Eradication of Staphylococcus aureus in Implant-Associated Osteomyelitis by an Injectable in situ-Forming Depot Antibiotics Delivery System.

BACKGROUND: Bone infections from Staphylococcus aureus are notoriously difficult to treat and have high recurrence rates. Local antibiotic delivery systems hold the potential to achieve high in situ antibiotic concentrations, which are otherwise challenging to achieve via systemic administration. Existing solutions have been shown to confer suboptimal drug release and distribution. Here we present and evaluate an injectable in situ-forming depot system termed CarboCell. The CarboCell technology provides sustained and tuneable release of local high-dose antibiotics. METHODS: CarboCell formulations of levofloxacin or clindamycin with or without antimicrobial adjuvants cis-2-decenoic acid or cis-11-methyl-2-dodecenoic acid were tested in experimental rodent and porcine implant-associated osteomyelitis models. In the porcine models, debridement and treatment with CarboCell-formulated antibiotics was carried out without systemic antibiotic administration. The bacterial burden was determined by quantitative bacteriology. RESULTS: CarboCell formulations eliminated S. aureus in infected implant rat models. In the translational implant-associated pig model, surgical debridement, and injection of clindamycin-releasing CarboCell formulations resulted in pathogen-free bone tissues and implants in 9/12, and full eradication in 5/12 pigs. CONCLUSIONS: Sustained release of antimicrobial agents mediated by the CarboCell technology demonstrated promising therapeutic efficacy in challenging translational models and may be beneficial in combination with the current standard of care.

Catheter-associated bladder mucosal trauma during intermittent voiding: An experimental study in pigs.

Objective: The objective of this study is to characterize bladder mucosal trauma associated with intermittent catheterization with conventional eyelet catheters (CECs) and to assess if a microhole zone catheter (MHZC) design concept reduces this adverse effect. Materials and Methods: A porcine model was developed to reflect human catheterization and bladder drainage. Nine pigs were randomized for catheterization with CEC (n = 6) or MHZC (n = 3). The bladder was drained repeatedly 20 times through the catheter. Cystoscopy was performed before and after the procedure, and bladders were analysed by histopathology. Two additional pigs were used for cystoscopy visualization of suction events in vivo. Cystoscopy, gross pathology, histopathological score, leucocyte infiltration, and intracatheter pressure at flow stops during voiding were compared for each group. Results: A significant higher pressure gradient was measured inside the CECs compared with MHZCs during flow stop. Consequently, CECs resulted in suction events inflicting bladder trauma characterized by loss of epithelium, oedema, haemorrhage, and neutrophil tissue infiltration. No significant trauma was identified when using MHZC. Conclusions: Considerable mucosal bladder trauma is inflicted by CECs which may be an overlooked risk factor for urinary tract infection. Catheters can be designed to minimize mucosal suction and reduce associated trauma. This may be a solution to reduce infection frequency and increase user comfort. Furthermore, the study demonstrates the potential of pigs as an attractive animal model for investigating urinary catheter performances.

Intraoperative tissue sampling for histology in chronic osteomyelitis shows high neutrophil infiltration centrally and low remains in debrided presumed infection-free regions.

INTRODUCTION: Histology of debrided bone tissue is a confirmatory diagnostic criterion for fracture related infection (FRI) and prosthetic joint infection (PJI). The aim of the present study was to describe the histopathology of the first and last debrided bone tissue in chronic osteomyelitis (CO) according to the international diagnostic guidelines for FRI and PJI. METHODS: 15 patients with CO were allocated to surgical treatment using a one-stage protocol including extensive debridement. Suspected infected bone tissue eradicated early in the debridement procedure was collected as a clearly infected sample (S1). Likewise, the last eradicated bone tissue was collected as a suspected non-infected sample (S2). The samples were processed for histology. HE-stained sections were patho-morphologically examinated. Immunohistochemistry with MAC-387 antibodies towards calprotectin was used for estimation of neutrophil granulocyte (NP) score (0, 1, 2 or 3). RESULTS: S1 samples showed a mean NP score of 2.6 (3 is confirmatory for infection). Following debridement, the NP score was significantly (p = 0.005) reduced to a mean NP score of 1.6. The S1 samples showed a mix of fibrovascular tissue, dense fibrosis, viable bone, bone necrosis and bone debris. S2 samples contained mostly viable bone tissue, however, often small fragments of necrotic bone or bone debris were present. CONCLUSION: The inflammatory response of CO still exists after debridement, although the response fades from the center. Therefore, sampling of debrided bone tissue for histology must be performed initially during surgery, otherwise there is a risk for underestimation of NP infiltration. The present results might also be highly relevant for FRI and PJI.

Rifampicin does not reduce moxifloxacin concentrations at the site of infection and may not improve treatment outcome of a one-stage exchange surgery protocol of implant-associated osteomyelitis lesions in a porcine model.

We aimed to evaluate moxifloxacin steady-state concentrations in infected bone and soft tissue and to explore the additive microbiological and pathological treatment effect of rifampicin to standard moxifloxacin treatment of implant-associated osteomyelitis (IAO). 16 pigs were included. On Day 0, IAO was induced in the proximal tibia using a susceptible Staphylococcus aureus strain. On Day 7, the pigs underwent one-stage exchange surgery of the IAO lesions and were randomized to receive seven days of intravenous antibiotic treatment of either rifampicin combined with moxifloxacin or moxifloxacin monotherapy. On Day 14, microdialysis was applied for continuous sampling (8 h) of moxifloxacin concentrations. Microbiological, macroscopical pathology, and histopathological analyses were performed postmortem. Steady-state moxifloxacin area under the concentration-time curve was lower in the combination therapy group in plasma (total) and subcutaneous tissue compartments (infected and noninfected) (p < 0.04), while no differences were found in bone compartments. No additional treatment effect of rifampicin to moxifloxacin treatment was found (p = 0.57). Conclusive, additive rifampicin treatment does not reduce moxifloxacin concentrations at the infection site. Rifampicin treatment may not be necessary in a one-stage exchange treatment of IAO. However, our sample size and treatment period may have been too small and short to reveal true clinical differences.

Evaluation of urine dipstick tests in experimental porcine urinary tract infection with uropathogenic Escherichia coli.

Urinary tract infection is a common disease in pigs and a major reason for sows to be culled. The disease, however, is difficult to diagnose due to lack of distinct clinical signs in the animals. We evaluated the diagnostic value of two commercial urine dipstick tests in 10 pigs using an experimental model of Escherichia coli urinary tract infection. Urine collected at baseline and 48 h after inoculation were analyzed. We show that dipstick tests positive of blood, leucocytes and particularly nitrite are very specific for E. coli UTI with a 100% positive predictive value.

Fibrinolytic and antibiotic treatment of prosthetic vascular graft infections in a novel rat model.

OBJECTIVES: We developed a rat model of prosthetic vascular graft infection to assess, whether the fibrinolytic tissue plasminogen activator (tPA) could increase the efficacy of antibiotic therapy. MATERIALS AND METHODS: Rats were implanted a polyethylene graft in the common carotid artery, pre-inoculated with approx. 6 log10 colony forming units (CFU) of methicillin resistant Staphylococcus aureus. Ten days after surgery, rats were randomized to either: 0.9% NaCl (n = 8), vancomycin (n = 8), vancomycin + tPA (n = 8), vancomycin + rifampicin (n = 18) or vancomycin + rifampicin + tPA (n = 18). Treatment duration was seven days. Approximately 36 hours after the end of treatment, the rats were euthanized, and grafts and organs were harvested for CFU enumeration. RESULTS: All animals in the control group had significantly higher CFU at the time of euthanization compared to bacterial load found on the grafts prior to inoculation (6.45 vs. 4.36 mean log10 CFU/mL, p = 0.0011), and both the procedure and infection were well tolerated. Vancomycin and rifampicin treatment were superior to monotherapy with vancomycin, as it lead to a marked decrease in median bacterial load on the grafts (3.50 vs. 6.56 log10 CFU/mL, p = 0.0016). The addition of tPA to vancomycin and rifampicin combination treatment did not show a further decrease in bacterial load (4.078 vs. 3.50 log10 CFU/mL, p = 0.26). The cure rate was 16% in the vancomycin + rifampicin group vs. 37.5% cure rate in the vancomycin + rifampicin + tPA group. Whilst interesting, this trend was not significant at our sample size (p = 0.24). CONCLUSION: We developed the first functional model of an arterial prosthetic vascular graft infection in rats. Antibiotic combination therapy with vancomycin and rifampicin was superior to vancomycin monotherapy, and the addition of tPA did not significantly reduce bacterial load, nor significantly increase cure rate.

Bacterial invasion of the submicron osteocyte lacuna-canaliculi network (OLCN): a part of osteomyelitis disease biology.

Two chronic osteomyelitis patients, a diabetic foot osteomyelitis patient and a fracture-related infection patient, all with staphylococci-positive microbiology, were examined to confirm the clinical relevance of bacterial invasion of the submicron osteocyte lacuna-canaliculi network (OLCN) in bone tissue. Based on immunohistochemistry and light microscopy both Staphylococcus aureus and Staphylococcus epidermidis were identified within the OLCN of all four patients. The findings consolidate that bacterial OLCN invasion is a clinically relevant part of osteomyelitis disease biology, which from experimental porcine infections, seems to be time depending. The microscopy pictures of the four patients significantly add to visualize the phenomenon of bacterial OLCN invasion.

Synthesis of the cyanobacterial halometabolite Chlorosphaerolactylate B and demonstration of its antimicrobial effect in vitro and in vivo.

Chlorosphaerolactylate B, a newly discovered antimicrobial halometabolite from the cyanobacterium Sphaerospermopsis sp. LEGE 00249 has been synthesized in three steps by using 12-bromododecanoic acid as starting material. A total of 0.5 g was produced for in vitro and in vivo antimicrobial efficacy testing. In vitro, the minimal inhibitory concentration (MIC) was estimated to be 256 mg/L for Staphylococcus aureus, while the minimal biofilm inhibitory concentration (MBIC) was estimated to be 74 mg/L. The in vivo study utilized a porcine model of implant-associated osteomyelitis. In total, 12 female pigs were allocated into 3 groups based on inoculum (n = 4 in each group). An implant cavity (IC) was drilled in the right tibia and followed by inoculation and insertion of a steel implant. All pigs were inoculated with 10 µL containing either: 11.79 mg synthetic Chlorosphaerolactylate B + 104 CFU of S. aureus (Group A), 104 CFU of S. aureus (Group B), or pure saline (Group C), respectively. Pigs were euthanized five days after inoculation. All Group B animals showed macroscopic and microscopic signs of bone infection and both tissue and implant harbored S. aureus bacteria (mean CFU on implants = 1.9 × 105). In contrast, S. aureus could not be isolated from animals inoculated with saline. In Group A, two animals had a low number of S. aureus (CFU = 6.7 × 101 and 3.8 × 101, respectively) on the implants, otherwise all Group A animals were similar to Group C animals. In conclusion, synthetic Chlorosphaerolactylate B holds potential to be a novel antimicrobial and antibiofilm compound.

Inflammatory proteins in infected bone tissue - An explorative porcine study.

OBJECTIVE: To explore the in situ inflammatory proteins in the local extracellular fluid of infected bone tissue. MATERIAL AND METHODS: Seven pigs went through a two-step surgery performing a traumatically implant-associated Staphylococcus aureus osteomyelitis in the proximal tibia. Five days later, microdialysis catheters (membrane cut off: 20 kDa) were placed in the implant cavity, infected and healthy cancellous bone, and infected and healthy subcutaneous tissue. Plasma samples were collected simultaneously. We employed an antibody-based proximity extension assay (Olink Inflammatory panel) for the measurement of inflammatory molecules within plasma and extracellular fluid of the investigated tissue compartments. RESULTS: A higher normalized protein expression in the infected bone tissue in comparison to healthy bone tissue was identified for proteins associated with angiogenesis and bone remodeling: OPG, TGFα, MCP-1, VEGFA, and uPA. Moreover, a parallel detectability of the systemic range of cytokines and chemokines as from the investigated local tissue compartments was demonstrated, indicating the same occurrence of proteins in the local environment as within plasma. CONCLUSION: An angiogenic and osteogenic inflammatory protein composition within the extracellular fluid of infected bone tissue was described. The findings support the current histopathological knowledge and, therefore, microdialysis may represent a valid method for sampling of material for protein investigation of the in vivo inflammatory composition within the extracellular environment in infected bone tissue.

The host response to bacterial bone infection involves a local upregulation of several acute phase proteins.

Bone infections often become chronic and can be difficult to diagnose. In the present study, the osseous gene expression of several acute phase proteins (APPs) during osteomyelitis was investigated in a porcine model of implant associated osteomyelitis (IAO) (sampled 5, 10 and 15 days after infection) and in slaughter pigs with spontaneous hematogenous osteomyelitis, and compared to gene expression in liver tissue. Furthermore, immunohistochemical (IHC) staining of the APP complement component C3 (C3) was performed on the porcine osteomyelitis lesions together with material from human patients with chronic osteomyelitis. In the porcine bone samples a local upregulation of the expression of several APP genes, including serum amyloid A (SAA) and C3, was observed during infection. In the liver, only C-reactive protein (CRP) and Inter-Alpha-Trypsin Inhibitor Heavy Chain 4 were significantly upregulated. Serum concentrations of CRP, SAA and haptoglobin were only upregulated at day 5 in infected animals of the IAO model. This indicates a limited systemic response to osteomyelitis. Similar numbers of positive IHC stained C3 leukocytes were found in human and porcine bone samples with chronic osteomyelitis, indicating a high transcriptional value of porcine models of osteomyelitis. The local upregulation of APPs could potentially be used for diagnosing osteomyelitis.

The inflammatory response to bone infection - a review based on animal models and human patients.

Bone infections are difficult to diagnose and treat, especially when a prosthetic joint replacement or implant is involved. Bone loss is a major complication of osteomyelitis, but the mechanism behind has mainly been investigated in cell cultures and has not been confirmed in human settings. Inflammation is important in initiating an appropriate immune response to invading pathogens. However, many of the signaling molecules used by the immune system can also modulate bone remodeling and contribute to bone resorption during osteomyelitis. Our current knowledge of the inflammatory response relies heavily on animal models as research based on human samples is scarce. Staphylococcus aureus is one of the most common causes of bone infections and is the pathogen of choice in animal models. The regulation of inflammatory genes during prosthetic joint infections and implant-associated osteomyelitis has only been studied in rodent models. It is important to consider the validity of an animal model when results are extrapolated to humans, and both bone composition and the immune system of pigs has been shown to be more similar to humans, than to rodents. Here in vivo studies on the inflammatory response to prosthetic joint infections and implant-associated osteomyelitis are reviewed.

In Vivo Gentamicin Susceptibility Test for Prevention of Bacterial Biofilms in Bone Tissue and on Implants.

The objective of this study was to set up an in vivo gentamicin susceptibility test for biofilm prevention in bone tissue and on implants. Twenty-five pigs were allocated to six groups. Pigs in group A (n = 6) were inoculated with saline. Pigs in groups B (n = 6), C (n = 3), D (n = 3), E (n = 3), and F (n = 4) were inoculated with 10 µl saline containing 104 CFU of Staphylococcus aureus Different concentrations based on the MIC of gentamicin for the specific strain were added to the 10-µl inoculum for groups C (160× MIC), D (1,600× MIC), E (16,000× MIC), and F (160,000× MIC). The inocula were injected into a predrilled tibial implant cavity, followed by insertion of a steel implant (2 by 15 mm). The pigs were euthanized after 5 days. In vitro, all the doses used were found to be bactericidal after up to 6 h. All implant cavities of pigs inoculated with bacteria and bacteria plus 160× MIC or 1,600× MIC of gentamicin were positive for S. aureus In animals in each of groups E (16,000× MIC) and F (160,000× MIC), 2/3 and 1/4 of the implant cavities were S. aureus positive, respectively. By grouping groups C and D (<10,000× MIC) and groups E and F (>10,000× MIC), a significant decrease in the number of implant-attached bacteria was seen only between the high-MIC-value group and group B. Histologically, it was demonstrated that 1,600×, 16,000×, and 160,000× MIC resulted in a peri-implant tissue reaction comparable to that in saline-inoculated animals. In vivo, the antimicrobial tolerance of the inoculated planktonic bacteria was increased by in vivo-specific factors of acute inflammation. This resulted in bacterial aggregation and biofilm formation, which further increased the gentamicin tolerance. Thus, susceptibility patterns in vitro might not reflect the actual in vivo susceptibility locally within a developing infectious area.

Combined Staining Techniques for Demonstration of Staphylococcus aureus Biofilm in Routine Histopathology.

Aim: Visualization of Staphylococcus aureus biofilm using histochemical staining and combined histochemistry (HC) and immunohistochemistry (IHC). Methods: The ability of S. aureus S54F9 to form biofilm was tested in vitro. Hereafter, infected bone tissue was collected from two different porcine models of osteomyelitis inoculated with S. aureus strain S54F9. The infection time was five and fifteen days, respectively. Twenty-five different histochemical staining protocols were tested in order to find the stains that could identify extracellular biofilm matrix. Protocols with an optimal visualization of biofilm extracellular matrix were combined with an immunohistochemical protocol based on a specific antibody against S. aureus. The combined protocols were applied to the tissue from the porcine models and to infected bone tissue from a child suffering from chronic staphylococcal osteomyelitis for more than a year. Results:S. aureus S54F9 showed an ability to form biofilm in vitro. Visualization of biofilm, i.e. bacterial cells and extracellular matrix in different colours, was seen when the immunohistochemical protocol was combined with Alcian Blue pH3, Luna and Methyl-pyronin green. The bacterial cells were red to light brown and the extracellular matrix either light blue, blue or orange depending on the histochemical stain. In the porcine models and the human case 10 and 90 percent, respectively, of the bacterial aggregates in a 100x magnification field displayed both the extracellular matrix and the bacterial cells simultaneously in two different colours. Conclusions: A combination of HC and IHC can be used to diagnose and characterise biofilm infections on a routine basis.

Single-dose bone pharmacokinetics of vancomycin in a porcine implant-associated osteomyelitis model.

The increasing incidence of orthopaedic methicillin-resistant Staphylococcus aureus (MRSA) infections represents a significant therapeutic challenge. Being effective against MRSA, the role of vancomycin may become more important in the orthopaedic setting in the years to come. Nonetheless, vancomycin bone and soft tissue penetration during infection remains unclear. In eight pigs, implant-associated osteomyelitis was induced on day 0, using a Staphylococcus aureus strain. Following administration of 1,000 mg of vancomycin on day 5, vancomycin concentrations were obtained with microdialysis for 8 h in the implant bone cavity, in cancellous bone adjacent to the implant cavity, in subcutaneous adipose tissue (SCT) adjacent to the implant cavity, and in healthy cancellous bone and healthy SCT in the contralateral leg. Venous blood samples were also obtained. The extent of infection and inflammation was evaluated by post-mortem computed tomography scans, C-reactive protein serum levels and cultures of blood and swabs. In relation to all the implant cavities, bone destruction was found. Ranging from 0.20 to 0.74, tissue penetration, expressed as the ratio of the area under the concentration-time curve from 0 to the last measured value, was incomplete for all compartments except for healthy SCT. The lowest penetration was found in the implant cavity. In conclusion, Staphylococcus aureus implant-associated osteomyelitis was found to reduce vancomycin bone penetration, especially in the implant cavity. These findings suggest that it may be unsafe to rely solely on vancomycin therapy when treating acute osteomyelitis. Particularly when metaphyseal cavities are present, surgical debridement seems necessary. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1093-1098, 2018.

Novel porcine model of implant-associated osteomyelitis: A comprehensive analysis of local, regional, and systemic response.

Pigs are favorable experimental animals for infectious diseases in humans. However, implant-associated osteomyelitis (IAO) models in pigs have only been evaluated using high-inoculum infection (>108 CFU) models in 1975 and 1993. Therefore, the aim of this paper was to present a new low inoculum porcine model of human IAO based on 42 experimental pigs. The model was created by drilling an implant cavity in the tibial bone followed by insertion of a small steel implant and simultaneous inoculation of Staphylococcus aureus bacteria (n = 32) or saline (n = 10). The infected pigs were either inoculated with 104 CFU (n = 26) or 102 and 103 CFU (n = 6). All animals were euthanized 5 days after insertion of implants. Pigs receiving the high-inoculum infections showed a significantly higher volume of bone lesion, number of neutrophils around the implant, concentrations of acute phase proteins in serum, and enlargement of regional lymph nodes. A positive correlation was present between a high number of surrounding neutrophils and high values of all other parameters. Furthermore, a threshold of 40 neutrophils per 10 high power fields for the histopathological diagnosis of high grade IAO was defined. IN CONCLUSION: This paper describes a novel low-inoculum S. aureus porcine model of IAO which was demonstrated to be reliable, reproducible and discriminative to human IAO, and represents a requested and valuable tool in orthopedic research. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2211-2221, 2017.

Early implant-associated osteomyelitis results in a peri-implanted bacterial reservoir.

Implant-associated osteomyelitis (IAO) is a common complication in orthopedic surgery. The aim of this study was to elucidate how deep IAO can go into the peri-implanted bone tissue within a week. The study was performed in a porcine model of IAO. A small steel implant and either 104 CFU/kg body weight of Staphylococcus aureus or saline was inserted into the right tibial bone of 12 pigs. The animals were consecutively killed on day 2, 4 and 6 following implantation. Bone tissue around the implant was histologically evaluated. Identification of S. aureus was performed immunohistochemically on tissue section and with scanning electron microscopy and peptide nucleic acid in situ hybridization on implants. The distance of the peri-implanted pathological bone area (PIBA), measured perpendicular to the implant, was significantly larger in infected animals compared to controls (p = 0.0014). The largest differences were seen after 4 and 6 days of inoculation, where PIBA measurements of up to 6 mm were observed. Positive S. aureus bacteria were identified on implants and from 25 µm to 6 mm into PIBA. This is important knowledge for optimizing outcomes of surgical debridement in osteomyelitis.

Effects of Implant-Associated Osteomyelitis on Cefuroxime Bone Pharmacokinetics: Assessment in a Porcine Model.

BACKGROUND: The prolonged antibiotic therapy that is often needed for successful management of osteomyelitis may be related to incomplete penetration of antibiotics into the target site. The objective of this study was to assess the effects of implant-associated osteomyelitis on cefuroxime penetration into bone. METHODS: Implant-associated osteomyelitis using a Staphylococcus aureus strain was induced in the right tibia in ten pigs. After five days and following administration of 1500 mg of cefuroxime, measurements of cefuroxime were obtained using microdialysis for eight hours in the implant-related bone cavity, in the adjacent infected cancellous bone and infected subcutaneous tissue, and in healthy cancellous bone and subcutaneous tissue in the contralateral leg. Measurements of the corresponding free plasma concentrations were also obtained. The extent of the infection was assessed by postmortem computed tomography (CT) scans and cultures of blood, swabs, and bone specimens. RESULTS: Bone destruction was found in the implant cavities. No structural bone changes in the adjacent infected cancellous bone were visible on CT scans. S. aureus was grown on culture of specimens from all implant cavities and from eight of ten swabs and seven of ten bone samples from the infected bone. The areas under the concentration-time curves for the different tissues differed significantly, with the lowest area under the curve found in the implant cavity (analysis of variance; p < 0.001). Although not as notable as for the implant cavity, cefuroxime penetration into infected cancellous bone was incomplete but comparable with that in healthy bone. Despite poorer tissue penetration, slightly increased time with concentrations above the minimal inhibitory concentration (MIC) was achieved in the implant cavity up to MICs of 2 mg/L compared with the other tissues, but the time was shorter for higher MICs. CONCLUSIONS: Cefuroxime penetration into infected cancellous bone was incomplete but comparable with that in healthy bone. The destructive bone processes associated with acute osteomyelitis reduced cefuroxime penetration further. CLINICAL RELEVANCE: These findings support the general clinical perception that fast diagnosis and early initiation of antibiotics before the development of implant-associated cavities is important in nonsurgical management of acute osteomyelitis.

Whole-Genome Sequence of Staphylococcus aureus S54F9 Isolated from a Chronic Disseminated Porcine Lung Abscess and Used in Human Infection Models.

We obtained a draft genome sequence of Staphylococcus aureus strain S54F9, which was isolated from a chronic disseminated porcine lung abscess and used in porcine infection models. Genes coding for a number of toxins, including enterotoxins and superantigen, were demonstrated in this strain.

Specific Antibodies to Staphylococcus aureus Biofilm Are Present in Serum from Pigs with Osteomyelitis.

BACKGROUND: The Achilles heel in osteomyelitis is that bacteria, primarily Staphylococcus aureus, grow as a biofilm in the bone lesions. MATERIALS AND METHODS: In the present study, we explored the serum level of specific antibodies to S. aurues biofilm in porcine models of osteomyelitis. RESULTS: Significantly increased levels of antibodies towards the specific biofilm antigen SA0688 were measured in serum from pigs with S. aureus-associated acute and chronic osteomyelitis 5-7 and 10-14 days after inoculation, respectively. Simultaneously with raised antibody levels, an increase in serum interleukin 6 (IL 6) levels was also seen. CONCLUSION: The observed biofilm-specific antibody response represents a T-helper cell 17 (Th17) response and potentially a T-helper cell 1 (Th1) response. This is in agreement with previous studies in mice and rabbits speculating that S. aureus induces a Th1- and Th17-biased adaptive immune response, instead of a protective Th2 response, in order to evade the immune system, resulting in a chronic infection.