Aim

In the current study we aim to characterize the use of cationic host defense peptides (HDPs) as alternative antibacterial agents to include into novel antibacterial coatings for orthopedic implants.

Staphyloccous aureus represent one the most challenging cause of infections to treat by traditional antibacterial therapies. Thanks to their lack of microbial resistance described so far, HDPs represent an attractive therapeutic alternative to antibiotics. Furthermore, HDPs have been showed to control infections via a dual function: direct antimicrobial activity and regulation of immune response. However, HDPs functions characterization and comparison is controversial, as changing test conditions or cell type used might yield different effects from the same peptide. Therefore, before moving towards the development of HDP-based coatings, we need to characterize and compare the immunomodulatory and antibacterial functions under the same conditions in vitro of 3 well-known cathelicidins: human LL-37, chicken CATH-2, and bovine-derived IDR-1018.

Currently, no clinical options are available to prevent infections on uncemented orthopedic implants. Therefore we investigated the efficacy of DAC-hydrogel (disposable antibacterial coating(1), Novagenit, Italy) as carrier for various agents to prevent infections in an in vivo implant-model.

Titanium rods were implanted in the left tibiae in New Zealand White rabbits. Prior to implantation, the implant bed was contaminated with 10∧5 colony forming units S. aureus.

In the experimental groups, the hydrogel was loaded prior to be coated on the rods with: 2%(w/v) vancomycin (Van2 group, N=6), 5%(w/v) vancomycin (Van5 group, N=6), 10%(w/v) bioactive glass (BonAlive, Finland) (BAG group, N=6), which is antibacterial(2) and osteoconductive(3), or 0.5%(w/v) N-acetyl cysteine (NAC group, N=6), which inhibits bacterial growth and decreases biofilm formation(4). In the control group, empty hydrogel was applied (Gel group, N=12)

Blood values were measured weekly. Following explantation on day 28, the anterior tibia was processed for bacterial culture. The posterior tibia and rod were used for measuring bone-implant contact using micro-CT and for histopathology.

Results of the experimental groups were compared to the Gel group results. The blood values in the Van2 and Van5 groups were lower on day 7. Moreover, culture results demonstrated less animals with an infection in both groups at day 28. In accordance, these groups showed lower grades for infection. Further, the Van2 group demonstrated more bone-implant contact. These results suggest that infection was reduced in the Van2 and Van5 groups. In contrast, blood values, histological grades, and bone-implant contact of the BAG and NAC groups were comparable with the Gel group. These results suggest that infection was not prevented in the BAG and NAC groups.

Local application of vancomycin-loaded DAC-hydrogel successfully reduced implant-related infections. Loading of the hydrogel with BAG or NAC did not prevent infection. It is possible that BAG in powder form, as used in the present study, dissolved before the antibacterial effect could take place. Instead, BAG granules may be a viable alternative. Next, it is possible that the NAC concentration was too low to prevent infections in an in vivo environment, although this concentration was proven effective in vitro for its antibacterial properties.

Correct diagnosis of infection is crucial for an adequate treatment of orthopedic implant-related infections. In the orthopedic field, infections can be difficult to diagnose(1). As a consequence, patients may suffer from an undiagnosed and untreated implant-related infection.

To solve this problem, we are searching for a diagnostic method to detect these so-called low-grade infections. The technique fluorescence in situ hybridization (FISH) can detect slow-growing and even dead bacteria. Further, as FISH results are available within an hour after tissue collection it is an ideal candidate for diagnostic purposes.

AIM: to evaluate the FISH technique for its potential to detect and identify orthopedic infections.

Sonication fluid (SF) was collected by sonicating retrieved implants(2) from 62 patients. All samples were subjected to bacterial culture for clinical diagnostics. In addition, a commercially available FISH kit (miacom diagnostics, Germany), specifically designed for blood analysis (hemoFISH Masterpanel), was used. The kit contained 16S rRNA probes (positive control), non-sense probes (negative control), probes for Staphylococcus spp., Staphylococcus aureus, Streptococcus spp., Streptococcus pneumoniae, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Enterobacteriaceae, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Acetinobacter spp., and Stenotrophomonas maltophilia. All FISH analyses were performed according to the protocol provided with the kit.

Culture and FISH results were compared, considering culture as the gold standard. Culture resulted in 27 positive and 35 negative samples. Comparing FISH (16S rRNA probe) with culture, 24 samples tested true-positive and 32 samples true-negative. Furthermore, 3 samples tested false-negative and 3 samples false-positive.

The species cultured with the highest incidence were Propionibacterium acnes and Staphylococcus epidermidis, both from 8 SF samples. As the kit did not contain a probe for Propionibacterium acnes, these strains were only detected by the 16S rRNA probe. In addition, the latter samples tested positive with the Staphylococcus spp. probe.

Interestingly, 3 samples tested positive with FISH that were culture negative. This result could indicate a higher sensitivity for detection of bacteria with FISH than with culture.

Before FISH can be used for diagnostic purposes, the technique needs to be optimized to prevent false-negative results, for use on other patient materials and for detection of bacterial strains relevant for the orthopedic field like Propionibacterium acnes.

In conclusion, FISH holds promise to be used as a diagnostic tool for identifying orthopedic infections.

A new type of metallic silver bone cement was previously shown to be effective against both antibiotic sensitive and resistant bacteria.

In this study the efficacy of silver bone cement in preventing methicillin- sensitive Staphylococcal infections was compared with plain and tobramycin-containing bone cement, in a rabbit contaminated implant bed model.

In 48 rabbits 0.6%-silver, 1%-silver, plain or tobramycin-loaded (tobra) PMMA bone cement (Simplex^®P; Howmedica, Ireland) was injected into the medullary canal of the right femur after contamination of the implant bed with 105, 106 or 107 colony forming units (CFU) of Staphylococcus aureus. After 14 days bone was collected, homogenised and plated on blood agar plates. After an overnight incubation the number of CFU’s was counted. Bone was also collected for pathological analysis.

The plain and silver cement rabbits were all infected, whereas with tobra cement only 2 rabbits (17%) were infected (p< 0.001). The number of bacteria cultured from bone adjacent to the cement, was 6.4±0.3 and 6.1±0.3 for the 0.6% and 1%-silver rabbits. For the rabbits with plain and tobra cement this was 6.2±0.2 (p> 0.95) and 0.0±0.0 (p< 0.001). Two tobra rabbits had a positive culture of a distal bone sample. Histological sections of plain, 0.6% and 1%-silver cement rabbits all showed signs of infection; these signs were absent in the tobra rabbits.

Silver cement was not effective in preventing infection. However, in the current model bacteria are present directly at and distant from the implant surface, whereas silver cement predominantly exhibits an antimicrobial effect at the direct cement surface. The non-eluting silver cement seems less useful in situations where there are also bacteria present in surrounding tissues, like revision surgery. Whether silver cement has relevance in preventing bacterial colonization of cement, for instance in late haematogenous infections, or not remains to be seen.