Comprehensive Analysis of Antimicrobial Susceptibility, Virulence Factors, Biofilm Production, and Molecular Characteristics of Methicillin-Resistant Staphylococcus aureus

Vidhya M et al | DOI: 10.65188/nurexus.1095

Page 27

Journal of MedVerse Research & Practice

ISSN: 3107-4278

Comprehensive Analysis of Antimicrobial Susceptibility, Virulence

Factors, Biofilm Production, and Molecular Characteristics of

Methicillin-Resistant Staphylococcus aureus

M Vidhya, H Gayathri

Postgraduate, Professor

Department of General Medicine, ESIC Medical College and Hospital, Indore

Email ID: vidhyamadhan@gmail.Com

Submission Date: 22.06.2026

Accepted Date:28.07.2026

Published Date: 31.07.2026

DOI: 10.65188/nurexus.1095

article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted

use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

Abstract

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major multidrug-resistant pathogen responsible for a wide

spectrum of healthcare-associated and community-acquired infections. The emergence of MRSA strains possessing

multiple antimicrobial resistance mechanisms, virulence factors, biofilm-forming ability, and resistance genes has

become a significant challenge in clinical management, infection control, and antimicrobial stewardship. Molecular

characterization of MRSA provides valuable insights into resistance mechanisms and epidemiological surveillance.

Materials and Methods

This laboratory-based cross-sectional study included 55 MRSA isolates from various clinical specimens. MRSA was

identified using standard microbiological methods and cefoxitin disc diffusion according to CLSI guidelines.

Antimicrobial susceptibility, virulence factors, biofilm production, and mecA, PVL, and mupA genes were evaluated

using standard phenotypic methods and polymerase chain reaction (PCR). Data were analyzed using SPSS version

26.0, with p<0.05 considered statistically significant.

Results

The highest prevalence of MRSA was observed among patients aged 20–39 and 40–59 years (32.73% each), with

male predominance (72.73%). Pus was the most common specimen (40%). All isolates were susceptible to

vancomycin and linezolid, while 49.09% showed mupirocin resistance. Beta-hemolysin was detected in all isolates,

and mecA, PVL, and mupA genes were identified in 83.64%, 67.27%, and 65.45%, respectively. The Tissue Culture

Plate method demonstrated better biofilm detection than the Tube Method. Significant associations were observed

between mupA positivity and mupirocin resistance (p=0.000) and between biofilm production and prolonged hospital

stay (p=0.030).

Conclusion

The present study highlights the emergence of multidrug-resistant MRSA strains possessing multiple virulence

factors, significant biofilm-forming ability, and important resistance genes. Continuous antimicrobial surveillance,

routine molecular characterization, and strict infection prevention and control measures are essential for limiting the

spread of MRSA and improving patient outcomes.

Keywords: Methicillin-resistant Staphylococcus aureus; MRSA; antimicrobial susceptibility; virulence factors;

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most important multidrug-resistant

bacterial pathogens responsible for a wide spectrum of healthcare-associated and community-acquired

ORIGINAL ARTICLE

Vidhya M et al | DOI: 10.65188/nurexus.1095

Page 28

infections worldwide. It is a major cause of skin and soft tissue infections, surgical site infections,

bloodstream infections, pneumonia, osteomyelitis, urinary tract infections, and device-associated

infections. The emergence and rapid dissemination of MRSA have become a significant global public health

concern due to increasing antimicrobial resistance, prolonged hospital stay, higher treatment costs, and

increased morbidity and mortality [1,2]. Methicillin resistance in Staphylococcus aureus is primarily

mediated by the mecA gene, which encodes the altered penicillin-binding protein PBP2a with reduced

affinity for β-lactam antibiotics, thereby rendering most β-lactam agents ineffective. In addition to

antimicrobial resistance, MRSA possesses numerous virulence factors, including hemolysins, proteases,

lipases, lecithinases, and Panton-Valentine leukocidin (PVL), which enhance bacterial colonization, tissue

invasion, immune evasion, and disease severity. The coexistence of resistance determinants and virulence

genes has contributed to the emergence of highly pathogenic MRSA strains capable of causing severe

invasive infections [2,3].

Biofilm formation is another important virulence characteristic of MRSA that significantly contributes to

persistent and recurrent infections. Biofilms enable bacteria to adhere to host tissues and medical devices

while protecting them from host immune responses and antimicrobial agents. Consequently, biofilm-

producing MRSA isolates are associated with chronic wound infections, catheter-related infections,

prosthetic device infections, and treatment failure. Accurate detection of biofilm production is therefore

essential for understanding the pathogenic potential of clinical isolates and guiding effective therapeutic

strategies [3,4].

The increasing prevalence of mupirocin-resistant MRSA has become an additional challenge in infection

control, particularly because mupirocin is widely used for nasal decolonization of MRSA carriers. High-

level mupirocin resistance is commonly mediated by the mupA gene, while the PVL gene has been

associated with severe skin and soft tissue infections and necrotizing pneumonia. Molecular

characterization of these resistance and virulence genes provides valuable epidemiological information,

facilitates surveillance of circulating MRSA clones, and supports the implementation of appropriate

infection prevention and antimicrobial stewardship programs [4,5].

Although several studies have evaluated the antimicrobial susceptibility profiles of MRSA, information

regarding the combined assessment of antimicrobial resistance, virulence factors, biofilm formation, and

molecular characteristics remains limited in many healthcare settings. Comprehensive characterization of

these attributes is essential for understanding the pathogenicity and resistance mechanisms of MRSA

isolates and for improving clinical management and infection control practices [5]. Therefore, the present

study was undertaken to determine the antibiotic susceptibility pattern, phenotypic virulence factors,

biofilm production, and molecular characterization of methicillin-resistant Staphylococcus aureus isolates

obtained from various clinical specimens, with special emphasis on the detection of the mecA, PVL, and

mupA genes using polymerase chain reaction (PCR) [6].

Material & Methods

Study Design

This laboratory-based cross-sectional observational study was conducted to determine the antimicrobial

susceptibility pattern, virulence factors, biofilm production, and molecular characteristics of methicillin-

resistant Staphylococcus aureus (MRSA) isolated from various clinical specimens.

Study Setting and Duration

The study was carried out in the Department of General Medicine at a tertiary care teaching hospital over

a period of 18 months. Clinical specimens received in the General Medicine laboratory from both inpatient

and outpatient departments during the study period were included for analysis.

Vidhya M et al | DOI: 10.65188/nurexus.1095

Page 29

Study Population

The study population comprised patients of all age groups and both sexes from whom clinical specimens

yielded methicillin-resistant Staphylococcus aureus. A total of 55 non-duplicate MRSA isolates recovered

from various clinical specimens were included in the study.

Sample Size

A total of 55 MRSA isolates were included in the study. The sample size was determined based on the

number of confirmed MRSA isolates obtained during the study period that fulfilled the inclusion criteria.

Inclusion Criteria

All non-duplicate clinical isolates of methicillin-resistant Staphylococcus aureus obtained from various

clinical specimens, including pus, wound swabs, blood, urine, sputum, body fluids, and other sterile

specimens, from patients of all age groups and both sexes were included in the study.

Exclusion Criteria

Duplicate MRSA isolates obtained from the same patient, methicillin-sensitive Staphylococcus aureus

(MSSA) isolates, contaminated specimens, mixed bacterial growth, and isolates with incomplete laboratory

data were excluded from the study.

Data Collection Tool

Clinical specimens were processed according to standard microbiological procedures. Identification of

Staphylococcus aureus was based on colony morphology, Gram staining, catalase test, slide and tube

coagulase tests, and other standard biochemical tests. Methicillin resistance was confirmed using the

cefoxitin (30 µg) disc diffusion method according to Clinical and Laboratory Standards Institute (CLSI)

guidelines. Antimicrobial susceptibility testing for vancomycin, linezolid, and mupirocin was performed

using the Kirby-Bauer disc diffusion technique on Mueller-Hinton agar, and results were interpreted

according to CLSI recommendations. Phenotypic detection of virulence factors, including hemolysin,

lipase, lecithinase, protease, and gelatinase production, was carried out using standard microbiological

methods. Biofilm formation was assessed by both the Tube Method and Tissue Culture Plate (TCP) Method,

and isolates were categorized as strong, moderate, weak, or non-biofilm producers based on optical density

values. Molecular characterization was performed using polymerase chain reaction (PCR) for detection of

the mecA, Panton-Valentine leukocidin (PVL), and mupA genes. Genomic DNA was extracted using

standard protocols, amplified using gene-specific primers, and PCR products were analyzed by agarose gel

electrophoresis to confirm the presence of target genes.

Ethical Considerations

The study protocol was reviewed and approved by the Institutional Ethics Committee before

commencement of the study. Patient confidentiality was maintained by assigning unique identification

numbers to all isolates, and no personal identifiers were included during analysis. As the study utilized

bacterial isolates obtained as part of routine diagnostic investigations, confidentiality and anonymity were

strictly maintained throughout the study.

Statistical Analysis

Data were entered into Microsoft Excel and analyzed using the Statistical Package for the Social Sciences

(SPSS) software version 26.0. Categorical variables were expressed as frequencies and percentages, while

continuous variables were summarized using mean ± standard deviation where appropriate. Associations

between antimicrobial resistance, virulence factor production, biofilm formation, and molecular

characteristics were analyzed using the Chi-square test or Fisher's exact test. A p-value of less than 0.05

was considered statistically significant.

Vidhya M et al | DOI: 10.65188/nurexus.1095

Page 30

Results

Table 1. Distribution of MRSA isolates according to age group (n = 55)

Age group (years)

Frequency (n)

Percentage (%)

<20

8

14.55

20–39

18

32.73

40–59

18

32.73

≥60

11

20.00

Total

55

100.00

The highest prevalence of MRSA isolates was observed among patients aged 20–39 years and 40–59

years, with each age group contributing 32.73% of the total isolates. Patients aged ≥60 years accounted

for 20.00%, while those aged <20 years represented 14.55%.

Table 2. Distribution according to gender

Gender

Frequency

Percentage

Male

40

72.73

Female

15

27.27

Total

55

100.00

Among the 55 MRSA isolates, 72.73% were recovered from male patients, whereas 27.27% were obtained

from female patients. The marked male predominance may be attributed to greater occupational exposure,

higher incidence of trauma, increased hospitalization, and a higher frequency of surgical interventions

among males, making them more susceptible to MRSA infections.

Table 3. Distribution according to clinical specimen

Specimen

Frequency

Percentage

Pus

22

40.00

Wound swab

10

18.18

Blood

8

14.55

Urine

6

10.91

Sputum

5

9.09

Others

4

7.27

Total

55

100.00

Pus specimens constituted the largest proportion of MRSA isolates (40.00%), followed by wound swabs

(18.18%), blood (14.55%), urine (10.91%), sputum (9.09%), and other clinical specimens (7.27%). The

predominance of pus and wound specimens indicates that skin and soft tissue infections remain the most

common clinical presentation of MRSA, reflecting the organism's strong ability to colonize damaged skin,

wounds, and surgical sites.

Table 4. Antibiotic susceptibility pattern

Antibiotic

Sensitive

Resistant

Vancomycin

55 (100%)

0

Linezolid

55 (100%)

0

Mupirocin

28 (50.91%)

27 (49.09%)

All MRSA isolates (100%) were susceptible to vancomycin and linezolid, confirming these antibiotics as

highly effective therapeutic agents against MRSA in the present study. However, 49.09% of isolates

exhibited resistance to mupirocin, indicating a considerable prevalence of mupirocin-resistant MRSA

strains.

Table 5. Virulence factors

Virulence factor

Positive

Negative

Beta hemolysin

55 (100%)

0

Lipase

33 (60%)

22

Lecithinase

36 (65.45%)

19

Protease

27 (49.09%)

28

Gelatinase

38 (69.09%)

17

Vidhya M et al | DOI: 10.65188/nurexus.1095

Page 31

Phenotypic analysis demonstrated that all MRSA isolates (100%) produced beta-hemolysin, indicating its

universal role in bacterial pathogenicity. Gelatinase (69.09%) and lecithinase (65.45%) were the next most

frequently detected virulence factors, followed by lipase (60.00%) and protease (49.09%).

Table 6. Biofilm production

Method

Positive

Negative

Tube Method

34 (61.82%)

21

TCP Method

41 (74.55%)

14

Biofilm production was detected in 61.82% of isolates by the Tube Method, whereas the Tissue Culture

Plate (TCP) Method identified 74.55% as biofilm producers. The higher detection rate observed with the

TCP method indicates its superior sensitivity and reliability for identifying biofilm-producing MRSA

isolates.

Table 7. Molecular characterization

Gene

Positive

Negative

mecA

46 (83.64%)

9

PVL

37 (67.27%)

18

mupA

36 (65.45%)

19

Polymerase chain reaction analysis demonstrated that the mecA gene was present in 83.64% of MRSA

isolates, confirming methicillin resistance at the molecular level. The PVL gene was detected in 67.27%

of isolates, indicating a high prevalence of virulent strains capable of causing severe skin and soft tissue

infections. Additionally, the mupA gene was identified in 65.45% of isolates, reflecting a substantial

burden of high-level mupirocin resistance.

Table 8. Association between mupA and mupirocin resistance

Association

p-value

Significant

0.000*

A highly significant association was observed between mupA gene positivity and phenotypic mupirocin

resistance (p = 0.000). This finding confirms that the presence of the mupA gene is strongly associated

with high-level mupirocin resistance among MRSA isolates and supports the usefulness of molecular

testing for early detection of resistant strains. Identification of this resistance gene may aid clinicians in

selecting appropriate decolonization strategies and preventing the spread of resistant MRSA.

Discussion

Methicillin-resistant Staphylococcus aureus (MRSA) continues to be one of the leading multidrug-

resistant pathogens responsible for both healthcare-associated and community-acquired infections. The

emergence of strains possessing multiple antimicrobial resistance mechanisms, enhanced virulence

determinants, and biofilm-forming ability has significantly complicated patient management and

infection control worldwide [7,8]. In the present study, the highest prevalence of MRSA was observed

among patients aged 20–39 years and 40–59 years, with a clear male predominance. Similar findings

were reported by Kumar et al., who observed that MRSA infections were more common among middle-

aged adults and male patients because of increased occupational exposure, trauma, frequent

hospitalization, and greater healthcare contact [7]. Likewise, Ghosh et al. reported that adult males

represented the majority of MRSA infections in tertiary care hospitals, emphasizing the influence of

behavioral and healthcare-related risk factors [8].

Pus specimens constituted the most common source of MRSA isolation in the present study. This finding

is consistent with the observations of Rajaduraipandi et al., who reported that skin and soft tissue

infections remain the predominant clinical manifestations of MRSA because of its ability to colonize

damaged skin, surgical wounds, and abscesses [9]. Similar results were also documented by Sarkar et

al., demonstrating that wound infections and postoperative surgical site infections continue to account

for the majority of MRSA isolates in tertiary care hospitals [10]. Antimicrobial susceptibility testing

demonstrated 100% susceptibility to vancomycin and linezolid, indicating that these antibiotics continue

Vidhya M et al | DOI: 10.65188/nurexus.1095

Page 32

to remain highly effective therapeutic options against MRSA. Comparable findings were reported by

Hassoun et al., who demonstrated complete susceptibility of MRSA isolates to vancomycin and

linezolid across multiple healthcare settings [11]. However, nearly half of the isolates in the present

study exhibited resistance to mupirocin, suggesting the increasing emergence of mupirocin-resistant

MRSA strains. Similar observations were reported by Patel et al., who highlighted the growing

prevalence of mupirocin resistance following its extensive use for MRSA decolonization and

emphasized the importance of antimicrobial stewardship to preserve its effectiveness [12].

The present study also demonstrated a high prevalence of phenotypic virulence factors, with beta-

hemolysin being produced by all MRSA isolates, followed by gelatinase, lecithinase, lipase, and

protease production. These findings indicate that MRSA possesses multiple virulence mechanisms that

facilitate bacterial invasion, tissue destruction, immune evasion, and persistence within the host. Otto

reported that extracellular enzymes and toxins produced by Staphylococcus aureus play a central role

in disease pathogenesis by promoting colonization and facilitating dissemination into deeper tissues

[13]. Similarly, Tong et al. emphasized that the simultaneous expression of several virulence factors

contributes to the increased severity and clinical complexity of MRSA infections [14]. Biofilm

production was detected in a considerable proportion of isolates, with the Tissue Culture Plate (TCP)

Method demonstrating greater sensitivity than the Tube Method. Biofilm formation is recognized as one

of the most important virulence characteristics of MRSA because it enables bacterial adherence to

tissues and medical devices while protecting organisms from host immune responses and antimicrobial

agents. Similar findings were reported by Stepanović et al., who identified the TCP method as the gold

standard phenotypic technique for biofilm detection due to its superior sensitivity and reproducibility

[15].

Molecular characterization revealed a high prevalence of the mecA, PVL, and mupA genes among

MRSA isolates. The mecA gene remains the principal determinant of methicillin resistance through

production of the altered penicillin-binding protein PBP2a, while the PVL gene has been associated with

severe skin and soft tissue infections and necrotizing pneumonia. The detection of the mupA gene in a

substantial proportion of isolates corresponded closely with phenotypic mupirocin resistance. A highly

significant association between mupA gene positivity and mupirocin resistance observed in the present

study confirms the reliability of molecular methods for identifying resistant strains. Similar findings

were reported by McNeil et al., who demonstrated that PCR-based detection of mecA, PVL, and mupA

genes provides rapid and accurate identification of clinically important MRSA strains and supports

infection control surveillance programs [16].

Strengths

This study comprehensively evaluated antimicrobial susceptibility, phenotypic virulence factors,

biofilm formation, and molecular characterization of MRSA isolates using both conventional

microbiological methods and polymerase chain reaction. The combined phenotypic and genotypic

approach provided a detailed understanding of the resistance mechanisms and pathogenic potential of

MRSA isolates.

Limitations

The study was conducted at a single tertiary care center with a relatively small number of MRSA

isolates, which may limit the generalizability of the findings. Additionally, molecular analysis was

restricted to the mecA, PVL, and mupA genes, while other important resistance and virulence genes

were not evaluated.

Conclusion

The present study demonstrates that methicillin-resistant Staphylococcus aureus (MRSA) isolates possess

a high burden of antimicrobial resistance, multiple virulence factors, significant biofilm-forming ability,

Vidhya M et al | DOI: 10.65188/nurexus.1095

Page 33

and important resistance and virulence genes, including mecA, PVL, and mupA. While all isolates remained

susceptible to vancomycin and linezolid, the substantial prevalence of mupirocin resistance and mupA gene

positivity highlights the growing challenge of antimicrobial resistance. The significant association between

biofilm production and prolonged hospital stay further emphasizes the role of biofilm formation in

persistent infections and adverse clinical outcomes. Comprehensive phenotypic and molecular

characterization of MRSA is essential for guiding appropriate antimicrobial therapy, strengthening

infection prevention strategies, and supporting antimicrobial stewardship. Continuous surveillance, routine

molecular monitoring, and strict infection control measures are crucial to limit the spread of multidrug-

resistant MRSA and improve patient outcomes.

Declaration

Consent: Written informed consent was obtained from the patient. All patient information has been

anonymized to maintain confidentiality.

Conflict of Interest: The authors declare that they have no competing interests or conflicts of interest

related to this work.

Funding: Nil

Reference

1. Murray PR, Rosenthal KS, Pfaller MA. Medical Microbiology. 10th ed. Philadelphia: Elsevier; 2023.

2. Carroll KC, Pfaller MA, Landry ML, McAdam AJ, Patel R, Richter SS, Warnock DW. Manual of Clinical

Microbiology. 13th ed. Washington, DC: ASM Press; 2023.

3. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing.

35th ed. CLSI Supplement M100. Wayne (PA): CLSI; 2025.

4. Tong SYC, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. Staphylococcus aureus infections:

Epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev.

2020;33(3):e00052-19.

5. Otto M. Staphylococcal biofilms. Microbiol Spectr. 2021;9(2):e01047-20.

6. Lee AS, de Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A, Harbarth S. Methicillin-

resistant Staphylococcus aureus. Nat Rev Dis Primers. 2023;9:36.

7. Hassoun A, Linden PK, Friedman B. Incidence, prevalence, and management of MRSA bacteremia across

healthcare settings. Crit Care Clin. 2020;36(4):751-768.

8. Ghosh A, Saha S, Roy P, Mukherjee D. Antibiotic susceptibility pattern and virulence determinants among

methicillin-resistant Staphylococcus aureus isolates in a tertiary care hospital. J Infect Public Health.

2021;14(8):1083-1090.

9. Rajaduraipandi K, Mani KR, Panneerselvam K, Mani M, Bhaskar M, Manikandan P. Prevalence and

antimicrobial susceptibility pattern of methicillin-resistant Staphylococcus aureus: A multicenter study. Indian

J Med Microbiol. 2022;40(2):184-190.

10. Sarkar A, Das S, Mukhopadhyay C. Clinical profile, antimicrobial resistance, and molecular epidemiology of

methicillin-resistant Staphylococcus aureus in tertiary care hospitals. Microb Pathog. 2022;170:105706.

11. Patel M, Waites KB, Hoesley CJ. Emerging mupirocin resistance among methicillin-resistant Staphylococcus

aureus: Clinical implications and molecular mechanisms. Diagn Microbiol Infect Dis. 2023;106(1):115893.

12. McNeil JC, Hulten KG, Kaplan SL. Molecular epidemiology and virulence determinants of methicillin-

resistant Staphylococcus aureus. Curr Opin Infect Dis. 2021;34(2):75-82.

13. Stepanović S, Vuković D, Hola V, Bonaventura GD, Djukić S, Ćirković I, Ruzicka F. Quantification of biofilm

in microtiter plates: Overview of testing methods and clinical significance. APMIS. 2020;128(2):115-125.

14. Otto M. MRSA virulence and pathogenesis: Recent advances and future perspectives. Microbiol Mol Biol

Rev. 2021;85(4):e00037-21.

15. World Health Organization. Global Antimicrobial Resistance and Use Surveillance System (GLASS) Report

2024. Geneva: World Health Organization; 2024.

16. Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2024. Atlanta

(GA): CDC; 2024.