Chronic Staphylococcal Osteomyelitis Research Articles

Systemic application of bone-targeting peptidoglycan hydrolases as a novel treatment approach for staphylococcal bone infection.

The rising prevalence of antimicrobial resistance in S. aureus has rendered treatment of staphylococcal infections increasingly difficult, making the discovery of alternative treatment options a high priority. Peptidoglycan hydrolases, a diverse group of bacteriolytic enzymes, show high promise as such alternatives due to their rapid and specific lysis of bacterial cells, independent of antibiotic resistance profiles. However, using these enzymes for the systemic treatment of local infections, such as osteomyelitis foci, needs improvement, as the therapeutic distributes throughout the whole host, resulting in low concentrations at the actual infection site. In addition, the occurrence of intracellularly persisting bacteria can lead to relapsing infections. Here, we describe an approach using tissue-targeting to increase the local concentration of therapeutic enzymes in the infected bone. The enzymes were modified with a short targeting moiety that mediated accumulation of the therapeutic in osteoblasts and additionally enables targeting of intracellularly surviving bacteria.

Induction of protective interferon-β responses in murine osteoblasts following Staphylococcus aureus infection.

The refractory and recurrent nature of chronic staphylococcal osteomyelitis may be due, at least in part, to the ability of Staphylococcus aureus to invade and persist within bone-forming osteoblasts. However, osteoblasts are now recognized to respond to S. aureus infection and produce numerous immune mediators and bone regulatory factors that can shape the host response. Type I interferons (IFNs) are best known for their antiviral effects, but it is becoming apparent that they impact host susceptibility to a wide range of pathogens including S. aureus. Here, we have assessed the local expression of IFN-β by specific capture ELISA in an established in vivo mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis, specific capture ELISAs, and/or immunoblot analyses, were then used to assess the expression of type I IFNs and select IFN stimulated genes (ISGs) in S. aureus infected primary murine osteoblasts. The effect of IFN-β on intracellular S. aureus burden was assessed in vitro following recombinant cytokine treatment by serial colony counts of liberated bacteria. We report the presence of markedly elevated IFN-β levels in infected bone tissue in a mouse model of staphylococcal osteomyelitis. RNA Tag-Seq analysis of S. aureus infected osteoblasts showed enrichment of genes associated with type I IFN signaling and ISGs, and elevated expression of mRNA encoding IFN-β and ISG products. IFN-β production was confirmed with the demonstration that S. aureus induces its rapid and robust release by osteoblasts in a dose-dependent manner. Furthermore, we showed increased protein expression of the ISG products IFIT1 and IFIT3 by infected osteoblasts and demonstrate that this occurs secondary to the release of IFN-β by these cells. Finally, we have determined that exposure of S. aureus-infected osteoblasts to IFN-β markedly reduces the number of viable bacteria harbored by these cells. Together, these findings indicate an ability of osteoblasts to respond to bacteria by producing IFN-β that can act in an autocrine and/or paracrine manner to elicit ISG expression and mitigate S. aureus infection.

Osteocytes Serve as a Reservoir for Intracellular Persisting Staphylococcus aureus Due to the Lack of Defense Mechanisms.

Chronic staphylococcal osteomyelitis can persist for long time periods causing bone destruction. The ability of Staphylococcus aureus to develop chronic infections is linked to its capacity to invade and replicate within osteoblasts and osteocytes and to switch to a dormant phenotype called small colony variants. Recently, osteocytes were described as a main reservoir for this pathogen in bone tissue. However, the mechanisms involved in the persistence of S. aureus within these cells are still unknown. Here, we investigated the interaction between S. aureus and osteoblasts or osteocytes during infection. While osteoblasts are able to induce a strong antimicrobial response and eliminate intracellular S. aureus, osteocytes trigger signals to recruit immune cells and enhance inflammation but fail an efficient antimicrobial activity to clear the bacterial infection. Moreover, we found that extracellular signals from osteocytes enhance intracellular bacterial clearance by osteoblasts. Even though both cell types express Toll-like receptor (TLR) 2, the main TLR responsible for S. aureus detection, only osteoblasts were able to increase TLR2 expression after infection. Additionally, proteomic analysis indicates that reduced intracellular bacterial killing activity in osteocytes is related to low antimicrobial peptide expression. Nevertheless, high levels of lipid mediators and cytokines were secreted by osteocytes, suggesting that they can contribute to inflammation. Taken together, our results demonstrate that osteocytes contribute to severe inflammation observed in osteomyelitis and represent the main niche for S. aureus persistence due to their poor capacity for intracellular antimicrobial response.

Implant-associated osteomyelitis: Development, characterisation, and application of a porcine model.

Implant-associated osteomyelitis: Development, characterisation, and application of a porcine model.

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.

Oral Antibiotic Therapy

Oral Antibiotic Therapy

Chronic bacterial osteomyelitis suppression of tumor growth requires innate immune responses

Clinical studies over the past several years have reported that metastasis-free survival times in humans and dogs with osteosarcoma are significantly increased in patients that develop chronic bacterial osteomyelitis at their surgical site. However, the immunological mechanism by which osteomyelitis may suppress tumor growth has not been investigated. Therefore, we used a mouse model of osteomyelitis to assess the effects of bone infection on innate immunity and tumor growth. A chronic Staphylococcal osteomyelitis model was established in C3H mice and the effects of infection on tumor growth of syngeneic DLM8 osteosarcoma were assessed. The effects of infection on tumor angiogenesis and innate immunity, including NK cell and monocyte responses, were assessed. We found that osteomyelitis significantly inhibited the growth of tumors in mice, and that the effect was independent of the infecting bacterial type, tumor type, or mouse strain. Depletion of NK cells or monocytes reversed the antitumor activity elicited by infection. Moreover, infected mice had a significant increase in circulating monocytes and numbers of tumor associated macrophages. Infection suppressed tumor angiogenesis but did not affect the numbers of circulating endothelial cells. Therefore, we concluded that chronic localized bacterial infection could elicit significant systemic antitumor activity dependent on NK cells and macrophages.

Long-Term Follow-Up Trial of Oral Rifampin-Cotrimoxazole Combination versus Intravenous Cloxacillin in Treatment of Chronic Staphylococcal Osteomyelitis

Oral therapies alternative to fluoroquinolones against staphylococcal chronic osteomyelitis have not been evaluated in comparative studies. Consecutive nonaxial Staphylococcus aureus chronic osteomyelitis cases were included in a comparative trial after debridement. Fifty patients were randomized: group A (n = 22) was treated with cloxacillin for 6 weeks intravenously plus 2 weeks orally (p.o.), and group B (n = 28) was treated with rifampin-cotrimoxazole for 8 weeks p.o. During follow-up (10 years), five relapses occurred: two (10%) in group A and three (11%) in group B. Foreign-body maintenance was associated with relapse (P = 0.016). Oral rifampin-cotrimoxazole treatment showed outcomes comparable to those for intravenous cloxacillin treatment.

Treatment of experimental osteomyelitis by liposomal antibiotics.

Traditional antibiotic therapy of staphylococcal osteomyelitis by a single drug or a drug combination is ineffective in producing complete sterilization of infected bones. The aim of this study was to develop a non-traditional delivery system of antibiotics for treatment of chronic experimental osteomyelitis. In the current work, ciprofloxacin and vancomycin were encapsulated in a cationic, anionic or neutral liposomal formulation. For prolonged circulation in serum, liposomal dispersions (<100 nm in diameter) were sonicated for different times (20, 40, 60 or 80 s), and tested for antibacterial activities. Liposomes sonicated for 40 s gave the highest antibacterial activities in vitro. Since cationic liposomes trapped the highest percentage of antibiotics, and enhanced antibacterial activity above that of the free drugs, they were used for therapeutic trials to treat chronic staphylococcal osteomyelitis induced in rabbits. Therapeutic trials with antibiotics given intravenously revealed that, free ciprofloxacin or vancomycin given alone for 14 days was ineffective in sterilizing bone. Combination therapy with free ciprofloxacin and vancomycin for 14 days was more effective. However, this group showed renal dysfunction and severe diarrhoea, which resulted in loss of 33.3% of treated animals. Treatment with liposomal forms of either drug for 7 days was ineffective. Meanwhile, combination therapy in liposomal form for 7 days was more effective. Complete sterilization of bone tissues on cultures (100% cure) was obtained only in the group treated for 14 days with the combination of both drugs in liposomal form. Moreover, liposomal formulations showed much lower nephrotoxicity and a lower incidence of severe diarrhoea than that induced by free drugs.

Chronic staphylococcal osteomyelitis: a new experimental rat model

A rat model of chronic staphylococcal osteomyelitis was developed. Fibrin glue (5 microliters) and Staphylococcus aureus (2 x 10(6) CFU/5 microliters) were inoculated into the proximal metaphysis of the tibia. The rats were killed at intervals of between 1 and 6 months, and the tibias were removed. Induced lesions were evaluated by radiographic, macroscopic, and histological examinations and bacterial counts. Roentgenograms revealed osteomyelitis in more than 90% of the tibias. Gross bone pathology revealed skeletal deformation, new bone formation, abscesses, and draining skin fistulas in more than 80% of cases. Histological examination revealed osteomyelitis in more than 90% of cases, and bacterial counts were positive in 86% of cases. Only fibrin glue (5 microliters) was inoculated into controls. Controls showed no osteomyelitic lesions, and counts were negative in seven of eight control tibias. The main feature of this model is the use of fibrin glue instead of the sclerosing agents and foreign bodies used in other models. The model reproduces lesions similar to those of human posttraumatic osteomyelitis and can be reliably used in pathophysiological and therapeutic studies.

Cefepime: overview of activity in vitro and in vivo.

Cefepime is a novel methoxyimino-aminothiazolyl cephalosporin with a quaternized N-methyl-pyrrolidine moiety at the 3' position conferring zwitterionic properties. Because of this the molecule penetrates the outer cell membrane of Gram-negative bacteria rapidly. In addition it is resistant to degradation by several plasmid and chromosomally-mediated beta-lactamases, for which it also shows very low affinity and no inducing capacity. It has good affinity for PBPs 2 and 3 of Escherichia coli and for PBP 3 of Pseudomonas aeruginosa. Its broad-spectrum of activity includes Gram-positive and Gram-negative pathogens. It is more active than cefotaxime or ceftazidime, against Enterobacteriaceae. The MIC90 for P. aeruginosa is higher than that of ceftazidime, but lower than those of cefpirome, cefoperazone and latamoxef. Other Gram-negative organisms, Haemophilus influenzae, Neiserria meningitidis, Neiserria gonorrhoeae, Moraxella catarrhalis are highly susceptible to cefepime. Among Gram-positive species methicillin-susceptible Staphylococcus aureus and coagulase-negative staphylococci, whether beta-lactamase producers or not, Streptococcus pneumoniae and Streptococcus pyogenes are susceptible. Cefepime is active against cefotaxime- and/or ceftazidime-resistant Enterobacteriaceae. Only strains of P. aeruginosa producing large amounts of beta-lactamase may be resistant to both ceftazidime and cefepime. In experimental infections such as meningitis, induced with various bacterial species in neonatal rats and chronic staphylococcal osteomyelitis in rabbits, cefepime has shown good efficacy.

Enzyme-linked immunosorbent assay for detection of antibodies to Staphylococcus aureus cell walls in experimental osteomyelitis.

An enzyme-linked immunosorbent assay was used to evaluate and compare the immunoglobulin G antibody response to Staphylococcus aureus cell walls of rabbits with either chronic staphylococcal osteomyelitis or subcutaneous abscesses. Osteomyelitis of the femur was produced by the intramedullary application of a sclerosing agent (3% sodium tetradecyl sulfate) and S. aureus. Radiographic evidence of osteomyelitis was observed in 10 of the 13 animals that survived the 10-week experimental period, and the diagnosis was confirmed by histopathology in 8 of the 10 instances. Abscess formation was initiated in a separate group of rabbits by the subcutaneous injection of S. aureus cells. All 10 of these rabbits subsequently developed abscesses, which usually resolved spontaneously within 3 to 5 weeks. Elevated levels of immunoglobulin G antibodies to the cell wall antigen were detected in 7 of 10 rabbits with osteomyelitis at 21 days postinfection, and these animals continued to display high antibody levels even at 59 days postinfection. In contrast, elevated levels of anti-cell-wall antibodies were only detected in 1 of 10 rabbits with subcutaneous abscesses. The enzyme-linked immunosorbent assay was found to be a rapid and sensitive serological technique for the detection of cell wall antibodies in this experimental osteomyelitis model and may be useful for the diagnosis of staphylococcal bone infections in humans.

Experimental chronic staphylococcal osteomyelitis in rabbits: treatment with rifampin alone and in combination with other antimicrobial agents.

The efficacy of rifampin alone and in combination with sisomicin, cephalothin, sisomicin and cephalothin, or trimethoprim was measured in an experimental model of chronic osteomyelitis due to Staphylococcus aureus in rabbits. Single-drug regimens used for 28 days were relatively ineffective (bone sterilization rate, 5%-55%). Combinations of rifampin with other agents were more effective (bone sterilization rate, 75%-100%). The combination of rifampin, sisomicin, and cephalothin given for only 14 days sterilized the bones of all treated rabbits. Staphylococci isolated from the bones of rabbits that had received rifampin alone or in combination with other antibiotics were highly resistant to rifampin; in contrast, these organisms remained sensitive to the other agents used. Results of in vitro studies of synergy correlated with in vivo results in some but not all instances. The results obtained with rifampin in the treatment of experimental chronic osteomyelitis caused by S. aureus suggest that clinical trials of this agent for the treatment of osteomyelitis in humans are warranted.

Immunoassay of acute phase reactants and Latex-CRP as activity tests in chronic staphylococcal osteomyelitis.

20 patients with chronic staphylococcal osteomyelitis were tested for 6 acute phase reactants: alpha 1-antitrypsin, orosomucoid, haptoglobin, ceruloplasmin, C-reactive protein (CRP) and antichymotrypsin during different phases of the disease. CRP was best correlated to clinical activity and in 3 cases CRP and ceruloplasmin increased a few weeks before a clinically apparent exacerbation of the osteomyelitis took place. A Latex-CRP slide method showed fairly good agreement with CRP assessed by immunoassay. Determination of CRP is a suitable test for following the activity of chronic osteomyelitis.

Assay of teichoic acid antibodies and antistaphylolysin in the diagnosis of Staphylococcal osteomyelitis.

Teichoic acid antibodies (TAA), measured by gel diffusion, and antistaphylolysin (ASa) values were determined in 46 osteomyelitis patients and 200 healthy controls. A positive TAA titer (greater than or equal to 4) was seen in 4/16 patients with acute hematogenous osteomyelitis caused by Staphylococcus aureus and in 7/22 patients with chronic staphylococcal osteomyelitis, while ASta was positive (greater than or equal to 2.0 IU/ml) in 9 patients in both groups all except 2 exceeding 4.0 IU/ml. No positive TAA titres or ASta values were seen in 8 patients with chronic nonstaphylococcal osteomyelitis. The rate of positive test results in 200 controls was 7% for TAA and 4% for ASta. Basing diagnosis on a positive value in either of the tests gave 21 positive results in 38 patients with staphylococcal osteomyelitis; 18 by ASta alone and 11 by TAA alone. Both tests were positive at the same time in 3 patients with acute and in 5 patients with chronic staphylococcal osteomyelitis but never in controls.

Teichoic acid antibodies in chronic staphylococcal osteomyelitis.

Gel-diffusion and the enzyme-linked immunosorbent assay (ELISA) were used to quantify and to identify the immunoglobulin class of teichoic acid antibodies in patients with chronic staphylococcal osteomyelitis and a wide variety of other infections. Teichoic acid antibodies were identified by gel-diffusion in 14 of 23 patients with staphylococcal endocarditis, six of 30 with staphylococcal bacteremia without endocarditis, four of 35 with staphylococcal skeletal infections, and one of 45 with nonstaphylococcal infections. None of the 20 patients with chronic staphylococcal osteomyelitis had positive gel-diffusion assays, even though many had had their infections for several years. The ELISA method was more sensitive than gel-diffusion in measuring teichoic acid antibodies, but was also much less specific. Teichoic acid antibodies were detected predominantly in the IgG fraction of serum. Our findings suggest that the presence and degree of antigenemia are more important than the duration of the staphylococcal infection in stimulating production of teichoic acid antibodies.

Lipolytic activity of Staphylococcus aureus strains from cases of human chronic osteomyelitis and other infections.

The fatty acid accumulation in a lipid emulsion broth was examined after growth of S. aureus strains incubated for 18 hours at 37 degrees C. Among 40 strains from acute infections and from nasal carriers, Tween 80-positive staphylococci in phage group III caused accumulation of significantly larger amounts of fatty acids than did Tween 80-positive strains in phage group I and Tween 80-negative strains in phage groups I and III. Eight out of 17 S. aureus strains isolated from chronic osteomyelitis gave rise to a low amount of, or total lack of, fatty acids in the lipid broth. Seven of these 8 strains were isolated from cases showing relapse after intensive and prolonged antibiotic treatment. The lipid metabolism of 4 strains showing fatty acid accumulation in lipid broth, ranging from zero to the highest value found in the investigation, was further studied. Results of examination of lipolytic action on trioleinphospholipid and incorporation of 14C-labelled oleic acid suggest that the degree of accumulation of fatty acids in lipid broth depends on variations in the lipolytic activity of the strains rather than on differences in incorporation of the produced fatty acids. The significance of fatty acid accumulation is discussed in relation to the prognosis of chronic staphylococcal osteomyelitis.

Antibiotics in the Treatment of Chronic Staphylococcal Osteomyelitis

Fourteen patients with chronic osteomyelitis were treated with oral penicillin, oxacillin, or cloxacillin at a dose of 5 gm/day, plus 0.5 gm of probenecid (Benemid) three or four times per day. Treatment was continued for 2 1/2 to six months. In ten patients, evidence of osteomyelitis disappeared and there has been no recurrence during follow-up periods varying from six months to 2 1/2 years. Significant hepatotoxicity was encountered in two patients; one similar patient, not in this series but receiving a similar regimen of antibiotic, developed leukipenia. Thus, patients with chronic osteomyelitis appear to benefit from such a therapeutic program, but they must be monitored closely for hepatic or hematologic toxicity.

The prognosis of chronic staphylococcal osteomyelitis after long-term antibiotic treatment.

Abstract41 patients with chronic staphylococcal osteomyelitis received antibiotic therapy for at least 6 months. They were observed for 1–5.5 years after the end of treatment. The frequency of recurrences after such long-term treatment (LTT) was significantly lower than before. Despite this improvement, 21 recurrences were diagnosed in 12 patients (29%) during the observation period. The recurrences did not appear to be associated with any particular characteristic of the patients. 16 of the recurrences were seen in 8 patients infected with staphylococci belonging to phage group I. Various aspects of the virulence of staphylococci were considered, but no particular factor could be regarded as decisive for the recurrence tendency. Half of the patients were treated surgically and/or locally with antibiotics during LTT. Especially sequestrectomy appeared to improve the prognosis.

A Paradigm for Human Chronic Osteomyelitis

The inoculation of Staphylococcus aureus into the tibial marrow cavity of rabbits following tibial fracture and rod insertion, as well as into the rodded tibia without fracture, results in chronic staphylococcal osteomyelitis in a high percentage of the animals. Staphylococci were easily recovered from the tibial marrow cavity for as long as eighteen months after onset of the infection. This model closely resembles the human disease in which chronic osteomyelitis develops as a complication of internal fixation devices, and provides a reliable method for evaluating various specific approaches to the treatment of this difficult problem in management.