Aim

Biofilm-related infections represent a recurrent problem in the orthopaedic setting. In recent years, great interest was directed towards the identification of novel molecules capable to interfere with pathogens adhesion and biofilm formation on implant surfaces. In this study, two stable forms of α-tocopherol, the hydrophobic acetate ester and the water-soluble phosphate ester, were tested in vitro as coating for titanium prostheses.

Culture examination is still considered the gold standard for diagnosis of bone and joint infections, including prosthetic ones, even if in up to 20–30% of cases, particularly prosthetic joint infections, it fails to yield microbial growth. To overcome this limitation, determination of markers of inflammation and or infection directly in joint fluid has been proposed.

Aim of this study was to evaluate the applicability of measurement of lecukocyte esterase (LE), C-reactive protein (CRP) and glucose in synovial fluid for diagnosis of bone and joint infections.

Synovial fluids from 80 patients were aseptically collected and sent to laboratory for microbiological cultures. After centrifugation at 3000 rpm for 10 minutes, pellet was used for cultures, while the surnatant was used for determination of LE, CRP and glucose. LE and glucose were evaluated by means of enzymatic colorimetric strips developed for urinanalysis. One drop of synovial fluid was placed on the LE and on the glucose pads and the results were read after about 120 seconds. A LE test graded + or ++, and a glucose test equal to trace or negative were considered suggestive for infection. CRP was measured by an automated turbidimetric method.

On the basis of clinical findings, microbiological, haematological and histological analyses patients were retrospectively divided into 2 groups. Group 1 comprised 19 infected patients (12 males, 7 females age: 70.6 ± 10.3 yrs, range: 47 – 88 yrs) while Group 2 included 61 aseptic patients (32 males and 29 females, age: 61.5 ± 16.3 yrs, range: 15 – 84). Sensitivity of the three tests was 89.5%. 84% and 73,7% for LE, CRP and glucose, respectively. Specificity was 98.4%, 88.5% and 70% for LE, CRP and glucose, respectively. Positive and negative predictive values were 94.4% and 96.8% for LE, 69.6% and 94.6% for CRP and 77.8% and 89.6% for glucose test. When LE was combined with CRP, sensitivity increased to 94.7%, while no differences were observed for LE combined with glucose.

Leukocyte esterase has proven to be a rapid, simple and inexpensive test to rule in or out bone and joint infections. Combination of its measurement with that of CRP increased sensitivity. In conclusion, the combination of leukocyte esterase and CRP may represent a simple and useful tool for diagnosis of bone and joint infections.

The role of biofilm in pathogenesis of several chronic human infections is widely accepted, as this structure leads pathogens to persist among the human body, being protected from the action of antibacterial molecules and drugs (1). It has been estimated that up to 65% of bacterial infections are caused by microorganisms growing in biofilms (2). Moreover, biofilm is involved in device-related orthopaedic bacterial infections, which are unaffected by vaccines and antibiotic therapies, constituting a serious problem for the human health care.

The aim of the present work was to evaluate the anti-biofilm action of a selected and patented lactobacillus strain (MD1) supernatant, both on the in-formation- biofilm and on mature biofilm produced by pathogenic bacteria.

MD1 was grown in BHI for 48 h at 37°C. After incubation, the sample was centrifuged for 5’ for 14,000 × g and the supernatant previously filtered and treated in order to obtain the anti-biofilm compounds (Special Supernatant – SS) was collected. Staphylococcus aureus and Pseudomonas aeruginosa strains were grown in BHI for 24h at 37°C. The anti-biofilm ability of the tested SS – lactobacillus strain was evaluated by a spectrophotometric method according to Christensen at al., following the incubation of pathogens and the “mature biofilm” with the lactobacillus supernatant. Confocal Laser Scanning Microscopy was used to confirm the data obtained from Crystal Violet Assay.

After the incubation of the SS with pathogens and mature biofilm, the formation of biofilm was inhibited and a significant disruption of the mature biofilm was observed. Interestingly, the same properties were observed also when the SS pH was neutralized to pH 6.5. In particular, the reduction of biofilm production and the disruption of mature biofilm was about 50–70% for all microorganisms.

The SS lactobacillus strain MD1 exhibited a relevant antibiofilm action against mature and in-formation-biofilm produced by S. aureus and P. aeruginosa strains tested in the study. Moreover, the antibiofilm action has been observed to be pH-independent, as when the supernatant was neutralized to pH 6.5, the reduction of pathogenic biofilm has been still observed. These promising results highlighted the possibility to use this SS-lactobacillus anti-biofilm property to develop a cost-effective and safety treatment able to reduce the impact of pathogenic biofilm on device-related orthopaedic bacterial infections.