Implant-associated Bone Infection Research Articles

Cutibacterium acnes invades submicron osteocyte lacuno-canalicular networks following implant-associated osteomyelitis.

Cutibacterium acnes, part of normal skin flora, is increasingly recognized as an opportunistic pathogen capable of causing chronic prosthetic joint infections (PJI) associated with total hip and knee arthroplasty. However, there is a paucity of literature examining the pathogenesis of C. acnes during PJI. To study this, we developed an implant-associated osteomyelitis murine model in which 8-10-week-old C57BL6 mice were subjected to transtibial implantation of titanium or stainless-steel L-shaped pins contaminated with C. acnes. Postsurgery, mice were killed on Days 14 and 28 for terminal assessments of (1) bacterial load in bone, implant, and internal organs (heart, spleen, kidney, and liver), (2) bone osteolysis (micro-CT), (3) abscess formation (histology), and (4) systematic electron microscopy (EM). In vitro scanning EM (SEM) confirmed that C. acnes can form biofilms on stainless-steel and titanium implants. In mice, C. acnes could persist for 28 days in the tibia. Also, we observed C. acnes dissemination to internal organs. C. acnes chronic osteomyelitis revealed markedly reduced bone osteolysis and abscess formation compared to Staphylococcus aureus infections. Importantly, transmission EM (TEM) investigation revealed the presence of C. acnes within canaliculi, demonstrating that C. acnes can invade the osteocyte lacuno-canalicular networks (OLCN) within bone. Our preliminary pilot study, for the first time, revealed that the OLCN in bone can be a reservoir for C. acnes and potentially provides a novel mechanism of why C. acnes chronic implant-associated bone infections are difficult to treat.

Preventing septic implant failures in osteoporotic hip fractures using antibiotic-loaded functionalized nanocement

Osteoporosis is a prevalent skeletal disorder and the most common cause of debilitating hip fractures, primarily affecting post-menopausal women and elderly people. Implants used to stabilize osteoporotic fragility fractures are susceptible to bacterial infections, which further deteriorate the implantationsite. In the current study, we developed a nanocement-based treatment for tackling hip implant infection in an osteoporotic rat model. The development of osteoporosis was confirmed by an 80% decrease in serum estradiol levels and a further reduced T-score value of −4.170 ± 0.411 for femur neck canal. Further, an infected K-wire was implanted in femoral neck canal to induce implant-associated osteomyelitis. In order to combat infection and promote bone healing, it was followed by debridement and implantation of rifampicin-loaded nanocement functionalized with various combinations of bone marrow MSCs (bMSC) exosomes, bone morphogenetic protein-2 (BMP-2) and zoledronic acid (ZA). The findings of this study demonstrated the potential of exosomes as an alternative for reducingBMP-2 dosage, and the treatment promoted successful bone regeneration withenhanced mechanical strength, and a significant decrease in bacterial load. Improved bone remodeling and matrix deposition at the implantation site were confirmed by DXA, micro-CT, and histology. Further, immunohistochemical staining showed increased expression of collagen I and CD31, which indicated bone matrix deposition and vascularization, respectively, while reduced expression of α-SMA and NOS2 confirmed a reduction in fibrosis and inflammation, respectively. Overall, our one-step treatment strategy has the potential to prevent implant-associated bone infection in osteoporotic hip fractures, promote bone formation, and enhance the mechanical strength of osteoporotic bone, thereby preventing subsequent secondary fractures.

PH-Responsive nanoplatform synergistic gas/photothermal therapy to eliminate biofilms in poly(L-lactic acid) scaffolds.

To date, implant-associated infection is still a significant clinical challenge, which cannot be effectively eliminated by single therapies due to the formation of microbial biofilms. Herein, a pH-responsive nanoplatform was constructed via the in situ growth of zinc sulfide (ZnS) nanoparticles on the surface of Ti3C2 MXene nanosheets, which was subsequently introduced in poly(L-lactic acid) (PLLA) to prepare a composite bone scaffold via selective laser sintering technology. In the acidic biofilm microenvironment, the degradation of ZnS released hydrogen sulfide (H2S) gas to eliminate the biofilm extracellular DNA (eDNA), thus destroying the compactness of the biofilm. Then, the bacterial biofilm became sensitive to hyperthermia, which could be further destroyed under near-infrared light irradiation due to the excellent photothermal property of MXene, finally achieving gas/photothermal synergistic antibiofilm and efficient sterilization. The results showed that the synergistic gas/photothermal therapy for the composite scaffold not only evidently inhibited the formation of biofilms, but also effectively eradicated the eDNA of the already-formed biofilms and killed 90.4% of E. coli and 84.2% of S. aureus under near infrared light irradiation compared with single gas or photothermal therapy. In addition, the composite scaffold promoted the proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells. Thus, the designed scaffold with excellent biofilm elimination and osteogenesis ability has great potential as an alternative treatment for implant-associated bone infections.

Interactions of Neutrophils with the Polymeric Molecular Components of the Biofilm Matrix in the Context of Implant-Associated Bone and Joint Infections

In the presence of orthopedic implants, opportunistic pathogens can easily colonize the biomaterial surfaces, forming protective biofilms. Life in biofilm is a central pathogenetic mechanism enabling bacteria to elude the host immune response and survive conventional medical treatments. The formation of mature biofilms is universally recognized as the main cause of septic prosthetic failures. Neutrophils are the first leukocytes to be recruited at the site of infection. They are highly efficient in detecting and killing planktonic bacteria. However, the interactions of these fundamental effector cells of the immune system with the biofilm matrix, which is the true interface of a biofilm with the host cells, have only recently started to be unveiled and are still to be fully understood. Biofilm matrix macromolecules consist of exopolysaccharides, proteins, lipids, teichoic acids, and the most recently described extracellular DNA. The latter can also be stolen from neutrophil extracellular traps (NETs) by bacteria, who use it to strengthen their biofilms. This paper aims to review the specific interactions that neutrophils develop when they physically encounter the matrix of a biofilm and come to interact with its polymeric molecular components.

Galleria mellonella as an alternative in vivo model to study implant-associated fungal infections.

Fungal implant-associated bone infections are rare but difficult to treat and often associated with a poor outcome for patients. Candida species account for approximately 90% of all fungal infections. In vivo biofilm models play a major role to study biofilm development and potential new treatment options; however, there are only a very few in vivo models to study fungi-associated biofilms. Furthermore, mammalian infection models are replaced more and more due to ethical restrictions with other alternative models in basic research. Recently, we developed an insect infection model with Galleria mellonella larvae to study biofilm-associated infections with bacteria. Here, we further expanded the G. mellonella model to study in vivo fungal infections using Candida albicans and Candida krusei. We established a planktonic and biofilm-implant model to test different antifungal medication with amphotericin B, fluconazole, and voriconazole against the two species and assessed the fungal biofilm-load on the implant surface. Planktonic infection with C. albicans and C. krusei showed the killing of the G. mellonella larvae at 5 × 105 colony forming units (CFU). Treatment of larvae with antifungal compounds with amphotericin B and fluconazole showed significant survival improvement against planktonic C. albicans infection, but voriconazole had no effect. Titanium and stainless steel K-wires were preincubated with C. albicans and implanted inside the larvae to induce biofilm infection on the implant surface. The survival analysis revealed significantly reduced survival of the larvae with Candida spp. infection compared to noninfected implants. The treatment with antifungal amphotericin B and fluconazole resulted in a slight and nonsignificant improvement survival of the larvae. The treatment with the antifungal compounds in the biofilm-infection model was not as effective as in the planktonic infection model, which highlights the resistance of fungal biofilms to antifungal compounds like in bacterial biofilms. Scanning electron microscopy (SEM) analysis revealed the formation of a fungal biofilm with hyphae and spores associated with larvae tissue on the implant surface. Thus, our study highlights the use of G. mellonella larvae as alternative in vivo model to study biofilm-associated implant fungal infections and that fungal biofilms exhibit high resistance profiles comparable to bacterial biofilms. The model can be used in the future to test antifungal treatment options for fungal biofilm infections.

ZnO nanoparticle modified chitosan/borosilicate bioglass composite scaffold for inhibiting bacterial infection and promoting bone regeneration

The treatment of implant-associated bone infection remains a significant clinical challenge. However, bone scaffolds with antimicrobial activity and osteoinductive properties can prevent these infections and improve clinical outcomes. In this study, borosilicate bioglass and chitosan composite scaffolds were prepared, and then the surface was modified with nano-zinc oxide. In vitro and in vivo experiments showed that the chitosan/borosilicate bioglass scaffolds have good degradation and osteogenic properties, while the oxidized Zinc scaffolds have better antibacterial properties.

Coating of bone implants with silica, hyperbranched polyethyleneimine, and gentamicin prevents development of osteomyelitis in a porcine model

The use of bone implants and prostheses has contributed to a revolution in modern medicine; however, in the beginning, not much was asked from the implant and prosthetic materials per se. Therefore, the next game-changer in orthopedic research will come from new material designs which for instance can aid in prevention of implant-associated bone infections. Here, we describe the development of a new sol-gel coating technique that can deliver an efficient antimicrobial surface coating on orthopedic implants. Gentamicin was stocked in a novel nanocomposite xerogel made from silica and hyperbranched polyethyleneimine. The xerogel was anchored inside a porous surface made by coating of bone implants with titanium microspheres. Thereby, only the small water-soluble gentamicin molecules diffused in an aqueous environment, i.e., just after surgical insertion and leaving behind a titanium scaffold for osseointegration. The novel xerogel coating prevented development of severe Staphylococcus aureus induced osteomyelitis in a porcine model, which untreated, replicated the pathology seen in stage 3A on the Cierny–Mader classification system for osteomyelitis in adults.

Skeletal infections: microbial pathogenesis, immunity and clinical management.

Osteomyelitis remains one of the greatest risks in orthopaedic surgery. Although many organisms are linked to skeletal infections, Staphylococcus aureus remains the most prevalent and devastating causative pathogen. Important discoveries have uncovered novel mechanisms of S. aureus pathogenesis and persistence within bone tissue, including implant-associated biofilms, abscesses and invasion of the osteocyte lacuno-canalicular network. However, little clinical progress has been made in the prevention and eradication of skeletal infection as treatment algorithms and outcomes have only incrementally changed over the past half century. In this Review, we discuss the mechanisms of persistence and immune evasion in S. aureus infection of the skeletal system as well as features of other osteomyelitis-causing pathogens in implant-associated and native bone infections. We also describe how the host fails to eradicate bacterial bone infections, and how this new information may lead to the development of novel interventions. Finally, we discuss the clinical management of skeletal infection, including osteomyelitis classification and strategies to treat skeletal infections with emerging technologies that could translate to the clinic in the future.

Evaluation of a syndromic panel polymerase chain reaction (spPCR) assay for the diagnosis of device‐associated bone and joint infections (BJI)

ObjectivePathogen detection is crucial for diagnosis and targeted therapy in implant-associated bone and joint infections (BJI). Culture-based microbiology regularly fails to identify causative pathogens. This study evaluated the diagnostic accuracy and clinical usefulness of a syndromic panel polymerase chain reaction (spPCR) assay targeting common BJI pathogens in tissue specimens from patients with implant-associated BJI. MethodsResults obtained by spPCR assay and a 16S rDNA PCR were compared with results obtained from a standard of care (SOC) culture-based diagnostics, serving as a gold standard. In total, 126 specimens obtained from 73 patients were analyzed. ResultsThe spPCR assay correctly identified 33/40 culture-positive samples (82.5 %) and was positive in 9/86 (10.5 %) culture-negative samples, resulting in an overall sensitivity of 84.6 % (95% confidence interval [CI] 68.79-93.59%) and specificity of 89.35% (95% CI 80.6-94.81%). The spPCR was more sensitive compared with the 16S rDNA PCR (37.5%). The spPCR identified pathogens in 7/51 (13.7%) SOC-negative patients. Re-evaluation of spPCR results in clinical context suggested their clinical significance. ConclusionAn spPCR assay targeting common pathogens causing implant-associated BJI may help to identify causative agents in culture-negative cases. As false-negative results are possible, spPCR assays appear as an add-on approach for pathogen detection in implant-associated BJI.

AmpC hyperproduction in a Cedecea davisae implant-associated bone infection during treatment: a case report and therapeutic implications

BackgroundData on antimicrobial resistance mechanisms are scanty for Cedecea spp., with very variable antibiotic resistance patterns documented. Here we report the first in vivo resistance evolution of a C. davisae clinical isolate in a patient with a complex hand trauma and provide insight in the resistance mechanism, leading to therapeutic implications for this pathogen.Case presentationCedecea davisae was isolated from a patient with hand trauma during a first surgical debridement. Six days after primary surgical treatment and under antimicrobial treatment with amoxicillin-clavulanic acid and later cefepime, follow up cultures yielded C. davisae which demonstrated a resistance development. The susceptible parental isolate and its resistant derivative were characterized by whole genome sequencing, ampC, ompC and ompF by RT- PCR. The resistant derivative demonstrated an A224G SNP in ampD, the transcriptional regulator of ampC, leading to a His75Arg change in the corresponding AmpD protein. AmpC transcription of the resistant derivative was 362-times higher than the susceptible isolate. Transcription levels of ompF and ompC were 8.5-fold and 1.3-fold lower, respectively, in the resistant derivative. Downregulation of OmpF putatively resulted from a mutation in the presumed promoter region upstream of the dusB-Fis operon, a proposed regulator for ompF.ConclusionsThis case demonstrates the in vivo resistance development of C. davisae within 7 days similar to that of the members of the Enterobacter cloacae complex. Our findings add valuable information for future therapeutic management of these opportunistic pathogens as they warrant the same empirical treatment as AmpC producers.

Silver-doped bioglass modified scaffolds: A sustained antibacterial efficacy

Implant-related bacterial infection is a serious complication, which even causes implant failure. Silver (Ag) nanoparticles are broadly used antibacterial agents due to their excellent antibacterial ability and broad-spectrum bactericidal property. However, the significance of burst release cannot be entirely ignored. In this study, Ag doped mesoporous bioactive glasses (Ag-MBG) nanospheres were synthesized using modified Stöber method, then incorporated into poly L-lactic acid (PLLA) matrix to prepare the composite scaffolds via selective laser sintering (SLS) technology. Herein, Mesoporous bioactive glasses (MBG) sol had many negatively-charged silicon hydroxyl groups, which could adsorb positively-charged Ag ions by electrostatic interaction and eventually form Si-O-Ag bonds into MBG. Moreover, MBG promoted osteoblast colonization due to its continuous release of Si ions. The results showed the Ag-MBG/PLLA scaffold could sustainedly release Ag ions for 28 days, and exhibited significantly antibacterial ability against Escherichia coli, its bacterial inhibition rate was over 80%. In addition, the composite scaffold also showed good cytocompatibility. It may be concluded that the prepared Ag-MBG/PLLA scaffold has great potential to repair implant-associated bone infection.

Safety of Tedizolid as Suppressive Antimicrobial Therapy for Patients With Complex Implant-Associated Bone and Joint Infection due to Multidrug-Resistant Gram-Positive Pathogens: Results From the TediSAT Cohort Study.

A prospective cohort study was conducted to evaluate long-term safety of tedizolid as suppressive antimicrobial treatment in patients with implant-associated bone and joint infection caused by multidrug-resistant gram-positive pathogens. Seventeen patients received tedizolid with a median duration of treatment of 6 months. No patients developed a serious adverse event.

Bacteriophage-Delivering Hydrogels: Current Progress in Combating Antibiotic Resistant Bacterial Infection

Antibiotic resistance remains as an unresolved global challenge in the health care system, posing serious threats to global health. As an alternative to antibiotics, bacteriophage (phage) therapy is rising as a key to combating antibiotic-resistant bacterial infections. In order to deliver a phage to the site of infection, hydrogels have been formulated to incorporate phages, owing to its favorable characteristics in delivering biological molecules. This paper reviews the formulation of phage-delivering hydrogels for orthopedic implant-associated bone infection, catheter-associated urinary tract infection and trauma-associated wound infection, with a focus on the preparation methods, stability, efficacy and safety of hydrogels as phage carriers.

Adjunctive Use of Phage Sb-1 in Antibiotics Enhances Inhibitory Biofilm Growth Activity versus Rifampin-Resistant Staphylococcus aureus Strains.

Effective antimicrobials are crucial for managing Staphylococcus aureus implant-associated bone infections (IABIs), particularly for infections due to rifampin-resistant S. aureus (RRSA). Failure to remove the implant results in persistent infection; thus, prolonged suppressive antibiotic therapy may be a reasonable alternative. However, a high incidence of adverse events can necessitate the discontinuation of therapy. In this scenario, commercial Staphylococcal bacteriophage Sb-1 combined with antibiotics is an option, showing a promising synergistic activity to facilitate the treatment of biofilm infections. Therefore, we evaluated the efficacy of the inhibitory activity of five antibiotics (doxycycline, levofloxacin, clindamycin, linezolid, and rifampin) alone or combined with phage Sb-1 (106 PFU/mL) in a simultaneous and staggered manner, to combat five clinical RRSA strains and the laboratory strain MRSA ATCC 43300 in 72 h by isothermal microcalorimetry. The synergistic effects were observed when phage Sb-1 (106 PFU/mL) combined with antibiotics had at least 2 log-reduction lower concentrations, represented by a fractional biofilm inhibitory concentration (FBIC) of <0.25. Among the antibiotics that we tested, the synergistic effect of all six strains was achieved in phage/doxycycline and phage/linezolid combinations in a staggered manner, whereas a distinctly noticeable improvement in inhibitory activity was observed in the phage/doxycycline combination with a low concentration of doxycycline. Moreover, phage/levofloxacin and phage/clindamycin combinations also showed a synergistic inhibitory effect against five strains and four strains, respectively. Interestingly, the synergistic inhibitory activity was also observed in the doxycycline-resistant and levofloxacin-resistant profile strains. However, no inhibitory activity was observed for all of the combinations in a simultaneous manner, as well as for the phage/rifampin combination in a staggered manner. These results have implications for alternative, combined, and prolonged suppressive antimicrobial treatment approaches.

Pseudomonas aeruginosa Implant-Associated Bone and Joint Infections: Experience in a Regional Reference Center in France.

Background: P. aeruginosa implant-associated bone and joint infections (BJI) is considered to be one of the most difficult to treat BJI. The data focusing specifically on this pathogen are sparse, and it seems difficult to extrapolate the results obtained with Enterobacteriaceae.Methods: We performed a retrospective observation study of all P. aeruginosa implant-associated BJI diagnosed at our institution from 2011 to 2018. We defined failure as any type of relapse, including persistence of the same P. aeruginosa, superinfection by another organism(s) or any other cause of relapse such as the need for a subsequent surgery. Nonparametric statistical methods were used to compare the study groups and Kaplan-Meier curves and multivariate Cox analysis and were used to detect determinants associated with treatment failure.Results: A total of 90 patients (62% men, median age 60 years IQR 47–72) including 30 (33%) prosthetic-joint infections and 60 (66%) other implant-associated BJIs were studied. Most of them were acute (62%). During the prolonged follow-up, (median 20 months; IQR 9–37), 23 patients (26%) experienced treatment failure. Optimal surgical treatment (DAIR for acute forms, explantation, 1-stage or 2-stage exchange for others) was significantly associated with a higher success rate in the univariate analysis (p = 0.003). Sixty-four (71%) patients received effective initial treatment against P. aeruginosa administered and 81 of them (90%) did for at least 3 weeks: both these parameters correlated with a higher success rate. In the multivariate Cox-analysis optimal surgical treatment, IV effective treatment of at least 3 weeks and treatment with ciprofloxacin for at least 3 months proved to be independently associated to a better outcome in patients with P. aeruginosa implant-associated BJI.Conclusion: P. aeruginosa implant-associated BJI is one of the most difficult-to-treat BJI, with a strong impact on the prognosis of the surgical strategy. An effective initial IV antibiotic treatment for at least 3 weeks seems to be required, followed by oral ciprofloxacin for a total duration of 3 months.

Complete Genome Sequence of Staphylococcus aureus EDCC 5398, a Clinical Isolate from Implant-Associated Bone Infection.

Here, we report the high-quality complete genome sequence of Staphylococcus aureus EDCC 5398, which was isolated from a patient with implant-associated bone infection. The assembled genome consists of 2,843,534 bp, with a G+C content of 33%; it has 2,739 coding sequences, belongs to sequence type 22, and contains mecA and balz genes, which contribute to methicillin resistance.

Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections

Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of β-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections.Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections.

Obesity/type 2 diabetes increases inflammation, periosteal reactive bone formation, and osteolysis during Staphylococcus aureus implant-associated bone infection.

Obese and type 2 diabetic (T2D) patients have a fivefold increased rate of infection following placement of an indwelling orthopaedic device. Though implant infections are associated with inflammation, periosteal reactive bone formation, and osteolysis, the effect of obesity/T2D on these complicating factors has not been studied. To address this question, C57BL/6J mice were fed a high fat diet (60% Kcal from fat) to induce obesity/T2D, or a control diet (10% Kcal from fat) for 3 months, and challenged with a transtibial pin coated with a bioluminescent USA300 strain of S. aureus. In the resulting infected bone, obesity/T2D was associated with increased S. aureus proliferation and colony forming units. RNA sequencing of the infected tibiae on days 7 and 14 revealed an increase in 635 genes in obese/T2D mice relative to controls. Pathways associated with ossification, angiogenesis, and immunity were enriched. MicroCT and histology on days 21 and 35 demonstrated significant increased periosteal reactive bone formation in infected obese/T2D mice versus infected controls (p < 0.05). The enhanced periosteal bone formation was associated with increased osteoblastic activity and robust endochondral ossification, with persistant cartilage on day 21 that was only observed in infected obesity/T2D. Osteolysis and osteoclast numbers in obesity/T2D were also significantly increased versus infected controls (p < 0.05). Consistent with an up-regulated immune transcriptome, macrophages were more abundant within both the periosteum and the new reactive bone of obese/T2D mice. In conclusion, we find that implant-associated S. aureus osteomyelitis in obesity/T2D is associated with increased inflammation, reactive bone formation, and osteolysis. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:1614-1623, 2018.

Orthopaedic biofilm infections.

Many infections of the musculoskeletal system are biofilm infections that develop on non-living surfaces. Microorganisms adhere either on dead bone (sequesters) or implants. As a rule for a curative concept, chronic osteomyelitis or implant-associated bone infection must be treated with a combination of surgery and antimicrobial therapy. If an implant is kept in place, or a new device is implanted before complete healing of infection, a biofilm-active antibiotic should be used. Rifamycins are active against biofilms of staphylococci, and fluoroquinolones against those of Gram-negative bacilli. In this review, the management of chronic osteomyelitis, periprosthetic joint infection and implant-associated osteomyelitis of long bones is presented.

TLR9 mediates S. aureus killing inside osteoblasts via induction of oxidative stress.

BackgroundStaphylococcus aureus is the principle causative pathogen of osteomyelitis and implant-associated bone infections. It is able to invade and to proliferate inside osteoblasts thus avoiding antibiotic therapy and the host immune system. Therefore, development of alternative approaches to stimulate host innate immune responses could be beneficial in prophylaxis against S. aureus infection. TLR9 is the intracellular receptor which recognizes unmethylated bacterial CpG-DNA and activates immune cells. Synthetic CpG-motifs containing oligodeoxynucleotide (CpG-ODNs) mimics the stimulatory effect of bacterial DNA.ResultsOsteoblast-like SAOS-2 cells were pretreated with CpG-ODN type-A 2216, type-B 2006, or negative CpG-ODN 2243 (negative control) 4 h before infection with S. aureus isolate EDCC 5055 (=DSM 28763). Intracellular bacteria were streaked on BHI plates 4 h and 20 h after infection. ODN2216 as well as ODN2006 but not ODN2243 were able to significantly inhibit the intracellular bacterial growth because about 31 % as well as 43 % of intracellular S. aureus could survive the pretreatment of SAOS-2 cells with ODN2216 or ODN2006 respectively 4 h and 20 h post-infection. RT-PCR analysis of cDNAs from SAOS-2 cells showed that pretreatment with ODN2216 or ODN2006 stimulated the expression of TLR9. Pretreatment of SAOS-2 cells with ODN2216 or ODN2006 but not ODN2243 managed to induce reactive oxygen species (ROS) production inside osteoblasts as measured by flow cytometry analysis. Moreover, treating SAOS-2 cells with the antioxidant Diphenyleneiodonium (DPI) obviously reduced S. aureus killing ability of TLR9 agonists mediated by oxidative stress.ConclusionsIn this work we demonstrated for the first time that CPG-ODNs have inhibitory effects on S. aureus survival inside SAOS-2 osteoblast-like cell line. This effect was attributed to stimulation of TLR9 and subsequent induction of oxidative stress. Pretreatment of infected SAOS-2 cells with ROS inhibitors resulted in the abolishment of the CPG-ODNs killing effects.