Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides, proteins, eDNA and their correlation with biofilm mass.

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R2 = 0.882), while the content of proteins turned out to be weakly (R2 = 0.465) and that of eDNA very weakly associated (R2 = 0.202) to the biofilm mass.

Is Treatment With Dithiothreitol More Effective Than Sonication for the Diagnosis of Prosthetic Joint Infection?

BACKGROUND: Prosthetic joint infection (PJI) is among the most-severe complications of a total joint arthroplasty. Identification of the causal organism is of paramount importance for successful treatment, and sonication of implants may aid in this identification. Dithiothreitol (DTT) treatment has been proposed as an alternative to sonication to improve diagnosis, reduce costs, and improve reliability of the procedure, but its efficacy remains poorly characterized. QUESTIONS/PURPOSES: (1) Are DTT and sonication more sensitive and/or more specific than standard cultures of tissue samples for the diagnosis of PJI? (2) Which test (DTT or sonication) is more sensitive when the clinician does not suspect infection before surgery? (3) Which test (DTT or sonication) is more sensitive when the clinician suspects infection before surgery? METHODS: Two hundred thirty-two patients undergoing revision of a knee or hip arthroplasty were prospectively evaluated in this randomized study. Cultures were performed on five tissue samples from each patient and on fluid obtained by prosthesis treatment in patients randomly assigned to sonication (117 patients) or DTT (115 patients). The reference standard against which cultures (on tissue samples and on fluids from sonication or DTT) were compared was the Musculoskeletal Infection Society definition of PJI. RESULTS: Cultures on sonication and DTT fluids provided higher sensitivity (89% and 91%, respectively) than those on standard cultures of tissue samples (79%; p < 0.001). Among patients in whom infection was not suspected before surgery, the sensitivity of DTT was greater than that for sonication and cultures on tissue samples (100% versus 70% and 50%; p < 0.001). Among patients in whom infection was suspected before surgery, the sensitivity of DTT and sonication were not greater than that for standard cultures (89% and 94% versus 86%). CONCLUSIONS: In this randomized study, we found no difference in sensitivity between DTT and sonication for the detection of PJI, and both of those tests were more sensitive than standard tissue cultures. Thus, cultures of sonication or DTT fluid should be considered important additional tools to standard cultures for definition of PJI and should be considered together with other criteria, especially in settings where infection is not suspected before revision surgery.Level of Evidence Level I, diagnostic study.

The Opportunistic Pathogen Staphylococcus warneri: Virulence and Antibiotic Resistance, Clinical Features, Association with Orthopedic Implants and Other Medical Devices, and a Glance at Industrial Applications.

In recent decades, the risk of developing opportunistic infections has increased in parallel with the ever-increasing number of people suffering from chronic immunosuppressive diseases or undergoing prosthetic surgery. Staphylococcus warneri is a Gram-positive and coagulase-negative bacterium. Usually found as a component of the healthy human and animal microbiota of the skin and mucosae, it can take on the role of an opportunistic pathogen capable of causing a variety of infections, ranging from mild to life-threatening, not only in immunocompromised patients but even, although rarely, in healthy people. Here, in addition to a concise discussion of the identification and distinguishing features of S. warneri compared to other staphylococcal species, a systematic overview of the findings from case reports and clinical studies is provided. The paper highlights the virulence and antibiotic resistance profiles of S. warneri, the different clinical contexts in which it has proven to be a serious pathogen, emphasizing its ability to colonize artificial prosthetic materials and its tropism for musculoskeletal and cardiovascular tissues. Some original data on orthopedic implant infections by S. warneri complement the discussion. Finally, from a different perspective, the paper addresses the possibilities of industrial exploitation of this bacterium.

Combining Multiple Spectroscopic Techniques to Reveal the Effects of Staphylococcus aureus Infection on Human Bone Tissues.

Osteomyelitis (OM) and periprosthetic joint infections (PJIs) are major public health concerns in Western countries due to increased life expectancy. Infections usually occur due to bacterial spread through fractures, implants, or blood-borne transmission. The pathogens trigger an inflammatory response that hinders bone tissue regeneration. Treatment requires surgical intervention, which involves the precise removal of infected tissue, wound cleansing, and local and systemic antibiotic administration. Staphylococcus aureus (SA) is one of the most common pathogens causing infection-induced OM and PJIs. It forms antimicrobial-resistant biofilms and is frequently found in healthcare settings. In this proof-of-concept, we present an approach based on multiple spectroscopic techniques aimed at investigating the effects of SA infection on bone tissue, as well as identifying specific markers useful to detect early bacterial colonization on the tissue surface. A cross-section of a human femoral diaphysis, with negative-culture results, was divided into three parts, and the cortical and trabecular regions were separated from each other. Two portions of each bone tissue type were infected with SA for one and seven days, respectively. Multiple techniques were used to investigate the impact of the infection on bone tissue, Brillouin-Raman microspectroscopy and attenuated total reflection Fourier transform infrared spectroscopy were employed to assess and develop a new noninvasive diagnostic method to detect SA by targeting the bone of the host. The results indicate that exposure to SA infection significantly alters the bone structure, especially in the case of the trabecular type, even after just one day. Moreover, Raman spectral markers of the tissue damage were identified, indicating that this technique can detect the effect of the pathogens' presence in bone biopsies and pave the way for potential application during surgery, due to its nondestructive and contactless nature.

Is an isolated positive sonication fluid culture in revision arthroplasties clinically relevant?

OBJECTIVES: The aim of this study was to investigate the clinical relevance of an isolated positive sonication fluid culture (SFC) in patients who underwent revision surgery of a prosthetic joint. We hypothesized that cases with a positive SFC have a higher rate of infection during follow-up compared with controls with a negative SFC. METHODS: This retrospective multicentre observational study was performed within the European Study Group of Implant-Associated Infections. All patients who underwent revision surgery of a prosthetic joint between 2013 and 2019 and had a minimum follow-up of 1 year were included. Patients with positive tissue cultures or synovial fluid cultures were excluded from the study. RESULTS: A total of 95 cases (positive SFC) and 201 controls (negative SFC) were included. Infection during follow-up occurred in 12 of 95 cases (12.6%) versus 14 of 201 controls (7.0%) (p = 0.125). In all, 79.8% of cases were with treated with antibiotics (76/95). Of the non-treated cases, 89% (17/19) had a positive SFC with a low virulent microorganism. When solely analysing patients who were not treated with antibiotics, 16% of the cases (3/19) had an infection during follow-up versus 5% of the controls (9/173) (p = 0.08). DISCUSSION: Although not statistically significant, infections were almost twice as frequent in patients with an isolated positive SFC. These findings require further exploration in larger trials and to conclude about the potential benefit of antibiotic treatment in these cases.

An Observational Prospective Clinical Study for the Evaluation of a Collagen-Hydroxyapatite Composite Scaffold in Hip Revision Surgery.

One of the greatest challenges of hip revision surgery is the need to restore extensive bone loss by creating a stable reconstruction with long-term durability. The present observational, investigator-initiated prospective study was carried out to evaluate the clinical and radiological results of the use of a commercial biomimetic collagen-hydroxyapatite composite biomaterial (RegenOss) applied in hip revision surgery. Thirty-three patients who underwent hip revision were included in this study, and 29 received up to 2 years of follow-up. The acetabulum was reconstructed using an uncemented hemispherical shell both with or without an iliac fixation stem. Functional recovery was assessed according to the Harris Hip Score (HHS) at the pre-hospitalisation check-up, and at 6-, 12-, and 24-month follow-ups. Radiological evaluation consisting of X-ray analyses (6, 12, and 24 month follow-ups) and CT scan exams (within 10 weeks post-surgery and at 12-month follow-up) were performed to evaluate the reduction in bone defect and new bone regeneration. All the patients reported a complete recovery and a considerable improvement in functional outcome assessed by the HHS, which was significantly higher at all the follow-ups than at pre-hospitalisation. Moreover, radiological assessments revealed good scaffold integration. Overall, collected data suggest that RegenOss is a valid and safe alternative to restoring acetabular bone loss in revision hip arthroplasty.

Commercial Bone Grafts Claimed as an Alternative to Autografts: Current Trends for Clinical Applications in Orthopaedics.

In the last twenty years, due to an increasing medical and market demand for orthopaedic implants, several grafting options have been developed. However, when alternative bone augmentation materials mimicking autografts are searched on the market, commercially available products may be grouped into three main categories: cellular bone matrices, growth factor enhanced bone grafts, and peptide enhanced xeno-hybrid bone grafts. Firstly, to obtain data for this review, the search engines Google and Bing were employed to acquire information from reports or website portfolios of important competitors in the global bone graft market. Secondly, bibliographic databases such as Medline/PubMed, Web of Science, and Scopus were also employed to analyse data from preclinical/clinical studies performed to evaluate the safety and efficacy of each product released on the market. Here, we discuss several products in terms of osteogenic/osteoinductive/osteoconductive properties, safety, efficacy, and side effects, as well as regulatory issues and costs. Although both positive and negative results were reported in clinical applications for each class of products, to date, peptide enhanced xeno-hybrid bone grafts may represent the best choice in terms of risk/benefit ratio. Nevertheless, more prospective and controlled studies are needed before approval for routine clinical use.

Nanodecoration of electrospun polymeric fibers with nanostructured silver coatings by ionized jet deposition for antibacterial tissues.

Silver-based nanomaterials are used as antibacterial agents in a number of applications, including wound dressing, where electrospun materials can effectively promote wound healing and tissue regeneration thanks to their biomimicry, flexibility and breathability. Incorporation of such nanomaterials in electrospun nonwovens is highly challenging if aiming at maximizing stability and antibacterial efficacy and minimizing silver detachment, without neglecting process straightforwardness and scalability. In this work nanostructured silver coatings were deposited by Ionized Jet Deposition (IJD) on Polylactic acid, a medical grade polyester-urethane and Polyamide 6,6 nanofibers. The resulting materials were thoroughly characterized to gain an in-depth view of coating morphology and substrate resistance to the low-temperature deposition process used. Morphology of silver coatings with well-cohesive grains having dimensions from a few tens to a few hundreds of nanometers was analyzed by SEM, TEM and AFM. TGA, DSC, FTIR and GPC showed that the polymers well withstand the deposition process with negligible effects on their properties, the only exception being the polylactic acid that resulted more susceptible to degradation. Finally, the efficacy against S. aureus and E. coli bacterial strains was demonstrated, indicating that electrospun fibers decorated with nanostructured silver by IJD represent a breakthrough solution in the field of antibacterial devices.

Demineralized bone matrix paste formulated with biomimetic PLGA microcarriers for the vancomycin hydrochloride controlled delivery: Release profile, citotoxicity and efficacy against S. aureus.

Infection and resulting bone defects caused by Staphylococcus aureus is one of the major issues in orthopaedic surgeries. Vancomycin hydrochloride (VaH) is largely used to manage these events. Here, a human derived bone paste supplemented with biopolymer microcarriers for VaH sustained delivery to merge osteoinductive and antimicrobial actions is described. In detail, different emulsion formulations were tested to fabricate micro-carriers of poly-lactic-co-glycolic acid (PLGA) and hydroxyapatite (HA) by a proprietary technology (named Supercritical Emulsion Extraction). These carriers (mean size 827 ± 68 µm; loading 47 mgVaH/gPLGA) were assembled with human demineralized bone matrix (DBM) to obtain an antimicrobial bone paste system (250 mg/0.5 cm3 w/v, carrier/DBM). Release profiles in PBS indicated a daily drug average release of about 4 µg/mL over two weeks. This concentration was close to the minimum inhibitory concentration and able to effectively inhibit the S. aureus growth in our experimental sets. Carriers cytotoxicity tests showed absence of adverse effects on cell viability at the concentrations used for paste assembly. This approach points toward the potential of the DBM-carrier-antibiotic system in hampering the bacterial growth with accurately controlled antibiotic release and opens perspectives on functional bone paste with PLGA carriers for the controlled release of bioactive molecules.

Panton-Valentine leukocidin gene detected in Staphylococcus aureus strain isolated from a knee arthroprosthesis infection.

This report focuses on the molecular characterization of a Staphylococcus aureus strain isolated from a knee arthroprosthesis infection and recognized retrospectively as a carrier of the Panton-Valentine leukocidin gene. The stored microbiological isolate, which belonged to the strain collection of the Research Unit on Implant Infections of the Rizzoli Orthopaedic Institute, was retrieved for molecular analysis. Genotyping was carried out, revealing an interesting profile. In addition to the positivity for the Panton-Valentine toxin gene, the results indicated that the isolate belonged to the agr III group and was endowed with bbp and cna genes, both encoding for staphylococcal adhesins that bind bone proteins. The strain had the mecA gene for methicillin resistance, even though it was unable to resist any of the beta-lactam or other antibiotics. Its gene configuration matched that of other community-acquired methicillin-resistant and methicillin-susceptible Staphylococcus aureus(CA-MRSA and CA-MSSA) strains which have recently been reported worldwide. As far as we know,this is the first report on a PVL-positive S. aureus strain associated with an orthopedic implant (knee arthroprosthesis) infection.

Sonication Improves the Diagnosis of Megaprosthetic Infections.

Limited data are available for the diagnosis of patients with tumors with infected endoprosthetic reconstructions. The purpose of this study was to evaluate whether sonication is effective for the diagnosis of infection and to compare it with tissue cultures. The files of 58 patients who underwent revision surgery for suspected infected endoprosthetic reconstructions were reviewed. Cultures were performed on 5 tissue samples obtained from each patient and on fluid obtained by sonication of the megaprosthesis. The sensitivity, specificity, and negative and positive predictive values of tissue and sonication fluid cultures were evaluated. Overall, tissue and sonication fluid cultures confirmed an infection in 42 of the 58 patients. In 36 of the 42 infected endoprosthetic reconstructions, tissue and sonication fluid cultures identified the same bacterial isolate. In 5 cases, a bacterial isolate was identified only in sonication fluid cultures, and in 1 case, a bacterial isolate was identified only in tissue cultures. The sensitivity and negative predictive value of sonication fluid cultures were statistically significantly better than those of tissue cultures, while the specificity and positive predictive value were not different between the 2 culture types. Compared with tissue cultures for the diagnosis of infected megaprostheses in patients with tumors, sonication fluid cultures are associated with a better sensitivity and negative predictive value and a similar specificity and positive predictive value. Therefore, sonication should be considered a useful adjunct for the optimal diagnosis and management of these patients. [Orthopedics. 2019; 42(1):28-32.].

Is sonication of antibiotic-loaded cement spacers useful in two-stage revision of prosthetic joint infection?

BACKGROUND: In a two-stage exchange protocol for prosthetic joint infection (PJI), bacteria surviving over the antibiotic-loaded cement spacers may cause the persistence of infection with renewed clinical symptoms following the surgery. Culture after sonication of removed prosthesis is more sensitive than conventional periprosthetic tissue culture for the microbiological diagnosis of PJI. The aim of this study was to assess whether sonication of the spacer at the time of the second-stage procedure may improve the diagnosis of persistent PJI. METHODS: We evaluated by microbiological culture the sonication fluid from 222 cement spacers implanted in a two-stage exchange protocol in 157 patients affected by PJI. A mean of 1.3 (range, 1-4) spacer per patient was performed. RESULTS: In 53 out of 222 spacers analyzed infection was confirmed according to the MSIS criteria. In 22 cases the infection was confirmed by both cultures on periprosthetic tissue and on sonication fluid from the spacers. In 23 cases persistent PJI was identified because of only cultures of periprosthetic tissue and 8 because of results obtained after spacer sonication. The sensitivity of periprosthetic tissue cultures was higher than that of cultures performed on sonication fluid (84.9% vs 56.6%, p < .001). CONCLUSIONS: Even though sonication of cement spacers has performances inferior than those reported for prosthesis, it can be considered a complementary method to unravel persistent infection during a two-stage exchange protocol for PJI.

Orthopedic implant infections: Incompetence of Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis to invade osteoblasts.

Septic failure is still the major complication of prosthetic implants. Entering host cells, bacteria hide from host immune defenses, shelter from extracellular antibiotics, and cause chronic infection. Staphylococcus aureus, the leading etiologic agent of orthopedic implant infections, is able to enter bone cells and induce osteoblast apoptosis, osteoclast recruitment, and highly destructive osteomyelitis. Staphylococcus epidermidis, Staphylococcus lugdunensis, and Enterococcus faecalis are opportunistic pathogens causative of implant-related infections. This study investigated the ability to internalize into osteoblastic MG63 cells of 22 S. epidermidis, 9 S. lugdunensis, and 21 E. faecalis clinical isolates from orthopedic implant infections. Isolates were categorized in clusters by ribotyping. Internalization assay was carried out by means of a microtiter plate-based method. S. epidermidis, S. lugdunensis, and E. faecalis strains turned out incompetent to enter osteoblasts, exhibiting negligible internalization into MG63 cells, nearly three orders of magnitude lower than that of S. aureus. Osteoblast invasion does not appear as a pathogenetic mechanism utilized by S. epidermidis, S. lugdunensis, or E. faecalis for infecting orthopedic implants. Moreover, it can be inferred that intracellularly active antimicrobials should not be necessary against implant infections caused by the three bacterial species. Finally, implications with the uptake of biomaterial microparticles by nonphagocytic cells are enlightened. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 788-801, 2016.

Use of sodium hypochlorite for skin antisepsis before inserting a peripheral venous catheter: a pilot study.

Although it can be prevented, catheter-related bacteremia is common and dangerous. The antiseptics most widely used during insertion of peripheral venous catheters (PVCs) include povidone iodine, alcohol, and chlorhexidine. Another widely used antiseptic is a solution of 0.057 g sodium hypochlorite. This pilot study explored the contamination rate of the PVC tip inserted after skin decontamination with sodium hypochlorite. Culture analysis of the tips of the PVCs inserted into the 42 participants showed 7 (16.7%) colonized catheters. The results of this pilot study suggest taking into serious consideration the assessment of this antiseptic in randomized experimental studies.

Staphylococcus lugdunensis, an aggressive coagulase-negative pathogen not to be underestimated.

The new emerging coagulase-negative pathogen Staphylococcus lugdunensis is responsible for severe cardiac and joint infections. Since the biochemical phenotypic systems designed for the identification of CoNS do not appear to be species specific and are hardly reliable for the discrimination of S. lugdunensis from other staphylococci, its precise identification requires fine molecular methods. The pathogenic mechanisms by which S. lugdunensis causes severe infections are not yet completely elucidated and in this review its virulence and toxic determinants are surveyed as well as its adhesins and biofilm production.

Total hip arthroplasty after excision arthroplasty: indications and limits.

We reviewed performed a retrospective review of our series of excision arthroplasty patients and identified 16 patients who had undergone the procedure for sepsis, and who we had subsequently subjected to revision to a total hip arthroplasty (THA). Mean follow-up was 96 +/- 15 months. There was a significant reduction in limb length discrepancy and a marked improvement in walking capability. Range of movement improved most in patients under 65 years of age. Patient selection is critical, because THA after excision arthroplasty is complex, and may have less satisfactory results primary surgery.

Biofilm extracellular-DNA in 55 Staphylococcus epidermidis clinical isolates from implant infections.

Biofilm formation is broadly recognized as an important virulence factor in many bacterial species implicated in implant-related opportunistic infections. In spite of a long history of research and many investigative efforts aimed at elucidating their chemical composition, structure, and function, the nature of bacterial biofilms still remains only partly revealed. Over the years, different extracellular polymeric substances (EPS) have been described that contribute functionally and structurally to the organization of biofilms. Recently extracellular DNA (eDNA) has emerged as a quantitatively conspicuous and potentially relevant structural component of microbial biofilms of many microbial species, Staphylococcus aureus and S. epidermidis among them. The present study aims at comparatively investigating the amount of eDNA present in the biofilm of 55 clinical isolates of S. epidermidis from postsurgical and biomaterial-related orthopedic infections. Quantification of eDNA was performed by a non-destructive method directly on bacterial biofilms formed under static conditions on the plastic surface of 96-well plates.

Characterization of 26 Staphylococcus warneri isolates from orthopedic infections.

Staphylococcus warneri is a coagulase negative Staphylococcus (CNS) commonly present in the flora of human epithelia and mucosal membranes. Over the last two decades, similarly to other CNS species, S. warneri has been reported as a new emerging pathogen, capable of causing serious infections usually in association with the presence of implant materials, but, at times, even in the absence of a foreign body and in patients considered immunocompetent. At present, there is still a lack of scientific data on the pathogenesis and epidemiology of this species. The present study investigated a collection of 26 clinical isolates derived from orthopedic infections, some associated with implant materials and others not. Automated ribotyping showed the existence of 5 distinct ribogroups. Except for the least numerous ribogroup consisting of a single isolate, all other ribogroups included at least one strain obtained from an infection not associated with implant materials, suggesting that putative virulence factors necessary for infections even in the absence of a foreign body could be transversal to most ribogroup categories. Orthopedic infections were found to involve S. warneri strains with low antibiotic resistance potential, differing in this respect from the strains isolated at neonatal intensive care units, where this species has been described to figure among the principal causative agents and exhibit an alarming profile of antibiotic resistance.