin resistance is shown in (Table/Fig 6) (10),(14),(15),(16),(17),(18).
A comparative analysis of the correlation between resistance to methicillin and CLI by different authors is presented in (Table/Fig 7) (10),(14),(15),(16),(17),(18).
The increased occurrence of the iMLSB phenotype in MRSA infections compared to MSSA infections indicates that CLI treatment for MSSA infections is effective in many cases, whereas it may result in treatment failure for MRSA infections. In summary, present study discovered a significant occurrence of the inducible CLI resistance phenotype in this area. Authors recommend that a D-test should be conducted whenever CLI is planned for use in S. aureus infections to ensure effective treatment of patients. Proper and effective utilisation of CLI can reduce the need for alternative medications such as vancomycin and help avoid the emergence of resistance to these drugs.
Limitation(s)
Emphasising the main constraints of this research, such as the lack of real-time PCR assessment of gene expression and the genetic similarity among ERY-resistant strains, is crucial.
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Conclusion |
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The high levels of MRSA and iMLSB phenotypes in S. aureus underscore the importance of including the D-test and methicillin resistance test in regular susceptibility testing for S. aureus control. The D-test is a viable additional option for identifying inducible CLI resistance in clinical laboratory practices. Furthermore, identifying the presence of the erm gene in staphylococcal strains that show positive results in the D-test could help establish a standardised procedure for the test.
Authors’ contribution: All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.
Data availability: All data generated or analysed during this study are included in the manuscript.
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| TABLES AND FIGURES | |
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