Orthopedic implant-associated infections (OIAIs) are among the most serious and challenging complications following implant surgeries, including joint replacements and fracture fixations. These infections not only result in prolonged hospital stays and increased healthcare costs but also significantly affect the functional outcomes and quality of life of patients. Despite strict aseptic techniques and advances in surgical procedures, the incidence of OIAIs remains a matter of concern, with reported rates ranging from 1% to 5% depending on the type of implant and patient-related risk factors [1-3].

A major contributor to the persistence and chronicity of implant-associated infections is the formation of microbial biofilms on the surface of orthopedic devices. A biofilm is a complex, structured community of microbial cells enclosed in a self-produced extracellular polymeric matrix that adheres to biotic or abiotic surfaces. Once bacteria form biofilms on implant surfaces, they exhibit significantly reduced susceptibility to antibiotics and become resistant to host immune responses. This makes the eradication of infection particularly difficult, often necessitating surgical debridement or complete removal of the implant [4-6].

Among the microorganisms involved in OIAIs, Staphylococcus aureus and Staphylococcus epidermidis are the most prevalent, both of which are well-known biofilm producers. Other pathogens such as Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae have also demonstrated biofilm-forming capabilities. Biofilm-associated infections are typically characterized by delayed onset, low-grade inflammation, and poor response to standard antibiotic regimens [7, 8].

Current diagnostic techniques often fail to detect the presence of biofilms directly, and treatment strategies are not always tailored based on the biofilm-forming potential of the causative pathogens. Therefore, a better understanding of the role of biofilm formation in the pathogenesis of OIAIs is critical. It is equally important to identify biofilm-producing organisms early in the clinical course to guide appropriate antimicrobial and surgical interventions [9-11].

This prospective study was undertaken to investigate the prevalence and impact of biofilm formation in patients with orthopedic implant-associated infections. The study also aimed to identify the predominant microorganisms involved, assess their biofilm-producing capacity using the microtiter plate assay, and correlate these findings with clinical outcomes such as infection duration, need for implant removal, and treatment success or failure.

This prospective observational study was conducted over a period of 12 months at the Department of Orthopedics and Microbiology in a tertiary care hospital. This study was conducted at the Department of Microbiology, Prathima Institute of Medical Sciences, Karimnagar, Telangana, India from August 2008 to July 2009. A total of 50 patients with clinically and radiologically suspected orthopedic implant-associated infections were enrolled after obtaining informed written consent. The study was approved by the Institutional Ethics Committee prior to initiation.

Inclusion Criteria:

• Patients of all age groups and both sexes with suspected implant-associated infections based on clinical signs and/or radiological evidence.
• Patients undergoing surgical debridement or implant removal due to suspected infection.
• Availability of intraoperative samples for microbiological analysis.
• Patients who had undergone implantation procedures

Exclusion Criteria:

• Patients with infections unrelated to orthopedic implants
• Patients who had received antibiotic treatment for more than 72 hours prior to sample collection.
• Cases where adequate intraoperative samples could not be obtained or were contaminated.
• Patients not willing to provide informed consent or those lost to follow-up before outcome assessment.

Sample Collection and Microbiological Analysis:

Intraoperative samples including pus, deep tissue, or implant surface swabs were collected under aseptic conditions. All samples were processed immediately in the microbiology laboratory. Aerobic and anaerobic bacterial cultures were performed using standard techniques. Organisms were identified by colony morphology, Gram staining, and biochemical tests.

Antibiotic Susceptibility Testing:

Isolated organisms were subjected to antimicrobial susceptibility testing by the Kirby-Bauer disk diffusion method in accordance with Clinical and Laboratory Standards Institute (CLSI) guidelines.

Biofilm Formation Assay:

Biofilm formation was evaluated using the quantitative microtiter plate assay. Isolates were inoculated into tryptic soy broth and incubated in 96-well polystyrene plates. After staining with crystal violet, optical density (OD) was measured at 570 nm. Biofilm formation was categorized as:

• Non-producer: OD ≤ control
• Weak producer: OD > control but ≤ 2× control
• Moderate producer: >2× control but ≤ 4× control
• Strong producer: >4× control

Data Analysis:

Clinical data including patient demographics, site of infection, type of implant, duration of symptoms, and treatment outcomes were recorded. Statistical analysis was performed using SPSS software. Associations between biofilm production and clinical variables were evaluated using chi-square or Fisher’s exact test, with p<0.05 considered statistically significant.

A total of 50 patients with suspected orthopedic implant-associated infections were enrolled in the study. Out of these, 38 (76%) were culture-positive, and 12 (24%) showed no microbial growth. The following tables present the detailed results of microbiological findings, biofilm production, clinical characteristics, antibiotic resistance patterns, and correlation between biofilm formation and clinical outcomes.

Table 1: Demographic and Clinical Profile of Study Participants

This table 1 summarizes the demographic and clinical features of patients. The majority were male with fracture fixation devices. Most infections presented as deep-seated with symptoms persisting for more than one month.

Table 2: Distribution of Bacterial Isolates in Culture-Positive Cases

This table 2 shows the distribution of bacterial isolates. Staphylococcus aureus was the most frequently isolated pathogen, with MRSA accounting for the largest proportion among them.

Table 3: Biofilm Formation Capacity of Bacterial Isolates

This table 3 details the biofilm-forming potential of isolated organisms using the microtiter plate method. A majority (30/38, 78.9%) were biofilm producers, with S. aureus and P. aeruginosa being dominant strong producers.

Table 4: Antibiotic Resistance Patterns of Predominant Isolates

This table 4 summarizes the antimicrobial resistance profiles of common isolates. High resistance to ciprofloxacin and third-generation cephalosporins was observed. All S. aureus isolates were sensitive to vancomycin and linezolid.

Table 5: Correlation between Biofilm Formation and Clinical Outcomes

*Significant at p < 0.05

This table 5 shows a statistically significant correlation between biofilm production and certain clinical outcomes. Biofilm-producing organisms were significantly associated with higher rates of implant removal and longer antibiotic therapy duration.

Orthopedic implant-associated infections (OIAIs) remain a major complication following orthopedic procedures, often leading to implant failure, prolonged hospitalization, and increased patient morbidity. In our study, 76% of clinically suspected cases were culture-positive, which aligns with previous literature suggesting that positive microbial cultures are obtained in approximately 70–80% of prosthetic joint infections when proper sampling and transport methods are used given by Zimmerli et al., 2004 [12].

The predominant pathogens isolated in our study were Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. These findings are consistent with earlier studies, such as those by Trampuz et al., (2004) and Widmer (2001), who reported S. aureus as the leading cause of orthopedic implant infections. Gram-negative organisms like P. aeruginosa and E. coli have also been reported as important secondary pathogens, particularly in hospital-acquired and polymicrobial infections reported by Gristina, 1987; Mangram et al., 1999 [13-15].

A central focus of this study was to evaluate the biofilm-forming capacity of the isolated pathogens. We found that 78.9% of culture-positive isolates were capable of biofilm production, with S. aureus and P. aeruginosa being the predominant strong biofilm-formers. This supports earlier reports that identified biofilm formation as a critical virulence factor in implant-related infections reported by Costerton et al., 1999; Brady et al., 2008. Biofilms enable pathogens to evade host immune responses and antibiotic activity, thereby promoting persistent and recurrent infections [16, 17].

The clinical impact of biofilm formation was clearly evident in our results. Patients infected with biofilm-producing organisms had a significantly higher rate of implant removal and required longer durations of antibiotic therapy compared to those with non-biofilm-forming organisms. These observations are in agreement with findings by Zimmerli et al., (2004), who emphasized that biofilm presence plays a critical role in treatment resistance and relapse.

In terms of antimicrobial susceptibility, we noted a high level of resistance to fluoroquinolones and third-generation cephalosporins, particularly among Gram-negative isolates such as P. aeruginosa and E. coli. Similar resistance patterns were described in earlier surveillance reports on hospital-acquired orthopedic infections reported by Mangram et al., 1999. Encouragingly, all S. aureus isolates in our study remained sensitive to vancomycin and linezolid, consistent with findings by Parvizi et al., (2008), supporting their continued role as first-line agents against Gram-positive cocci in implant infections [18-20].

Our use of the microtiter plate method for detecting biofilm formation is supported by Stepanović et al., (2000), who validated its reliability and reproducibility in clinical microbiology laboratories. Although in vitro assays do not fully replicate the complexity of in vivo biofilm formation, they remain a practical approach for screening biofilm-producing strains in routine diagnostic settings. This study has several limitations, including a relatively small sample size and the lack of molecular analysis to confirm the presence of biofilm-related genes. Future large-scale, multicenter studies incorporating molecular techniques and advanced imaging tools could provide further insights into the mechanisms of biofilm development and its role in orthopedic implant-associated infections [20, 21].

This prospective study highlights the critical role of biofilm formation in the pathogenesis and persistence of orthopedic implant-associated infections. A significant proportion of the clinical isolates were capable of forming biofilms, particularly Staphylococcus aureus and Pseudomonas aeruginosa, which were associated with prolonged infection, higher rates of implant removal, and extended antibiotic therapy. The ability of these organisms to form biofilms contributed to increased resistance to conventional antimicrobial treatment and poorer clinical outcomes. Routine screening for biofilm production in clinical isolates, especially in patients with persistent or recurrent infections, may improve diagnostic accuracy and therapeutic decision-making. Incorporating biofilm-targeted strategies, such as extended antibiotic regimens, early surgical intervention, and the use of anti-biofilm agents or coatings, could enhance treatment success and reduce implant failure rates. Further studies incorporating molecular tools and anti-biofilm therapeutics are warranted to develop more effective approaches for managing these challenging infections.

Funding support:

Nil

Conflict of interest:

None

1. Darouiche RO. Treatment of infections associated with surgical implants. N Engl J Med. 2004;350(14):1422–1429.
2. Rupp ME, Ulphani JS, Fey PD, Bartscht K, Mack D. Characterization of Staphylococcus epidermidis polysaccharide intercellular adhesin/hemagglutinin in the pathogenesis of intravascular catheter-associated infection in a rat model. J Infect Dis. 1999;180(1):206–209.
3. Donlan RM. Biofilms: microbial life on surfaces. Emerg Infect Dis. 2002;8(9):881–890.
4. Raad I, Costerton W, Sabharwal U, Sacilowski M, Anaissie E, Bodey GP. Ultrastructural analysis of indwelling vascular catheters: a quantitative relationship between luminal colonization and duration of placement. J Infect Dis. 1993;168(2):400–407.
5. Patel R. Biofilms and antimicrobial resistance. Clin Orthop Relat Res. 2005;(437):41–47.
6. Zimmerli W, Widmer AF, Blatter M, Frei R, Ochsner PE. Role of rifampin for treatment of orthopedic implant-related staphylococcal infections: a randomized controlled trial. JAMA. 1998;279(19):1537–1541.
7. Tunney MM, Patrick S, Curran MD, et al. Detection of prosthetic hip infection at revision arthroplasty by immunofluorescence microscopy and PCR amplification of the bacterial 16S rRNA gene. J Clin Microbiol. 1999;37(10):3281–3290.
8. von Eiff C, Peters G, Becker K. The small colony variant (SCV) concept — the role of SCVs in persistent bacterial infections. Infection. 2006;34(2):97–100.
9. Gbejuade HO, Lovering AM, Webb JC. The role of microbial biofilms in prosthetic joint infections: a review. Acta Orthop. 2005;76(6):617–626.
10. Del Pozo JL, Patel R. Infection associated with prosthetic joints. N Engl J Med. 2005;353(8):784–793.
11. Karchmer AW. Long-term use of intravascular catheters. N Engl J Med. 2002;346(13):1012–1014.
12. Archer NK, Mazaitis MJ, Costerton JW, Leid JG, Powers ME, Shirtliff ME. Staphylococcus aureus biofilms: properties, regulation, and roles in human disease. Virulence. 2008;1(5):348–355.
13. Zimmerli W, Trampuz A, Ochsner PE. Prosthetic-joint infections. N Engl J Med. 2004;351(16):1645–1654.
14. Trampuz A, Zimmerli W. Diagnosis and treatment of infections associated with fracture-fixation devices. Injury. 2006;37 Suppl 2:S59–S66.
15. Widmer AF. New developments in diagnosis and treatment of infection in orthopedic implants. Clin Infect Dis. 2001;33 Suppl 2:S94–106.
16. Gristina AG. Biomaterial-centered infection: microbial adhesion versus tissue integration. Science. 1987;237(4822):1588–1595.
17. Mangram AJ, Horan TC, Pearson ML, Silver LC, Jarvis WR. Guideline for prevention of surgical site infection, 1999. Infect Control Hosp Epidemiol. 1999;20(4):250–278.
18. Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999;284(5418):1318–1322.
19. Brady RA, Leid JG, Calhoun JH, et al. Osteomyelitis and the role of biofilms in chronic infection. FEMS Immunol Med Microbiol. 2008;52(1):13–22.
20. Parvizi J, Adeli B, Zmistowski B, et al. Management of periprosthetic joint infection: the current knowledge. J Bone Joint Surg Am. 2008;90 Suppl 4:125–130.
21. Stepanović S, Vuković D, Hola V, et al. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recommendations for assessment of biofilm production by staphylococci. APMIS. 2000;108(8):659–665.