Current Knowledge on Biomaterials for Orthopedic Applications Modified to Reduce Bacterial Adhesive Ability.

A significant challenge in orthopedics is the design of biomaterial devices that are able to perform biological functions by substituting or repairing various tissues and controlling bone repair when required. This review presents an overview of the current state of our recent research into biomaterial modifications to reduce bacterial adhesive ability, compared with previous reviews and excellent research papers, but it is not intended to be exhaustive. In particular, we investigated biomaterials for replacement, such as metallic materials (titanium and titanium alloys) and polymers (ultra-high-molecular-weight polyethylene), and biomaterials for regeneration, such as poly(ε-caprolactone) and calcium phosphates as composites. Biomaterials have been designed, developed, and characterized to define surface/bulk features; they have also been subjected to bacterial adhesion assays to verify their potential capability to counteract infections. The addition of metal ions (e.g., silver), natural antimicrobial compounds (e.g., essential oils), or antioxidant agents (e.g., vitamin E) to different biomaterials conferred strong antibacterial properties and anti-adhesive features, improving their capability to counteract prosthetic joint infections and biofilm formation, which are important issues in orthopedic surgery. The complexity of biological materials is still far from being reached by materials science through the development of sophisticated biomaterials. However, close interdisciplinary work by materials scientists, engineers, microbiologists, chemists, physicists, and orthopedic surgeons is indeed necessary to modify the structures of biomaterials in order to achieve implant integration and tissue regeneration while avoiding microbial contamination.

Novel Silver-Functionalized Poly(ε-Caprolactone)/Biphasic Calcium Phosphate Scaffolds Designed to Counteract Post-Surgical Infections in Orthopedic Applications.

In this study, we designed and developed novel poly(ε-caprolactone) (PCL)-based biomaterials, for use as bone scaffolds, through modification with both biphasic calcium phosphate (BCP), to impart bioactive/bioresorbable properties, and with silver nitrate, to provide antibacterial protection against Staphylococcus aureus, a microorganism involved in prosthetic joint infections (PJIs). Field emission scanning electron microscopy (FESEM) showed that the samples were characterized by square-shaped macropores, and energy dispersive X-ray spectroscopy analysis confirmed the presence of PCL and BCP phases, while inductively coupled plasma-mass spectrometry (ICP-MS) established the release of Ag+ in the medium (~0.15-0.8 wt% of initial Ag content). Adhesion assays revealed a significant (p < 0.0001) reduction in both adherent and planktonic staphylococci on the Ag-functionalized biomaterials, and the presence of an inhibition halo confirmed Ag release from enriched samples. To assess the potential outcome in promoting bone integration, preliminary tests on sarcoma osteogenic-2 (Saos-2) cells indicated PCL and BCP/PCL biocompatibility, but a reduction in viability was observed for Ag-added biomaterials. Due to their combined biodegrading and antimicrobial properties, the silver-enriched BCP/PCL-based scaffolds showed good potential for engineering of bone tissue and for reducing PJIs as a microbial anti-adhesive tool used in the delivery of targeted antimicrobial molecules, even if the amount of silver needs to be tuned to improve osteointegration.

Single versus Double Skin Preparation for Infection Prevention in Proximal Humeral Fracture Surgery.

Preoperative skin preparation plays a major role in preventing postoperative infections. This study aims to compare a single skin preparation (povidone iodine) with a double skin preparation (chlorhexidine gluconate followed by povidone iodine). Forty patients affected by proximal humeral fracture were included in the study. The day of surgery the two skin preparation strategies were performed in the same shoulder, divided into two areas, at the level of the deltopectoral approach. Skin swabs were collected from each area and subjected to microbiological analysis. Both skin preparations significantly reduced the positive culture rate. Coagulase-negative staphylococci (CoNS) dropped from 92.5% to 40% and to 7.5% after the single and double skin preparation (p<0.001), respectively. The positivity rate was reduced from 50% to 17.5% (p=0.002) and from 27.5% to 0% (p= 0.001) for Propionibacterium acnes and Staphylococcus aureus, respectively, with no difference between the two preparations. The double skin preparation had a more significant effect on bacterial load against CoNS compared to the single skin preparation (p<0.001 versus p= 0.015). In conclusion, both the approaches reduced S. aureus and P. acnes skin load, whereas the double skin preparation is more effective than the single one against CoNS. In light of our findings, preoperative strategies able to reduce bacterial load could potentially increase the final efficacy of perioperative traditional skin preparations.

Unexpected Listeria monocytogenes detection with a dithiothreitol-based device during an aseptic hip revision.

Prosthetic joint infection diagnosis is often difficult since biofilm-embedded microorganisms attach well to the prosthetic surfaces and resist their detection by conventional methods. DL-dithiothreitol has been described as a valid method for biofilm detachment on orthopedic devices. We report the case of an occasional detection of Listeria monocytogenes in a non immuno-compromised patient with a preoperative diagnosis of aseptic loosening. The infection diagnosis due to such rare bacteria was made postoperatively, thanks to a DL-dithiothreitol-based device. This may be considered a feasible approach for the microbiological analysis of prosthetic joint infection, considering that a prompt diagnosis of such biofilm-associated infections could bring some advantages, such as an early and appropriate antibiotic therapy administration and a reduction of undiagnosed infections.

Effect of surgical delay on bacterial colonization in proximal humeral fractures.

Postoperative infection is a severe complication after proximal humeral fracture surgical treatment. The aim of this study was to determine if the surgical delay could modify the number and type of bacteria on the surgical site. A two stages study was set up. In the first stage the effect of delay was simulated in 20 patients affected by proximal humeral fracture treated conservatively. In a second stage, the effect of delay was measured in 20 patients that underwent surgery. In stage 1, three skin culture swabs were taken in correspondence of the deltopectoral approach, the day of the fracture (day 0), the day after (day 1), and five days after fracture (day 5). In stage 2, skin swab cultures were taken the day of trauma and immediately before surgery and cultured on various media suitable for aerobic and anaerobic bacteria. The number of bacteria increased over the course of the study, from day 0 to day 5, both considering the total number of colony-forming units and individual species of pathogen bacteria. The second stage of the study confirmed these data. An increasing number of bacteria was observed in patients that underwent surgery later than 2 days from trauma. The delay of surgery increased bacterial colonization of the skin in the deltopectoral approach area including common pathogenic bacteria such as Staphylococcus aureus, coagulase-negative staphylococci and Propionibacterium acnes. This might justify the correlation between delay to surgery and risk of infection. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:942-948, 2016.

Application of multiple laboratory tests for Mycobacterium avium ssp. paratuberculosis detection in Crohn's disease patient specimens.

The difficulties involved in detecting and enumerating Mycobacterium avium subsp. paratuberculosis (MAP) as a pathogen potentially involved in Crohn's disease (CD) are well known. This study aimed to improve this situation through the application of multiple laboratory diagnostic tests to detect and isolate this bacterium from different specimens collected from CD-patients and non-CD subjects as controls. A total of 120 samples (terminal ileum and colon biopsies, blood and stool) were obtained from 19 CD-patients and from 11 individuals who did not have a clinicopathological diagnosis of CD (non-CD controls) attending for ileocolonoscopy. All samples were processed by staining techniques, culture on both solid and liquid media, and Insertion Sequence 900/F57 real-time PCR. The MAP frequency in CD-patients was found in a significantly greater proportion than in non-CD subjects; the most positive samples were biopsies from CD-patients tested by real-time PCR. MAP detection in biopsies, and in the other samples, by applying multiple and validated laboratory diagnostic tests, could be a marker of active infection, supporting MAP involvement in CD.

Incidence and risk factors for acute infection after proximal humeral fractures: a multicenter study.

BACKGROUND: The rate of acute infection after surgery for proximal humeral fractures is not known. The aims of this study were to report the incidence and to analyze the risk factors for infection after proximal humeral fracture treatment. MATERIALS AND METHODS: We report a retrospective multicenter study of 452 proximal humeral fractures. Data were modeled by use of univariate and/or linear regression analyses to determine the odds ratio (OR). A logistic regression analysis was used to check for demographic and other characteristics with the potential to confound a true association between risk factors and infection. RESULTS: The mean age was 62.1 years, and 314 patients were female patients. Of the patients, 18 (4%) had an acute infection. The factors that correlated with infection were length of surgery (OR, 1.009; P = .05), preoperative skin preparation with chlorhexidine gluconate (OR, 0.13; P = .008), and prophylactic antibiotic (OR, 10.73; P = .03). The delay to surgery was close to achieving significance (OR, 1.71; P = .06). CONCLUSION: This study suggests that washing the shoulder with chlorhexidine gluconate and avoiding the use of first-generation cephalosporin in favor of more effective prophylactic therapy are effective at reducing the risk of infection after treatment for proximal humeral fractures.

Bacteriological findings in radicular cyst and keratocystic odontogenic tumour fluids from asymptomatic patients.

OBJECTIVE: In this study the potential presence of bacteria in radicular cyst (RC) and keratocystic odontogenic tumour(KCOT) fluids from clinically asymptomatic patients was investigated. MATERIALS AND METHODS: Cyst fluids were collected by needle aspiration from 16 patients with asymptomatic osteolytic lesions (10 RCs and 6 KCOTs) undergoing surgery. All samples were transferred into tubes containing pre-reduced transport medium, delivered to the microbiology laboratory and processed within 1h. The cysts, surgically enucleated, were sent for standard histopathological examination. Cyst fluid samples were cultured on selective and differential media in anaerobic (for about 2 weeks) and aerobic (for 24-48 h) conditions to detect viable microorganisms. After incubation, the colonies were counted, Gram-stained and identified by biochemical tests. RESULTS: Cultures were positive for the presence of bacteria in 15 (9 RCs, 6 KCOTs) out of 16 cases. RCs and KCOTs generally yielded low bacterial counts (10(2)-10(4) CFU/ml) and were predominantly colonized by obligate anaerobes (64%), whereas less commonly by facultative anaerobes (36%). No significant differences in the detection frequencies of obligate and facultative anaerobes were evidenced between RCs and KCOTs. Propionibacterium acnes was the most common obligate anaerobe recovered both in RC and KCOT fluids. Among facultative anaerobes, Gemella morbillorum was more frequently isolated in KCOTs, whereas Staphylococcus spp. in RCs. CONCLUSIONS: Bacteria may be present and persist within fluids of clinically asymptomatic jaw cystic lesions. The influence of bacteria and latent bacterial infection within cystic jaw lesions should be reconsidered in odontogenic cyst progression.

Positive interaction of thyme (red) essential oil with human polymorphonuclear granulocytes in eradicating intracellular Candida albicans.

The essential oils have started to be recognized for their potential antimicrobial role only in recent years. Clinical experience showed that the efficacy of antimicrobial agents depends not only on their direct effect on a given microorganism but also on the functional activity of the host immune system. Since data on the effects of essential oils on the innate immune system are scanty and fragmentary, the aim of this study was to evaluate the influence of thyme (red) essential oil (EO), at subinhibitory/inhibitory concentrations, on intracellular killing activity by human polymorphonuclear granulocytes (PMNs) against Candida albicans. In order to provide a frame of reference for the activity of this EO, its in vitro killing activity in the absence of PMNs was also evaluated.Results showed that EO at subminimal inhibitory (subMIC)/minimal inhibitory (MIC) concentrations significantly enhanced intracellular killing of C. albicans in comparison with EO-free controls and was comparable to the positive control (fluconazole). In in vitro killing assays without PMNs, we observed progressive growth of the yeast cells in the presence of EO subMIC/MIC concentrations. A positive antifungal interaction with phagocytes could explain why this EO, which appeared to be only fungistatic in time-kill assays, had efficacy in killing yeast cells once incubated with PMNs.

Functional and phenotypical alterations of polymorphonuclear cells in Sézary syndrome patients.

Sézary syndrome (SS), the leukemic variant of cutaneous T-cell lymphoma (CTCL), has a poor prognosis and infections represent the most frequent cause of death. Polymorphonucleate granulocytes (PMNs) constitute an essential part of the innate immune system: their phagocytic and killing activity against pathogens is mediated by the interactions between Toll-like receptors (TLRs) and the Pathogen-associated molecular patterns (PAMPs). The aim of this study was to investigate PMN functional activity and phenotype in SS patients and their correlation with the onset of infectious complications. This prospective study enrolled 18 consecutive SS patients; PMN functional activity was evaluated by phagocytosis and intracellular killing tests towards Klebsiella pneumoniae. Flow-cytometry was applied to analyze PMN phenotype. PMNs from SS patients displayed a reduced phagocytic activity and intracellular killing against K. pneumoniae at 30 min and 60 min, more pronounced in SS patients with recurrent infections. CD11b and CD66b median fluorescence intensity (MFI) was significantly higher in SS than in healthy subjects, whereas CD62L MFI was decreased. No significant differences in TLR2, 4, 8 and 9 percentage expression or MFI were found. An increased TLR5 percentage expression was documented. The impairment in PMN functional activities in SS could favour the immune-suppression and raise infection risk.

Validation and standardization of IS900 and F57 real-time quantitative PCR assays for the specific detection and quantification of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease and may contribute to the onset and development of Crohn's disease in humans. Rapid detection of Map is fundamental because of its reported isolation from pasteurized milk and its potential for transmission through environmental sources. In this study, we developed two independent real-time quantitative PCR assays targeting the IS900 genetic insertion sequence and the F57 sequence, which proved capable of detecting and quantifying Map DNA. Validation and standardization of the developed methods were performed by evaluating diagnostic trueness, precision, and accuracy of the techniques. Specificity of the IS900 and F57 methods was verified in both in silico and experimental studies. The assays were found to be very accurate and precise with high repeatability and reproducibility. Moreover, the two real-time assays were very specific for Map, discriminating most of mycobacterial and nonmycobacterial species.

Schizophyllum commune: an unusual of agent bronchopneumonia in an immunocompromised patient.

We report a case of bronchopneumonia due to Schizophyllum commune in an immunocompromised patient. While this fungus rarely causes disease in humans, it has been reported in association with several clinical entities and lung disorders. A 59-year-old white man with a gastric carcinoma was admitted to S. Giovanni Battista Hospital (Turin, Italy). Three days after the admission, he developed a bronchopneumonia, which was diagnosed through the use of X-ray and showed an abnormal infiltrative shadow. Samples of bronchial aspirate were collected for laboratory microbiological investigation. Direct microscopic examination of these specimens revealed the presence of numerous septate, hyaline hyphae and rare clamp connections. Sabouraud Dextrose Agar and Columbia agar plus 5% blood media inoculated with portions of the same specimens yielded, after 4-5 days of incubation at 25 degrees C and 37 degrees C, a cottony white mould. The fungus was identified on the basis of its macroscopic and microscopic morphology. The macroscopic examination of the colony showed raised, curved, fan-shaped and shell-like basidiocarps. The microscope examination revealed the presence of hyaline, septate hyphae with clamp connections and short, thin spicules. The fungal isolate was identified as S. commune. The patient was cured after therapy with intravenous fluconazole (600 mg twice daily for over six weeks).

Microbial adherence on various intraoral suture materials in patients undergoing dental surgery.

PURPOSE: Sutures used in oral surgery should avoid or limit bacterial adhesion and proliferation to those parts exposed to oral fluids. Hence, microbial colonization on various intraoral suture materials from patients undergoing dental surgery was compared. PATIENTS AND METHODS: During dentoalveolar surgery, various suture materials were used in 60 patients, who were randomly divided into 5 groups of 12. In each group, silk was placed intraorally in association with a different type of suture (ie, Supramid, Synthofil, Ethibond Excel, Ti-cron, Monocryl) at the same site to compare microbial colonization intraindividually. Eight days postoperatively, the sutures were removed, and adhered micro-organisms were isolated, counted, and identified through enzymatic activities and fermentation of sugars. RESULTS: In all 60 patients, silk sutures exhibited the smallest affinity toward the adhesion of bacteria compared with considerable proliferation with nonresorbable multifilament sutures (Supramid, Synthofil, Ethibond Excel, Ti-cron). On the contrary, the microbial load was significantly lower when absorbable monofilament Monocryl was used. A greater quantity of bacteria was found on nonresorbable sutures than on absorbable ones, and nearly 2 times more facultative anaerobic bacteria were isolated in total. CONCLUSIONS: Our results show that bacteria adhere with different affinity to various types of suture materials. Absorbable silk and Monocryl exhibited the smallest number of adherent bacteria. Colonization by pathogens on sutures leads to the recommendation that sutures should be removed as early as possible after surgery is performed, to eliminate or to limit the reservoir for oral pathogens. This recommendation is dependent on whether the suture is absorbable.