In modern era, Prosthetic replacements and Implant surgery has become one of the commonest Orthopaedic operation which alleviates the pain and improve the mobility in damaged joints. But post-operative infection is a devastating complication 1. Detection of infection after Prosthetic replacements and Implant surgery is a challenge for management and prevention of complications 2. Infection can extend the patients' hospital stay, cost of treatment, increased mortality and morbidity, increased risk of readmission and debridement 3, 4. The prevalence of orthopaedic implant site infection reported in India is about 2.6%. 5 These infections are classified in to three stages, i.e., early (less than two weeks), delayed (2 to 10 weeks) and late (more than 10 weeks) infection. 6

The sources of the pathogens can be either endogenous or exogenous, but most of them are from the endogenous bacterial flora under certain favourable conditions 7, 8. The common micro-organisms causing infections are Staphylococcus aureus, coagulase negative Staphylococcus spp (CONS), Enterococci, Escherichia coli, Klebsiella, Proteus mirabilis and Pseudomonas 9. Implant-associated infections are the result of adhesion of bacteria to implant surface and subsequent biofilm formation at the implantation site. Once biofilm is formed it is difficult by host defence and ordinary antimicrobial therapy to remove it 10.

These days, most of the bacterial isolates are showing resistance to the major first line drugs. However, each hospital has different microbial flora and show different antibiotic susceptibility pattern so that empirical antibiotic therapy. Hence, this study was conducted to evaluate the prevalence of causative organisms, their antibiotic sensitivity pattern and their ability to form biofilm over the implants used in Orthopaedic surgeries.

The present study was conducted in the department of Microbiology, Adichunchanagiri institute of Medical sciences for a period of 3 years from September 2019 to August 2022. Sample size was calculated using prevalence study formula n=Z²P(1−P)/d², where n = Sample size, Z = statistic for a level of confidence (1.96 for 95% confidence level), P = Expected prevalence or proportion, and d = Precision. 11

The study group comprised a total of 100 patients who had undergone orthopaedic implant or prosthetic surgery and presented with signs and symptoms of infection. Informed written consent from patients and ethical clearance from the institution were obtained for the study. The patients particular were recorded on a prescribed proforma which included name, age, sex, diagnosis, comorbidity, smoking history, nutritional status, type of implant as variables.

All patients were given first generation cephalosporins as a standard prophylactic intravenous antibiotic in the operating room. Patients were observed for postoperative wound infection till discharge. The follow up was done up to three months according to a protocol, first visit after two weeks and subsequent visits on monthly basis.

The diagnosis of infection was based clinical observations. Orthopaedician collected the samples from the discharges which were adjacent to the infected implant and tissue, using sterile cotton swabs or a sterile disposable syringe and was immediately transported to the laboratory for culture and antibiotic sensitivity testing. All the samples were subjected to Gram staining and ZN staining. For isolation of aerobic bacteria, the sample was inoculated onto blood agar and MacConkey agar and incubated at 37 °C for 24 to 48 hours. For isolation of anaerobic bacteria, the sample was inoculated in Robertson's cooked meat broth. 12

All positive cultures were processed for the identification of isolates, antibiotic susceptibility tests and biofilm detection.

Antibiotic susceptibility testing was carried out by Kirby Bauer disc diffusion method as per Clinical Laboratory Standards Institute guidelines (CLSI), with Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922 as control strains 13. All the confirmed S. aureus isolates and coagulase negative Staphylococcus spp. (CONS) strains were screened for methicillin resistance using Cefoxitin (30µg) disc (HiMedia Lab. Pvt Ltd), Enterococcus isolates were tested for vancomycin sensitivity by using Vancomycin MIC strip (HiMedia Lab. Pvt Ltd). All Gram negative organisms were screened for ESBL production by phenotypic confirmatory combined-disc test 12, 14.

Congo red method was used for the detection of biofilm with S. epidermidis ATCC 35984 and S. epidermidis ATCC12228 as positive and negative controls respectively. Isolates were considered as strongly positive if there is a black colony with dry crystalline consistency. 12, 14

Among 100 Patients, majority 84 (84%) were male and 16 (16%) were female. Majority of patients (25%) were of 31-40-year age group followed by 41-50 year (22%).

Out of 100 patients investigated, 62 (62%) were with intra-medullary nail, 32 (32%) with plate and 6 (6%) with K-wire.

Among 100 patients, 72 (72%) were culture positive and 28 (28%) were culture negative.

Out of 72 culture positive patients, 31 specimens yield Gram positive cocci and 41 specimens yield Gram negative bacilli. Staphylococcus aureus 17 (26%) is the most common organism isolated in culture followed by Pseudomonas, Klebsiella, Proteus, E. coli, Citrobacter.

Among 72 isolates, 44 (61%) are biofilm producers and 28 (39%) were non-biofilm producers. Out of 44 biofilm producers, S. aureus 13 (30%) is the most common.

All Gram-positive isolates were susceptible to linezolid, vancomycin and Teicoplanin. Most of the Gram-negative isolates showed maximum susceptibility to Imipenem and piperacillin-tazobactam.

Out of 19 S. aureus isolates, 17 (89%) were MRSA strains. Out of 41 Gram- negative bacilli, 15 (37%) were ESBL producing strain with Klebsiella 6 (40%) being major organism followed by Pseudomonas 4 (26%) and E. coli 3 (20%). All Enterococcal isolates were sensitive to Vancomycin, no VRE (Vancomycin resistant Enterococci) was isolated.

Despite great advance in antimicrobial therapy, Orthopaedic implant site infection are the major cause of treatment failure and morbidity in patients. Implant-related infections continue to pose a problem for the Orthopaedician.

In this study, the prevalence of implant site infection from clinically suspected case was 72% (Figure 2), which is less compared to studies of Suneet T, et al. (77.98%), Anisha Fernandez, et al. (84%) and more compared to Gomez et al. (60%) 15, 16, 17. Many studies conducted in India reported prevalence rate between 60-95%, this variation could be due to misdiagnosing and under-reporting of cases 15.

In the present study, majority of patients were from 31- 40-year age group, which is in concordance with other studies 15, 18, this may be due to high activity of the individuals for their daily routine work and have high risk to sustain injury and road traffic accidents.

In our study, among culture positive isolates, majority were Gram negative bacilli (41%) compared to Gram positive cocci (31%), these finding is similar to the studies of Suneet T, et al. (60% & 38.80%) and Khosravi, et al. (64.5% &33.5%) 15, 19. Staphylococcus aureus (26%) was the most common organism isolated in culture followed by Pseudomonas (24%), which correlates with the studies of Khosravi, et al. (28.35%), Suneet T, et al. (28.35%) and Jain, et al. (26.6%) 15, 19, 20. Many studies have reported Klebsiella has second most common isolate, this variation in different isolates could be due to different nosocomial pathogens present in operating theatre of hospitals. 20, 21, 22

In this study, isolated organisms showed resistance to common used antibiotics. Most of the S. aureus were sensitive to linezolid, vancomycin, Teicoplanin and Pipericillin-tazobactum. CoNS were highly sensitive to linezolid, vancomycin, Teicoplanin (100%) followed by Pipericillin-tazobactum (80%), whereas Enterococci showed 100% susceptibility to most of the antibiotics. Among S. aureus, the high level resistance to commonly used drugs may be due to presence of MRSA (89%) and biofilm production (30%). Higher prevalence of MRSA has been reported by Jain, et al. (40%) and Satya Chandrika V, et al. (64%) 20, 23. Hence, vancomycin, linezolid, Teicoplanin can be used in the regimen for treatment of orthopaedic implant infection, especially in case of MRSA and VRE. In case of Gram negative organisms, majority were susceptible to Imipenem and piperacillin-tazobactam and resistance to common used antibiotics, this may be due to the presence of ESBL and biofilm production.

In our study, among 72 isolates, 44 (61%) showed biofilm production. S. aureus 13(30%) was the most common isolate. Majority of S. aureus were MRSA strains whereas, among Gram negative bacilli 15 (37%) were ESBL producers and resistant to commonly used antibiotics. The prevalence of ESBL is1-3 similar to the study of Anisha Fernandes, et al. (31.7%) 16. Our study showed increase in prevalence of Gram negative organism infection with multidrug resistance. This high level of resistance could be due to production of biofilm and it leads to long term antimicrobial therapy, prolonged stay in hospital, increased cost, morbidity, treatment failure and removal or replacement of implants and prosthesis.

There are few limitations of this study. Even though the study period is for 3 years, we have followed up postoperative patients for only 3 months, but in implant surgeries infection can develop 1 year long after surgery and demographic characteristics of hospital population may be changed.

Treating the Orthopaedic implant associated infection is a great challenge. Improper administration of antibiotics will lead to antibiotic resistance, hinder the cure and prolongs the hospital stay, there by increases the morbidity and economic burden to the patient.

It is high time that all clinical laboratories should start detecting the resistant profile routinely. So early intervention by proper selection of antibiotics according to culture and sensitivity plays a key role. MRSA is the commonest isolate in our study. So prevention of Orthopaedic Implant associated infection, screening of MRSA carrier in hospital worker, pre-operatively in patients and adequate intra-operative and post-operative measures should be taken to prevent MRSA infection.

A strict adherence to the antibiotic policy of the institution and multidisciplinary cooperation involving the orthopaedic surgeon and Microbiologists will reduce the incidence of orthopaedic implants site infection.