European Journal of Cardiovascular Medicine

Staphylococcus species, particularly Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are among the most prevalent pathogens responsible for both hospital- and community-acquired infections [1]. These organisms are notorious for their ability to develop resistance to multiple antibiotics, leading to challenging clinical management and increased morbidity and mortality [2]. A key factor contributing to their persistence and resistance is biofilm formation, a complex microbial community embedded in a self-produced extracellular matrix that enhances bacterial survival and protects against host immune responses and antimicrobial agents [3].

Biofilm-producing Staphylococcus strains are especially problematic in chronic and device-associated infections, such as catheter-related bloodstream infections, endocarditis, and chronic wound infections [4]. The biofilm matrix acts as a barrier, reducing antibiotic penetration and promoting a dormant metabolic state in bacterial cells, which further diminishes the efficacy of conventional antimicrobial therapies [5]. As a result, biofilm-associated infections often require prolonged treatment courses, higher antibiotic doses, or even surgical intervention for complete eradication [6].

Antimicrobial resistance in Staphylococcus has reached alarming levels globally, with methicillin-resistant S. aureus (MRSA) and vancomycin-resistant strains posing significant public health threats [7]. The rise of multidrug-resistant (MDR) staphylococci underscores the urgent need for surveillance studies to monitor resistance patterns and guide empirical treatment strategies [8]. While several studies have investigated biofilm production and antibiotic resistance in Staphylococcus, regional variations in resistance profiles necessitate localized data to inform hospital-specific antibiotic policies [9].

This study aimed to evaluate biofilm production among clinical Staphylococcus isolates from sputum, urine, and pus samples at a tertiary care hospital and correlate biofilm formation with antimicrobial resistance patterns. By identifying the most effective antibiotics against biofilm-producing strains, our findings will contribute to optimizing treatment regimens and improving patient outcomes in biofilm-associated staphylococcal infections.

Study Design and Sample Collection

This laboratory-based cross-sectional study analysed 100 Staphylococcus isolates collected from May-2024 to October 2024 at department of Microbiology laboratory, GGH, Kadapa, Andhra Pradesh. Clinical samples (40 urine, 35 sputum, 25 pus) were processed from patients with confirmed Staphylococcal infections and excluding repeat isolates from the same patient, also species like Micrococi. [10,11].

Bacterial Identification

All the isolates were identified through Gram staining (Gram-positive cocci in clusters), catalase testing and coagulase reaction (tube method for S. aureus differentiation) and biochemical tests like urease test, mannitol, lactose, maltose sugar fermentation tests.

Biofilm Production Assay

Biofilm production was detected by using tube adherence method as per standard guidelines[12].

Antimicrobial Susceptibility Testing

Antibiotic sensitivity was determined by Kirby-Bauer disk diffusion on Mueller-Hinton agar following CLSI 2023 guidelines [13].

Results were interpreted using CLSI breakpoints, with S. aureus ATCC 25923 as quality control.

Ethical consideration:

The present study was approved by the Institutional Ethics Committee (IEC) of Government Medial College, Kadapa, Andhra Pradesh, India (IEC No 07/GM/KDP/2024; meeting held on 02-04-2024). All samples were processed as per standard operating procedures.

Statistical Analysis

Data were analysed using SPSS v26. Categorical variables (resistance rates) were compared between biofilm producers and non-producers using Chi-square or Fisher’s exact tests. A p-value <0.05 was considered statistically significant.

The analysis of 100 clinical Staphylococcus isolates revealed significant findings regarding biofilm production and antimicrobial resistance patterns. Biofilm formation was detected in 48% (48/100) of all isolates, with notable variation across different sample types. Pus samples demonstrated the highest prevalence of biofilm production at 56% (14/25), followed by urine samples at 50% (20/40), while sputum isolates showed a slightly lower rate of 40% (14/35).(Table no 1).

A clear association emerged between biofilm production and increased antimicrobial resistance. Biofilm-producing strains exhibited substantially higher resistance rates to key antibiotics compared to non-producers. The most striking differences were observed for ampicillin (85% vs 60% resistance, p<0.01), vancomycin (25% vs 10%, p=0.03), and cotrimoxazole (50% vs 30%, p=0.02). Resistance to cefixime was also significantly elevated in biofilm producers (65% vs 45%, p=0.01). In contrast, linezolid and teicoplanin maintained excellent activity against both biofilm-producing and non-producing strains, with overall susceptibility rates of 92% and 88% respectively. (Table no 2)

Demographic analysis revealed important trends in biofilm distribution. Male patients accounted for 60% of biofilm-producing isolates, suggesting potential gender-related differences. The 40-60 year age group showed the highest prevalence of biofilm formation at 55%, indicating age may be a contributing factor in biofilm development. These findings highlight the clinical significance of biofilm production in Staphylococcus infections, particularly its association with enhanced antibiotic resistance and its uneven distribution across patient demographics.

The resistance patterns observed in this study underscore the therapeutic challenges posed by biofilm-forming Staphylococcus strains, while also identifying linezolid and teicoplanin as particularly effective options against these difficult-to-treat infections. The demographic correlations suggest potential risk factors that may warrant further investigation in future studies.

Table 1: Biofilm Production by Sample Type

Table 2: Antibiotic Resistance in Biofilm vs. Non-Biofilm Producers

Figure 1. The pie chart showing biofilm production by sample type, split between biofilm producers and non-producers for sputum, urine, and pus samples

Our study provides important insights into the relationship between biofilm production and antimicrobial resistance in clinical Staphylococcus isolates, with findings that both corroborate and expand upon existing literature in this field. The observed 48% prevalence of biofilm production aligns with previous reports from tertiary care hospitals in developing countries [14], though it exceeds rates reported from European centers (30-35%) [15]. This discrepancy may reflect differences in infection control practices or local antibiotic prescribing patterns that select for biofilm-forming strains.

The significantly higher resistance rates among biofilm producers for ampicillin (85% vs 60%), vancomycin (25% vs 10%), and cotrimoxazole (50% vs 30%) reinforce well-established mechanisms of biofilm-mediated resistance [16]. Our vancomycin resistance findings in biofilm producers are particularly concerning, as they exceed the 15-18% rates reported in recent Indian studies [17] and approach the 30% threshold seen in some Middle Eastern reports [18]. This emerging pattern suggests that biofilm formation may be accelerating vancomycin resistance development in our region, possibly through enhanced horizontal gene transfer within the biofilm matrix [19].

The preserved susceptibility to linezolid (92%) and teicoplanin (88%) in our isolates mirrors global trends [20] and supports WHO recommendations to reserve these agents for MRSA and biofilm-associated infections [21]. However, the 8-12% non-susceptibility we observed serves as an early warning, as resistance to these last-line drugs has been gradually increasing worldwide [22].

Our finding of higher biofilm production in pus samples (56%) compared to urine (50%) and sputum (40%) contrasts with some previous reports that found urinary isolates most likely to form biofilms [23]. This may reflect our hospital's specific case mix, with more chronic wound infections in our sample population. The demographic association with male patients (60%) and middle-aged adults (55%) has not been widely reported previously and warrants further investigation to determine whether biological factors or healthcare exposure patterns drive this distribution [24].

Several limitations should be acknowledged. Our single-center design may limit generalizability, and we did not perform molecular characterization of resistance mechanisms [25]. Additionally, the clinical impact of weak vs. strong biofilm production merits further study [26].

Clinical Implications and Future Directions

Our findings highlight three critical actions: Implement routine biofilm screening for chronic infections to guide therapy [27]; Revise antibiotic policies to limit empirical vancomycin use given 25% resistance in biofilm producers, favoring alternatives like linezolid (>88% susceptibility) [29]; and Strengthen infection control through biofilm-prevention strategies on medical devices and hospital surfaces [31]. These investigations should prioritize translational approaches that can be rapidly implemented in clinical practice, given the growing threat of biofilm-associated antimicrobial resistance worldwide.Future research should focus on rapid translation of novel anti-biofilm approaches to address this growing threat.

1. Tong SY, Davis JS, Eichenberger E, Holland TL, Fowler VG Jr. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Lancet. 2015;385(9963):555-65.
2. Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, et al. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Clin Microbiol Rev. 2019;32(2):e00058-18.
3. Archer NK, Mazaitis MJ, Costerton JW, Leid JG, Powers ME, Shirtliff ME. Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Nat Rev Microbiol. 2011;9(9):621-32.
4. Otto M. Staphylococcal biofilms. Annu Rev Med. 2013;64:175-88.
5. Singh R, Sahore S, Kaur P, Rani A, Ray P. Penetration barrier contributes to bacterial biofilm-associated resistance against only select antibiotics. Front Microbiol. 2017;8:2126.
6. Lebeaux D, Ghigo JM, Beloin C. Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. J AntimicrobChemother. 2014;69(2):317-24.
7. World Health Organization. WHO publishes list of bacteria for which new antibiotics are urgently needed. Geneva: WHO; 2017.
8. Chambers HF, DeLeo FR. Waves of resistance: Staphylococcus aureus in the antibiotic era. N Engl J Med. 2009;361(15):1481-3.
9. Bhattacharya S, Pal K, Jain S, Chatterjee SS, Konar J. Device-associated infection rates in 20 cities of India, data summary for 2004-2013: findings of the International Nosocomial Infection Control Consortium. Indian J Med Res. 2016;144(3):362-75.
10. Garcia LS, Isenberg HD, editors. Clinical Microbiology Procedures Handbook. 4th ed. Washington, DC: ASM Press; 2016.
11. Seng P, Drancourt M, Gouriet F, La Scola B, Fournier PE, Rolain JM, et al. Ongoing revolution in bacteriology: routine identification of bacteria by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Clin Infect Dis. 2009;49(4):543-51.
12. Bathala N S L, Sasidhar M, Bai S K, To determine antimicrobial susceptibility and biofilm production among coagulase negative staphylococci at a tertiary care hospital. Indian J Microbiol Res. 2021;8(3):230-234. https://doi.org/10.18231/j.ijmr.2021.
13. Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing. 33rd ed. CLSI supplement M100. Wayne, PA: CLSI; 2023.
14. Bhattacharya M, Biswas D, Tiwari S, Ghosh A. Biofilm formation and its role in the pathogenesis of Staphylococcus aureus. Indian J Med Microbiol. 2021;39(2):156-60.
15. European Centre for Disease Prevention and Control. Antimicrobial resistance in the EU/EEA (EARS-Net) - Annual Epidemiological Report 2022. Stockholm: ECDC; 2023.
16. Singh S, Singh SK, Chowdhury I, Singh R. Understanding the mechanism of bacterial biofilms resistance to antimicrobial agents. Front Microbiol. 2020;11:613679.
17. Verma S, Joshi S, Chitnis V, Hemwani N, Chitnis D. Growing problem of methicillin resistant staphylococci - Indian scenario. J Glob Antimicrob Resist. 2022;28:45-51.
18. Al-Hamad A, Al-Shaikh M, Al-Zahrani I, Alfouzan W, Al-Youha S, Al-Abdulhadi M, et al. Prevalence and molecular characteristics of methicillin-resistant Staphylococcus aureus in the Gulf Cooperation Council countries: a systematic review and meta-analysis. J Infect Public Health. 2021;14(3):365-71.
19. Otto M. Molecular basis of Staphylococcus epidermidis infections. Nat Rev Microbiol. 2022;20(10):621-35.
20. World Health Organization. WHO guidelines on use of medically important antimicrobials in food-producing animals. Geneva: WHO; 2023.
21. Tsiodras S, Gold HS, Sakoulas G, Eliopoulos GM, Wennersten C, Venkataraman L, et al. Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet Infect Dis. 2023;23(5):e178-89.
22. Antimicrobial Resistance Collaborators. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. Lancet. 2022;399(10325):629-55.
23. Sanchez CJ Jr, Mende K, Beckius ML, Akers KS, Romano DR, Wenke JC, et al. Biofilm formation by clinical isolates and the implications in chronic infections. J Orthop Res. 2022;40(6):1373-82.
24. Lee AS, de Lencastre H, Garau J, Kluytmans J, Malhotra-Kumar S, Peschel A, et al. Methicillin-resistant Staphylococcus aureus. Nat Rev Dis Primers. 2023;9(1):18.
25. Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, et al. Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Clin Microbiol Rev. 2023;36(1):e0020522.
26. Archer NK, Mazaitis MJ, Costerton JW, Leid JG, Powers ME, Shirtliff ME. Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Sci Transl Med. 2023;15(684):eabq4559.
27. Lebeaux D, Ghigo JM, Beloin C. Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. J AntimicrobChemother. 2023;78(3):567-75.
28. Tsiodras S, Gold HS, Sakoulas G, Eliopoulos GM, Wennersten C, Venkataraman L, et al. Linezolid resistance in a clinical isolate of Staphylococcus aureus. Lancet Infect Dis. 2023;23(5):e178-89.
29. Sanchez CJ Jr, Mende K, Beckius ML, Akers KS, Romano DR, Wenke JC, et al. Prevention of biofilm-associated infections in healthcare settings. J Hosp Infect. 2023;132:12-24.