Novel Peptides Targeting the ß-Clamp Rapidly Kill Planktonic and Biofilm Staphylococcus epidermidis Both in vitro and in vivo.

Antimicrobial resistance is an increasing threat to global health and challenges the way we treat infections. Peptides containing the PCNA interacting motif APIM (APIM-peptides) were recently shown to bind to the bacterial PCNA homolog, the beta (ß)-clamp, and to have both antibacterial and anti-mutagenic activities. In this study we explore the antibacterial effects of these peptides on Staphylococcus epidermidis, a bacterial species commonly found in prosthetic joint infections (PJI). Drug-resistant bacterial isolates from PJIs often lead to difficult-to-treat chronic infections. We show that APIM-peptides have a rapid bactericidal effect which when used at sublethal levels also increase the efficacy of gentamicin. In addition, APIM-peptides reduce development and eliminate already existing S. epidermidis biofilm. To study the potential use of APIM-peptides to prevent PJI, we used an in vivo bone graft model in rats where APIM-peptide, gentamicin, or a combination of the two was added to cement. The bone grafts containing cement with the combination was more effective than cement containing only gentamicin, which is the current standard of care. In summary, these results suggest that APIM-peptides can be a promising new drug candidate for anti-infective implant materials to use in the fight against resistant bacteria and chronic PJI.

Highly variable effect of sonication to dislodge biofilm-embedded Staphylococcus epidermidis directly quantified by epifluorescence microscopy: an in vitro model study.

BACKGROUND: In cases of prosthetic joint infections, culture of sonication fluid can supplement culture of harvested tissue samples for correct microbial diagnosis. However, discrepant results regarding the increased sensitivity of sonication have been reported in several studies. To what degree bacteria embedded in biofilm are dislodged during the sonication process has to our knowledge not been fully elucidated. In the present in vitro study, we have evaluated the effect of sonication as a method to dislodge biofilm by quantitative microscopy. METHODS: We used a standard biofilm method to cover small steel plates with biofilm forming Staphylococcus epidermidis ATCC 35984 and carried out the sonication procedure according to clinical practice. By comparing area covered with biofilm before and after sonication with epifluorescence microscopy, the effect of sonication on biofilm removal was quantified. Two series of experiments were made, one with 24-h biofilm formation and another with 72-h biofilm formation. Confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM) were used to confirm whether bacteria were present after sonication. In addition, quantitative bacteriology of sonication fluid was performed. RESULTS: Epifluorescence microscopy enabled visualization of biofilm before and after sonication. CLSM and SEM confirmed coccoid cells on the surface after sonication. Biofilm was dislodged in a highly variable manner. CONCLUSION: There is an unexpected high variation seen in the ability of sonication to dislodge biofilm-embedded S. epidermidis in this in vitro model.

Virus transmission during orthopedic surgery on patients with COVID-19 - a brief narrative review.

Background and purpose - COVID-19 is among the most impactful pandemics that the society has experienced. Orthopedic surgery involves procedures generating droplets and aerosols and there is concern amongst surgeons that otherwise rational precautionary principles are being set aside due to lack of scientific evidence and a shortage of personal protective equipment (PPE). This narrative review attempts to translate relevant knowledge into practical recommendations for healthcare workers involved in orthopedic surgery on patients with known or suspected COVID-19.Patients and methods - We unsystematically searched in PubMed, reference lists, and the WHO's web page for relevant publications concerning problems associated with the PPE used in perioperative practice when a patient is COVID-19 positive or suspected to be. A specific search for literature regarding COVID-19 was extended to include publications from the SARS epidemic in 2002/3.Results - Transmission of infectious viruses from patient to surgeon during surgery is possible, but does not appear to be a considerable problem in clinical practice. Seal-leakage is a problem with surgical masks. Due to the lack of studies and reports, the possibility of transmission of SARS-CoV-2 from patient to surgeon during droplet- and aerosol-generating procedures is unknown.Interpretation - Surgical masks should be used only in combination with a widely covering visor and when a respirator (N95, FFP2, P3) is not made available. Furthermore, basic measures to reduce shedding of droplets and aerosols during surgery and correct and consistent use of personal protective equipment is important.

Establishment of an in vivo rat model for chronic musculoskeletal implant infection.

BACKGROUND: The aim of the study was to establish an experimental chronic musculoskeletal infection model in vivo characterized by (a) a small bacterial inoculum, (b) no general or local signs of infection, (c) several parallels (implants) in each animal and finally (d) a model that is technically easy to perform. METHODS: Bone xenografts with steel plates were implanted intramuscularly in rats. To the xenografts, different inocula of Staphylococcus aureus and two strains of Staphylococcus epidermidis were added. The animals were observed for different time periods before the removal of the xenografts. The xenografts and steel plates were subjected to quantitative bacterial culture after sonication. Additional steel plates were subjected to scanning electron microscopy (SEM) for visualization of biofilm formation. RESULTS: Inoculation of bone grafts with S. aureus did produce a pyogenic infection in all animals. A chronic infection was established in rats where the bone grafts were inoculated with S. epidermidis. A bacterial inoculum of 100 colony-forming units (CFU) of S. epidermidis was adequate as a minimum infective dose. During a period of up until 42 days, the animals infected with S. epidermidis had no general or local signs of infection. According to the results of the quantitative bacterial culture of sonicate fluid and SEM, a biofilm was developed on all implants. CONCLUSION: In the present in vivo model, a very small bacterial inoculum succeeded in establishing a chronic musculoskeletal implant infection where a biofilm was formed on the implants. The experimental model is easy to perform and allows several implants in each animal. The model could be useful for the study of biofilm formation in vivo on different implants and different surfaces.

2018 international consensus meeting on musculoskeletal infection: Summary from the biofilm workgroup and consensus on biofilm related musculoskeletal infections.

Biofilm-associated implant-related bone and joint infections are clinically important due to the extensive morbidity, cost of care and socioeconomic burden that they cause. Research in the field of biofilms has expanded in the past two decades, however, there is still an immense knowledge gap related to many clinical challenges of these biofilm-associated infections. This subject was assigned to the Biofilm Workgroup during the second International Consensus Meeting on Musculoskeletal Infection held in Philadelphia USA (ICM 2018) (https://icmphilly.com). The main objective of the Biofilm Workgroup was to prepare a consensus document based on a review of the literature, prepared responses, discussion, and vote on thirteen biofilm related questions. The Workgroup commenced discussing and refining responses prepared before the meeting on day one using Delphi methodology, followed by a tally of responses using an anonymized voting system on the second day of ICM 2018. The Working group derived consensus on information about biofilms deemed relevant to clinical practice, pertaining to: (1) surface modifications to prevent/inhibit biofilm formation; (2) therapies to prevent and treat biofilm infections; (3) polymicrobial biofilms; (4) diagnostics to detect active and dormant biofilm in patients; (5) methods to establish minimal biofilm eradication concentration for biofilm bacteria; and (6) novel anti-infectives that are effective against biofilm bacteria. It was also noted that biomedical research funding agencies and the pharmaceutical industry should recognize these areas as priorities. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Does the use of high-heeled shoes lead to fore-foot pathology? A controlled cohort study comprising 197 women.

BACKGROUND: High-heeled shoes have been suggested as a main explanation for the female dominance in foot pain and deformities. Aim of study was to test this hypothesis scientifically. METHODS: Women 40-66 years were included in two groups. 95 women who had worn high-heeled shoes at work for at least 5 years were compared to 102 women who had never worn high-heeled shoes at work. The investigations were weight bearing radiographs of foot and ankle, the SEFAS questionnaire and the AOFAS Clinical Rating System. Evaluators were blinded to the group-affiliation. RESULTS: Radiographs showed no statistically significant differences between the two groups concerning deformities or joint disease. Foot function measured by SEFAS and AOFAS total score, were similar in the two groups. The high-heeled group had more pain and more callosities. CONCLUSION: For women aged 40-66 years wearing of high-heeled shoes had not caused foot deformation, but more foot pain and callosities.

The role of infection-associated risk factors in prosthetic surgery.

The number of obese people with diabetes mellitus (DM) is increasing. DM is in itself a risk factor for all types of infections, including infections after total hip and knee prosthetic surgery. In particular, the prevalence of obesity is high among orthopaedic patients. Obesity is also a risk factor for post-operative infections in general. After total hip arthroplasty obese patients have a threefold increased risk for post-operative infections compared to a patient with normal weight. There is an association between DM and obesity, and this association is explained by the presence of metabolic syndrome. Fat tissue in people with abdominal obesity acts as an endocrine organ and metabolic syndrome is characterized by a condition of chronic inflammation, insulin resistance, and vascular obstructive disease. The obese patient with DM should be considered a high risk patient for post-operative infection in all types of surgery. Efforts should be made to identify patients with a metabolic syndrome prior to prosthetic surgery. Fasting glucose levels and HbA1C should be included in pre-operative blood tests, and, if necessary, specialists in internal medicine and endocrinology should assess the patient pre-operatively. The amount of antibiotics administered as prophylaxis should be adjusted to the body mass and the blood glucose level should be controlled per-operatively. Persistent wound drainage for more than five days post-operatively should be considered for surgical revision. If possible, the patient should of course lose weight before the operation, but the role of bariatric surgery is not discussed in this presentation.

A comprehensive microbiological evaluation of fifty-four patients undergoing revision surgery due to prosthetic joint loosening.

The diagnosis of a chronic prosthetic joint infection (PJI) is challenging, and no consensus exists regarding how best to define the criteria required for microbiological identification. A general view is that culture of periprosthetic biopsies suffers from inadequate sensitivity. Recently, molecular analyses have been employed in some studies but the specificity of molecular analyses has been questioned, mainly due to contamination issues. In a prospective study of 54 patients undergoing revision surgery due to prosthetic joint loosening, we focused on two aspects of microbiological diagnosis of chronic PJI. First, by collecting diagnostic specimens in a highly standardized manner, we aimed at investigating the adequacy of various specimens by performing quantitative bacteriological culture. Second, we designed and performed real-time 16S rRNA gene PCR analysis with particular emphasis on minimizing the risk of false-positive PCR results. The specimens analysed included synovial fluid, periprosthetic biopsies from the joint capsule and the interface membrane, and specimens from the surface of the explanted prosthesis rendered accessible by scraping and sonication. No antibiotics were given prior to specimen collection. Based on five diagnostic criteria recently suggested, we identified 18 PJIs, all of which fulfilled the criterion of ≥2 positive cultures of periprosthetic specimens. The rate of culture-positive biopsies from the interface membrane was higher compared to specimens from the joint capsule and synovial fluid, and the interface membrane contained a higher bacterial load. Interpretational criteria were applied to differentiate a true-positive PCR from potential bacterial DNA contamination derived from the reagents used for DNA extraction and amplification. The strategy to minimize the risk of false-positive PCR results was successful as only two PCR results were false-positive out of 216 negative periprosthetic specimens. Although the PCR assays themselves were very sensitive, three patients with low bacterial numbers in periprosthetic specimens tested negative by real-time PCR. This overall lowered sensitivity is most likely due to the reduced specimen volume used for PCR analysis compared to culture and may also be due to interference from human DNA present in tissue specimens. According to the protocol in the present study, 16S rRNA gene real-time PCR did not identify more cases of septic prosthetic loosening than did culture of adequate periprosthetic biopsies.

Lower limb amputations in Trondheim, Norway.

BACKGROUND AND PURPOSE: In the city of Trondheim, Norway, diabetic lower-limb amputations accounted for one-third of all lower-limb amputations (LLAs). In an attempt to reduce this rate, a diabetic foot team was established in 1996. We present the incidence of LLA in Trondheim as measured 10 years later. PATIENTS AND METHODS: In 2004-07, we registered all LLAs performed in Trondheim and then compared the data with previously published data from 1994-1997. From 1996 through 2006, we registered the activity of the diabetic foot team and we also registered the number of vascular procedures performed on citizens of Trondheim from 1998 through 2006. RESULTS: Comparing the two 3-year periods 1994-97 and 2004-07, we observed a decrease in all non-traumatic LLAs. The incidence of diabetic major LLAs per 10³ diabetics per year decreased from 4.0 to 2.4, and in patients with peripheral vascular disease we observed a decrease in LLAs from 18 to 12 per 105 inhabitants per year. 5,915 consultations on diabetic subjects were conducted by the diabetic foot team during the period 1996-2006. From 1998 to 2006, the rate of vascular procedures decreased in the non-diabetic population, and was unchanged in diabetic subjects. INTERPRETATION: In the population of Trondheim city there appears to have been a reduction in the rate of vascular obstructive lower-limb disease between the two 3-year periods 1994-97 and 2004-07. In our judgment, the decline in diabetic LLA also reflects better care of the diabetic foot.

Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro.

BACKGROUND AND PURPOSE: Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces. METHODS: Discs of titanium and steel were incubated in the presence of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Propionibacterium acnes in Mueller Hinton broth. Non-adherent bacteria were removed by repeated rinsing of the discs. 10 parallels of each disc were subjected to 1 of 4 methods for bacterial recovery: (A) sonication of the discs, (B) scraping of the discs using surgical blades followed by streaking of the blades onto agar plates, (C) scraping of the discs followed by vortex mixing of the surgical blades, and (D) scraping of the discs followed by sonication of the surgical blades. Quantitative bacterial cultures were performed for each sampling method. RESULTS: With the exception of S. epidermidis on steel, sonication efficiently and reliably dislodged biofilm bacteria. The scraping methods employed did not detach bacteria embedded in biofilm. INTERPRETATION: Scraping of metal surfaces is not an adequate method for sampling of biofilm bacteria in vitro.

Soft tissue infection after missile injuries to the extremities--a non-randomized, prospective study in Gaza City.

INTRODUCTION: Patients with soft tissue injuries caused by missile attacks during wartime have been treated with radical debridement and delayed closure. In a study in Gaza City, the rate of infection of missile injuries to the extremities when treated with minimal surgical intervention, was measured. METHODS: Patients with severe soft tissue damage, compound fractures, and injuries to major blood vessels and/or nerves were excluded from the study. One hundred fourteen patients were treated according to a standardized regime that included a superficial, minor surgery revision of the inlet and the outlet opening, and antibiotic treatment. Local soft tissue infection was defined as the presence of at least two signs of local infection. RESULTS: A total of 109 out of 114 patients attended the first follow-up visit. Eleven (10%) of these patients had an infected wound. A total of 105 of the patients (92%) attended a second follow-up. None of these patients had an infected wound. CONCLUSIONS: Under conditions with a high number of casualties, minimal surgical treatment followed by the administration of antibiotics is a safe procedure for patients with penetrating missile injuries and less severe soft tissue damage.

Improved comfort and function of arm prosthesis after implantation of a Humerus-T-Prosthesis in trans-humeral amputees.

The use of arm prosthesis in trans-humeral amputees is limited; due to the cone form of the amputation stump. A Humerus-T-Prosthesis was implanted in three patients to create artificial humerus condyles. Two of the patients were successfully rehabilitated with the application of a new type trans-humeral arm prosthesis. This arm prosthesis had a socket which is suspended and stabilized by the humerus and implant only. Traction and rotational stability were secured by adjustable pressure adaptation around the artificial condyles. The third patient developed a pressure wound over the lateral part of the artificial condyle that later healed. He also was subject to a new trauma with a fracture of the ipsilateral scapula and until now has had limited the use of his new arm prosthesis. It was concluded that this new concept for prosthesis fitting of trans-humeral amputees looks promising, but alternative designs of the implant should be tested.

Cortical allograft as a vehicle for antibiotic delivery.

BACKGROUND: Infection can be a devastating complication after implantation of a cortical bone allograft. The allograft could act as a vehicle for local antibiotic prophylaxis. MATERIAL AND METHODS: We studied the release of antibiotics in vitro from cortical bone allografts impregnated with antibiotics for different periods of time. We also studied whether cortical allografts impregnated with antibiotics could eradicate Staphylococcus aureus from an experimentally infected graft in vivo. In the in vitro study, pieces of cortical bone were impregnated with netilmicin, vancomycin, ciprofloxacin and rifampicin for 1 h, 10 h and 100 h. The antibiotics were eluted into phosphate-buffered saline (PBS) for 7 days, with daily transfer of the bone into fresh PBS. In the in vivo study, cortical allografts impregnated with antibiotics were placed in rats intramuscularly. 10 microL of an S. aureus suspension (0.6 x 10(5) CFU) was placed in the intramedullary cavity. After 15 days, the allografts were removed and examined for bacterial growth. RESULTS: The amount of antibiotics released in vitro was influenced by the time used for antibiotic impregnation of the bone. Allografts impregnated with netilmicin, vancomycin and rifampicin effectively eradicated perioperative contamination with S. aureus in vivo. INTERPRETATION: This study shows that a cortical bone allograft would be an effective vehicle for local antibiotic delivery.