Infective endocarditis (IE) remains a serious and life-threatening condition characterized by microbial colonization of the endocardial surface, particularly cardiac valves. Despite advances in antimicrobial therapy and surgical interventions, IE continues to pose diagnostic and therapeutic challenges, with substantial morbidity and mortality rates globally [1]. A key factor contributing to the persistence and recurrence of IE is the ability of certain bacterial pathogens to form biofilms—a complex aggregation of microorganisms encased in a self-produced extracellular matrix that adheres to surfaces, such as damaged heart valves or prosthetic material [2].

Biofilm formation confers a significant survival advantage to bacteria by promoting resistance to host immune responses and antimicrobial agents, often resulting in chronic and relapsing infections [3]. Among the diverse pathogens implicated in IE, Staphylococcus aureus, coagulase-negative staphylococci, Enterococcus spp., and viridans group streptococci are particularly notorious for their biofilm-forming capabilities [4,5].

Biofilms also impede antibiotic penetration and facilitate the exchange of resistance genes, thereby exacerbating the challenge of eradicating infection in valve tissues [6].

Evaluating the biofilm-forming potential of bacterial isolates in IE cases can provide critical insight into pathogenesis, antibiotic resistance patterns, and prognosis. Despite the recognized clinical significance of biofilms, few routine diagnostic laboratories assess biofilm production as part of the microbiological workup for IE.

The present study aims to evaluate the prevalence and intensity of biofilm formation among bacterial isolates obtained from cardiac valve infections and to assess the correlation between biofilm production and antimicrobial resistance. This investigation may help inform more targeted therapeutic approaches and improve clinical outcomes in patients with IE.

Study Design and Setting: This was a prospective, observational study conducted over a period of 18 months in the Department of Microbiology in collaboration with the Department of Cardiology at a tertiary care hospital in India.

Sample Collection: Cardiac valve specimens (native or prosthetic) were collected aseptically during valve replacement surgeries performed for infective endocarditis (IE). A total of 86 specimens were processed. All samples were transported to the microbiology laboratory immediately in sterile containers under aseptic conditions.

Microbiological Processing: Each valve specimen was homogenized using sterile tissue grinders. The homogenate was inoculated onto blood agar, MacConkey agar, and brain-heart infusion (BHI) broth and incubated aerobically and anaerobically at 37°C for 24–48 hours. Growth was identified using standard microbiological methods including Gram staining, colony morphology, biochemical reactions, and VITEK 2 system (bioMérieux, France), wherever necessary.

Antimicrobial Susceptibility Testing (AST): AST was performed using the Kirby-Bauer disk diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentrations (MICs) were determined for selected antibiotics using E-test strips or broth microdilution method for multidrug-resistant isolates.

Detection of Biofilm Formation: All bacterial isolates were evaluated for biofilm production using the microtiter plate (MTP) assay method. Briefly, overnight cultures of each isolate were diluted in tryptic soy broth (TSB) supplemented with 1% glucose and incubated in 96-well flat-bottomed polystyrene plates at 37°C for 24 hours. After incubation, wells were washed, fixed, stained with 0.1% crystal violet, and the absorbance was measured at 570 nm using a microplate reader. Based on optical density (OD) values, biofilm production was categorized as: non-biofilm producer, weak, moderate, or strong biofilm producer according to established cut-offs.

Data Analysis: Data were analyzed using SPSS version 26.0. Descriptive statistics were used to present frequency, percentage, and mean ± standard deviation. Association between biofilm formation and antibiotic resistance was assessed using the chi-square test or Fisher's exact test. A p-value < 0.05 was considered statistically significant.

A total of 86 bacterial isolates were recovered from cardiac valve specimens (Table 1). Among these, Staphylococcus aureus was the most commonly identified pathogen, accounting for 32.6% of the isolates, followed by coagulase-negative staphylococci (CoNS) (23.3%) and Enterococcus spp. (14.0%). Gram-negative organisms such as Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli were also represented, though less frequently.

As shown in Table 2, 77.9% of isolates demonstrated the ability to form biofilms, with 33.7% categorized as strong producers, 24.4% as moderate, and 19.8% as weak biofilm formers. Notably, 22.1% of isolates did not exhibit biofilm formation.

Table 3 provides a breakdown of biofilm formation by bacterial species. Strong biofilm formation was predominantly observed in S. aureus (14/28, 50%) and CoNS (9/20, 45%), while Enterococcus spp. and Pseudomonas aeruginosa exhibited a moderate level of biofilm-forming capacity. Gram-negative bacilli such as Klebsiella pneumoniae and Acinetobacter baumannii showed relatively poor biofilm formation, with most being non-producers or weak producers.

Importantly, Table 4 highlights a statistically significant association between biofilm formation and multidrug resistance (MDR) (p = 0.032). Among the 67 biofilm-producing isolates, 38 (56.7%) were MDR, in contrast to only 6 (31.6%) among the 19 non-biofilm producers. This suggests that biofilm production may contribute to enhanced antimicrobial resistance in infective endocarditis pathogens.

Table 1: Distribution of Bacterial Isolates from Cardiac Valve Specimens (N = 86)

Table 2: Biofilm Formation among Bacterial Isolates (N = 86)

Table 3: Biofilm Formation by Bacterial Species

Table 4: Association Between Biofilm Production and Multidrug Resistance (MDR)

Chi-square p-value = 0.032 (statistically significant)

This study highlights the significant role of biofilm formation in bacterial isolates associated with cardiac valve infections. Among the isolates analyzed, a substantial proportion exhibited strong or moderate biofilm-producing ability, particularly among Staphylococcus aureus and coagulase-negative staphylococci, aligning with previous studies that have established these organisms as predominant biofilm formers in infective endocarditis (IE) cases [7]. Biofilms not only enhance bacterial adherence to valve tissues but also contribute to persistent infection by shielding the organisms from host immune mechanisms and antibiotics.

The high rate of biofilm production among Gram-positive organisms, particularly S. aureus, is consistent with findings from Elgharably et al., who demonstrated that S. aureus biofilm production leads to aggressive valvular destruction and higher complication rates [8]. Similarly, a study by Boles and Horswill emphasized the genetic regulation of biofilm formation in staphylococci and its direct implication in therapeutic failures [9].

Interestingly, our study also identified biofilm formation in Enterococcus faecalis isolates, which has been increasingly reported in literature as an emerging cause of prosthetic valve endocarditis [10]. Enterococcal biofilms are known to exhibit inherent resistance to β-lactams and aminoglycosides, further complicating management strategies [11]. Moreover, the correlation between multidrug resistance and strong biofilm production observed in our isolates aligns with previous reports suggesting that biofilm-producers are often more resistant to multiple antibiotic classes, thereby necessitating prolonged or combination therapy [12].

The standard methods used to detect biofilm formation, including the tissue culture plate (TCP) assay, remain useful in identifying phenotypic expression of biofilms. However, newer diagnostic modalities such as confocal laser scanning microscopy and real-time PCR for biofilm-associated genes may provide more robust insights into the biofilm-forming capabilities and resistance gene profiles of these pathogens [13].

Overall, the data underscore the need for routine evaluation of biofilm formation in microbiological analyses of cardiac valve specimens. Early identification of biofilm-producing pathogens may aid clinicians in selecting more effective antimicrobial regimens and considering timely surgical interventions where indicated [14]. This approach is especially relevant in cases involving prosthetic material, where biofilms are more likely to persist and result in relapsing infections [15].

This study highlights the high prevalence of biofilm formation among bacterial isolates from cardiac valve infections, particularly in Staphylococcus aureus and coagulase-negative staphylococci. Strong biofilm-producing strains were frequently associated with increased antimicrobial resistance, complicating the management of infective endocarditis. The significant correlation between biofilm formation and multidrug resistance underscores the clinical relevance of biofilm detection in guiding therapeutic decisions. Routine assessment of biofilm-forming capacity in cardiac valve isolates could be a valuable adjunct in tailoring antimicrobial strategies. Further research into anti-biofilm therapies may help improve outcomes in these difficult-to-treat infections.

1. Cahill TJ, Prendergast BD. Infective endocarditis. Lancet. 2016;387(10021):882-93.
2. Lebeaux D, Ghigo JM, Beloin C. Biofilm-related infections: bridging the gap between clinical management and fundamental aspects of recalcitrance toward antibiotics. Microbiol Mol Biol Rev. 2014;78(3):510-43.
3. Stewart PS, William Costerton J. Antibiotic resistance of bacteria in biofilms. Lancet. 2001;358(9276):135-8.
4. Donlan RM. Biofilms and device-associated infections. Emerg Infect Dis. 2001;7(2):277-81.
5. Raad I. Intravascular-catheter-related infections. Lancet. 1998;351(9106):893-8.
6. Lewis K. Riddle of biofilm resistance. Antimicrob Agents Chemother. 2001;45(4):999-1007.
7. Hasannejad Bibalan M, Pourbabaee M, Khorvash F, Zareian-Jahromi S, Yazdanpanah M. Biofilm Formation in Clinical Isolates of Staphylococcus aureus from Endocarditis Patients. Arch Clin Infect Dis. 2021;16(1):e109329.
8. Elgharably H, Hussain ST, Shrestha NK, Blackstone EH, Pettersson GB. Current hypotheses in cardiac surgery: biofilm in infective endocarditis. Semin Thorac Cardiovasc Surg. 2016;28(1):56-59.
9. Boles BR, Horswill AR. Staphylococcus aureus biofilm formation and its regulation. Future Microbiol. 2011;6(3):361-73.
10. Dahl A, Iversen K, Tonder N, Hoest N, Arpi M, Dalsgaard M, et al. Prevalence of Infective Endocarditis in Enterococcal Bacteremia. J Am Coll Cardiol. 2019;74(2):193-201.
11. Arias CA, Murray BE. The rise of the Enterococcus: beyond vancomycin resistance. Nat Rev Microbiol. 2012;10(4):266-78.
12. Cepas V, López Y, Muñoz E, Rolo D, Ardanuy C, Martí S, et al. Relationship between biofilm formation and antimicrobial resistance in Gram-negative bacteria. Microb Drug Resist. 2019;25(1):72-79.
13. Oliveira M, Nogueira CL, Silva Júnior A, et al. Comparative analysis of methods for biofilm detection in Staphylococcus spp. isolated from human clinical specimens. J Microbiol Methods. 2022;193:106438.
14. Murdoch DR, Corey GR, Hoen B, et al. Clinical presentation, etiology, and outcome of infective endocarditis in the 21st century: the International Collaboration on Endocarditis–Prospective Cohort Study. Arch Intern Med. 2009;169(5):463-473.
15. Pericas JM, Llopis J, Muñoz P, et al. A contemporary picture of enterococcal endocarditis. J Am Coll Cardiol. 2015;66(9):968-978.