Aim. A significant number of patients undergoing shoulder arthroplasty surgery have C acnes contamination at the end of the primary surgery. The objective of this study is to determine whether patients with C acnes contamination at the end of their primary shoulder surgery have a worse prognosis than those who end up without C. acnes contamination. Method. Prospective study including all patients who underwent a reverse shoulder prosthesis from January 2015 to December 2018. In all of them, 5 to 12 cultures were performed during primary surgery. The patients underwent surgery for shoulder arthritis secondary to rotator cuff tears, acute fracture of the proximal humerus, and sequelae of fracture of the proximal humerus. Exclusion criteria included the existence of previous surgeries on the affected shoulder, the presence of signs of infection, having received infiltrations and / or complementary invasive examinations (Arthro-MRI and Arthro-CT). Follow-up from 2 to 5 years. Functional assessment according to the Constant Functional Scale. All complications were also recorded. Results. 162 patients were included. Of these, 25 had positive cultures for C. acnes at the end of primary shoulder surgery. Average age of 74.8 years. 136 women and 26 men. 75.9% Shoulder arthritis secondary to rotator cuff tears, 13.6% acute fractures and 10.5% sequelae of fractures. There were no differences between patients with C. acnes and those without C. acnes regarding age and indication for surgery. Predominance of men in the group with positive C. acnes (p <0.001). No differences at 2 and 5 years in the Constant functional scale between the two groups (2 years, 59.6 vs 59.2 p 0.870) (5 years, 62.4 vs 59.5 p 0.360). Significant differences regarding the number of complications (p 0.001). Patients without C. acnes had 1 aseptic loosening of the metaglene and patients with C. acnes had 2 infections, 1 dislocation, and 1 revision surgery. Patients with contamination by C. acnes had more comorbidities (p 0.035) than patients without contamination. Conclusions. Patients with C acnes contamination at the end of primary surgery do not have functional differences when compared with patients without contamination at 2 and 5 years, but they have a higher number of complications in the medium term

Background. Although described as a commensal bacterium with low pathogenicity, Cutibacterium acnes involvement has been reported in many clinical entities: infections associated with devices, such as shoulder prosthetic joint infections, osteosynthesis, breast implants or cerebrospinal fluid shunts. Various studies show that C. acnes grows as a biofilm, contributing to its persistence by allowing its escape from the action of the immune system and antibiotics. Purpose. Our aim was to assess the activity of different active substances (erythromycin, clindamycin, doxycycline and Myrtacine. ®. ) on eight different well-characterized C. acnes strains after growth in biofilm mode. Methods. Eight susceptible strains of C. acnes were selected for this study, including two reference strains (ATCC6919 and ATCC11827) and six clinical strains. All C. acnes strains were studied using two different methods to study the biofilm production at different time points: the BioFilm Ring Test. ®. technique (early stages of adhesion) and the Crystal Violet (CV) method (mature biofilm). In a second step, the impact of different active substances (erythromycin, clindamycin, doxycycline and Myrtacine. ®. ) was studied. For the CV technique, two types of tests were performed: preventive tests (addition of active substances and bacteria at the same time) and curative challenge tests (addition of active substances on a biofilm already formed after 48h). Transmission electron microscopy was performed to investigate the morphology modifications. Results. C. acnes isolates from phylotypes IA. 1. and IA. 2. , seem to produce more mature biofilm in the first stages of adhesion than other phylotypes. Curative assays were performed to evaluate the efficacy of antibiotics and Myrtacine. ®. on mature biofilm. Significant efficacy of Myrtacine. ®. at 0.03% was observed for C. acnes strains. Moreover, the combination of Myrtacine. ®. and doxycycline appears to decrease the total biofilm biomass. The effect of doxycycline as a preventive measure was minimal. On the contrary, a similar use of Myrtacine. ®. as early as 0.001% showed significant efficacy with a significant decrease in total biofilm biomass for all C. acnes strains. Transmission electron microscopy revealed a significantly decreased biofilm growth in treated bacteria with Myrtacine. ®. compared to untreated bacteria. Moreover, the total number of bacteria decreased as the concentration of Myrtacine. ®. increased suggesting also an antimicrobial effect. Conclusion. These results confirm the difference in biofilm producing ability depending on C. acnes phylotypes. These results suggest that Myrtacine. ®. may be a promising alternative antibacterial and anti-biofilm agent like peroxide de benzoyle to prevent shoulder prosthetic joint infection involving planktonic and biofilm C. acnes

Aim. Cutibacterium acnes is a major skin commensal that may also act as an opportunistic pathogen. Findings of C. acnes in tissue cultures obtained during arthroplasty revision surgery are difficult to interpret, since they may represent true infection or contamination. This study investigated whether C. acnes isolates obtained from prosthetic joint infections (PJIs) were related and shared common genomic traits that might correlate with clinical courses and patient outcomes. Method. C. acnes isolates from revision surgery of patients with PJIs of the hip, shoulder, and knee were characterized using molecular methods to determine sequence type (ST) and the presence of virulence determinants (CAMP factors, dermatan sulfate-binding adhesion 1, hyaluronidase lyase, and linear plasmid). A standardized review of the patients’ medical charts was performed. Results. The study included 37 patients with C. acnes culture-positive tissue samples where multiple isolates of C. acnes belonged to the same ST. Most of the isolates belonged to phylotype IA. 1. Phylogenetic analysis of virulence determinants revealed no shared pattern among PJI isolates. Seven patients had a polymicrobial infection. Exchange revision was performed in 70% of the patients, and >50% of all patients received antibiotic treatment for ≥3 months. Failure was noted in seven patients, all of whom had shoulder PJIs. Conclusions. No specific ST or any identifiable unique feature among virulence determinants were found among C. acnes isolated from PJIs of hips and shoulders. The majority of all included patients had low inflammatory markers and were treated successfully, even when the infection consisted of a polymicrobial infection

Introduction and Objective. Found in bone-associated prosthesis, Cutibacterium acnes (C. acnes) is isolated in more than 50% of osteoarticular prosthesis infections, particularly those involving shoulder prostheses. Ongoing controversies exist concerning the origin of C. acnes infection. Few reports construct a reasonable hypothesis about probable contaminant displaced from the superficial skin into the surgical wound. Indeed, despite strict aseptic procedures, transecting the sebaceous glands after incision might result in C. acnes leakage into the surgical wound. More recently, the presence of commensal C. acnes in deep intra-articular tissues was reported. C. acnes was thus detected in the intracellular compartment of macrophages and stromal cells in 62.5% of the tested patients who did not undergo skin penetration. Among bone stromal cells, mesenchymal stem cells (MSCs) are predominantly found in bone marrow and periosteum. MSCs are the source of osteogenic lines of cells capable of forming bone matter. In this study, the pathogenicity of C. acnes in bone repair context was investigated. Materials and Methods. Human bone marrow derived MSCs were challenged with C. acnes clinical strains harvested from non-infected bone site (Cb). The behaviour of Cb strain was compared to C. acnes took from orthopaedic implant-associated infection (Ci). The infective capabilities of both strains was determined following gentamicin-based antibiotic protection assay. The morphology and ultrastructural analysis of infected MSCs was performed respectively through CLSM pictures of Phalloidin. ®. stained MSCs cytoskeleton and DAPI labelled Cb, and transmission and scanning electron microscopies. The virulence of intracellular Ci and Cb (Ci-MSCs and Cb-MSCs) was investigated by biofilm formation on non-living bone materials; and the immunomodulatory response of infected MSCs was investigated (PGE-2 and IDO secretion detected by ELISA). Bone cells (osteoblasts and PMA differentiated macrophages) were then challenged with Cb-MSCs and Ci-MSCs. Intracellular accumulation of ROS within infected macrophages was assessed by flow cytometry after 2 h of infection and the catalase production by Cb-MSC and Ci-MSC was evaluated. Statistical analyses were performed using Mann & Whitney test. Results. Following MSCs infection by C. acnes, the rate of viable bacteria inside MSCs was about 4% and 6% for Cb and Ci, respectively. Cb showed however a lower invasiveness in comparison to Ci (0.6-fold, p=0.01), confirming the higher pathogenicity of Ci. The ultrastructural and morphology analysis of infected MSCs confirmed the presence of bacteria free in MSCs cytoplasm, localized between F-actin fibers of MSCs, which preserved their elongated morphology. Considering the high level of secreted immunomodulatory mediators (PGE-2 and IDO), our results suggest that Cb-infected MSCs could promote a transition of macrophages from a primarily pro-inflammatory M1 to a more anti-inflammatory M2 phenotype. In comparison with Cb, Cb-MSCs increased significantly the formation of biofilm on TA6V and PEEK but reduced the biofilm formation on 316L SS. Ci-MSCs showed a significant increase in biofilm formation on PEEK vs Ci, while no difference in biofilm formation was noticed on TA6V and 316L SS. Regarding the ability of MSCs bacteria to infect osteoblasts, our results showed a higher infective capabilities of Cb-MSCs versus Cb (>2-fold, p=0.02), while no difference was noticed between Ci and Ci-MSCs. Along with an increase in catalase production by Cb-MSCs, we noticed its higher persistence to macrophage degradation. Conclusions. Taken together, our results demonstrate a shift in commensal Cb to pathogenic following infection. Indeed, Cb- MSCs acquires features that (i) increase biofilm formation on orthopedic based materials, (ii) increase the osteoblast infection and (iii) develop resistance to the macrophage degradation, through the increase of catalase production. Overall, these results showed a direct impact of C. acnes on bone marrow derived MSCs, providing new insights into the development of C. acnes during implant-associated infections

Aim. Cutibacterium acnes (C. acnes) is the most cultured organism implicated in periprosthetic shoulder infections. Nevertheless, the clinical significance of its persistence on the skin surface and in the deep layers during shoulder arthroplasty surgery remains still unknown. The purpose of this study was to know if the C. acnes isolate present in deep tissues at the end of a primary shoulder arthroplasty could be responsible for shoulder arthroplasty infection. Method. Prospective study including 156 patients undergoing primary shoulder arthroplasty. In all the patients included 5 to 12 tissue samples were obtained and were specifically cultured to detect C. acnes presence. DNA was extracted from the C. acnes colonies selected with the QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen, Hilden, Germany). Libraries were prepared using Nextera XT kit (Illumina) and sequenced in an Illumina MiSeq sequencer. Sequencing files were pre-processed using The Microbial Genome Atlas pipeline. Samples that failed on QC analysis were discarded for further analysis. Isolate nucleotide distances were calculated using Genome-based distance matrix calculator from the enveomics collection. Comparative genomic analysis was performed between intra- and inter-patients’ isolates. Data analysis was performed using R 3.6.3. Results. For twenty-seven out of 156 patients (17.31%), C. acnes was present at the end of the primary surgery. Two of these patients (both male) developed a C. acnes periprosthetic shoulder infection after 6 and 4 months from the primary surgery. DNA from the C. acnes responsible for the periprosthetic infection was further analysed by whole genome sequencing (WGS). Average Nucleotide Identity (ANI) value was assessed, measuring the nucleotide-level genomic similarity between genome pairs. We found a clear ANI clustering in two major groups which corresponded, mainly, to the associated phylotype (97%–98% ANI). Moreover, when analysing both isolates that developed a periprosthetic shoulder infection, we found that all the revision-surgery isolates clustered nearer to their corresponding primary-surgery isolates (99.4% of similarity) than to the other independent bacterial isolates, supporting the causal relationship between the initial and the delayed infection. Conclusions. C. acnes present at the end of the primary surgery can be the cause of early- or delayed-periprosthetic joint infections in shoulder arthroplasty, revealing the potential route of infection. Therefore, efforts must be made in terms of antibiotic prophylaxis and skin preparation to limit infections of total shoulder arthroplasties

Aim. The aim of our study was to analyze putative genes for virulence factors of Cutibacterium isolates obtained from implant-associated infections. Methods. We analyzed 64 isolates of Cutibacterium spp. (C. acnes (53/64), C. avidum (6/64), C. granulosum (4/64), C. namnetense (1/64)) using NextSeq 550 (Illumina, San Diego, CA, USA) and performed genomic analysis of 24 genes associated with virulence factors (VFs) of C. acnes previously reported in the literature. Most isolates were obtained from implant-associated infections (IAI) between 2012–2021 at the Institute of Microbiology and Immunology, Faculty of Medicine, Ljubljana. Additionally, we included the first C. namnetense isolated in our laboratory from surgical site infection. Results. C. acnes and C. namnetense have the highest number of VFs among those examined. The VFs gntK (shikimate kinase) and HYL-IB / II (hyaluronate lyase) are absent in phylotype IA. 1. (sequence types (ST) A, C, D according to the SLST scheme). Repressor gene of porphyrin synthesis, deoR is present in all Cutibacterium spp. isolates. The phylotypes II and IB show a similar distribution of VFs, with the presence of the VFs rcsB (compound for biofilm formation) and HYL-IA (hyaluronate lyase), which are absent in other C. acnes phylotypes and other Cutibacterium spp. In phylotypes IA. 1. and IB, the sequence of genes encoding VFs dsA1 and dsA2 does not have 100% genomic coverage, possibly indicating homologs between species. The isolates of C. acnes and C. namnetense possess all three CAMP (1,2,4) factors, which are not detected in other Cutibacterium spp. However, further analysis revealed species-specific CAMP factors in C. avidum and C. granulosum. Both species also have similar other genes for VFs, mainly encoding heat shock proteins and lipases, while VFs related to biofilm production are mostly absent (rcsB, ytpA). Conclusion. We found several differences in the distribution of VFs among Cutibacterium spp. isolated from IAI

Aim. Cutibacterium acnes is a significant cause of late-onset spinal implant infection (SII). In addition, usual preoperative prophylactic measures may be insufficient to prevent C. acnes operating site colonisation and infection, as demonstrated for prosthetic shoulder surgery. However, little information is available regarding risk factors for SII due to this microorganism. The aims of this study were to determine the characteristics of and risk factors for C. acnes SII. Method. we conducted a retrospective unmatched case-control study including all adult patients treated for mono and polymicrobial C. acnes SII during 2010–2015. Controls were randomly selected among patients diagnosed with SII due to other microorganisms during the same period. Results. Fifty-nine patients with C. acnes SII were compared with 59 controls. There was no difference in sex distribution (39% vs 53% men). Patients with C. acnes SII were younger (median age 42 vs. 65, p< 0.001), thinner (median body mass index (BMI) 21 vs. 25 kg/m. 2. , p< 0.001), and presented a better health status (ASA score≤ 2, 83% vs. 65%, p= 0.015; and presence of immunosuppression, 3% vs. 27%, p= 0.002). Patients with C. acnes SII were more likely to experience delayed/late infections (i.e. diagnosed >3 months post-instrumentation, 66% vs. 22%, p< 0.001) and to be instrumented for scoliosis (83% vs. 27%, p< 0.001) with an extended osteosynthesis (median number of fused vertebrae 12 vs. 5, p< 0.001). However, 20 C. acnes SII (34%) developed early (≤3 months) after instrumentation. The clinical presentation was significantly more indolent in the C. acnes group (presence of fever, 27% vs. 61%, p= 0.001; wound inflammation 39% vs. 61%, p< 0.001 and median C-reactive protein level 38 vs. 146 mg/L). Mixed C. acnes SII were diagnosed on 24 occasions (41%), 22 of which involving both C. acnes and staphylococcal strains. In the multivariate logistic regression model, factors independently associated with the development of SII involving C. acnes were age less than 65 (adjusted odds ratio [aOR] 7.13, 95% CI [2.44–24.4], p= 0.001), BMI< 22kg/m. 2. (aOR 3.71 [1.34–10.7], p= 0.012) and a number of fused vertebrae >10 (aOR 3.90 IC 95% [1.51–10.4], p= 0.005). Conclusions. There were significant differences between SII involving C. acnes and those involving other microorganisms. We identified a specific profile of patients at increased risk of developing C. acnes SII. These findings could contribute to improve both the prevention and treatment of such infections

Aim. Cutibacterium acnes is involved in chronic/low-grade pathologies such as prosthetic joint infection (PJI) or sarcoidosis. During these diseases, granulomatous structures are frequently observed. In this study, we induced a physiological granulomatous reaction in response to different well-characterized clinical C. acnes isolates in order to investigate the cellular process during the granulomatous formation. Method. An acne C. acnes ATCC6919 isolate (clonal complex (CC) 18, phylotype IA1), a PJI C. acnes BL clinical isolate (CC36, phylotype IB) and a sarcoidosis C. acnes S8 strain (CC28, phylotype IA. 2. ) were included and co-culture with PBMC. Cellular aggregation was followed daily using light microscopy. At various time points of incubation (day 3 and day 7), granuloma structures were processed for microscopic observation, colony forming unit enumeration after Triton ×100 lysis to release the internalized bacteria (bacterial load within the granulomas), as well as for flow cytometric analysis (detection of CD4, CD8 and NK lymphocytes). Results. All C. acnes isolates generated granulomatous structures in our experimental conditions. The bacterial burden was better controlled by granulomas induced by sarcoidosis C. acnes isolate. PJI C. acnes isolate, belonging to CC36, promoted the recruitment of CD8. +. lymphocytes inside the granuloma. At the opposite, acne and sarcoidosis C. acnes isolates, belonging, respectively, to phylotype IA. 1. /CC18 and phylotype IA. 2. /CC28 generated a higher number of granuloma and promoted the recruitment of CD4. +. lymphocytes inside the granuloma. Conclusions. Our results provide new arguments supporting the role of C. acnes in the development of infections and new explanations concerning the mechanisms underlying PJI due to C. acnes. This model appears to be a possible alternative assay to animal models for studying the immune response to C. acnes infection

Aim. The aim of this study was to confirm that Mirra's criterion (≥ 5 Polymorphonuclears (PMNs) per field in 5 high power fields (HPFs)) is not adequate for diagnosis of chronic bone and joint infections (BJIs) due to Cutibacterium acnes (C. acnes). The second objective was to determine if plasma-cell infiltration, that is a classical marker of chronic inflammation, could be useful for the diagnosis of chronic BJIs due to C. acnes. Methods. We retrospectively selected 25 patients from 2009 to 2013 with chronic BJIs due to C. acnes. In addition of Mirra's criterion, the number of plasma-cells (≥5 plasma-cells/5 HPFs, defined as “CRIOAc Lyon's criterion”) was implemented in the histopathological analysis. Patients were defined as infected, if at least one of the two criteria were present. Results. According to Mirra's and CRIOAc Lyon's histopathological criteria, positive histology was observed in respectively 12 (48%) and 16 (64%) cases. In 1 case the samples were not analyzable. Considering the 12 cases with negative Mirra's criterion, high plasma-cell infiltration (≥5 plasma-cells/5 HPFs; Figure 1) was observed in 6 cases (50%), and low plasma-cells infiltration (2–5 plasma-cells/5 HPFs) was observed in 5 other cases (42%). Conclusions. Mirra's criterion is not an adequate criterion to defining chronic BJIs [1, 2]. In our study, more cases of chronic BJIs due to C. acnes have been diagnosed using CRIOAc Lyon's criterion than Mirra's criterion. Adding CRIOAc Lyon's criterion might restore some histopathological diagnosis of chronic BJIs due to C. acnes, when a clinical chronic BJI is suspected. For any tables or figures, please contact the authors directly

Aim. Cutibacterium acnes, a skin commensal, is responsible for 5–10% of prosthetic joint infections (PJI). All current microbiological definitions of PJI require two or more identical commensal isolates to be recovered from the same procedure to diagnose PJI and rule out contamination. Unlike coagulase negative staphylococci, C.acnes shows a highly stereotypical susceptibility profile making impossible to phenotypically assess the clonal relationship of isolates. In order to determine the clonal relationship of multiple C.acnes isolates recovered from arthroplasty revisions, we analyzed by multi-locus sequence typing (MLST) C.acnes isolates grown from orthopedic device-related infections (ODRI) in a reference center for bone and joint infection. Methods. Laboratory records from January 2009 to January 2014 were searched for monomicrobial C.acnes ODRI with growth of C. acnes in at least 2 intraoperative and/or preoperative samples. Clinical, biological and demographic information was collected from hospital charts. All corresponding isolates biobanked in cryovials (−80°C) were subcultured on anaerobic blood agar, and identification confirmed by MALDI-TOF-MS. C.acnes isolates were typed using the MLST scheme described by Lomholt et al. Plasmatic pre-operative C-reactive protein (CRP) levels were determined using DimensionEXL (Siemens). A threshold of 10 mg/L was used to determine serologically positive ODRIs from negatives. Results. Over a 5-year period, 37 cases of monomicrobial C.acnes ODRI were diagnosed in our center. Among these 37 cases, 113/153 C.acnes isolates were cryopreserved. 110/113, corresponding to 36/37 cases, were typed by MLST: 14/36 (39%) ODRI cases were found to feature isolates belonging to two or more different STs and were qualified to be heteroclonal whereas 22/36 (61%) of ODRI cases were found to feature isolates belonging to the same ST and were qualified to be homoclonal. Homoclonal infections were significantly more likely to have elevated CRP levels compared to heteroclonal cases (p=0.0011, Fisher test). Patients with only two positive intraoperative samples had significantly lower CRP values than patients with three or more positive intraoperative samples (12,7mg/L vs 67mg/L; p=0,01, homoscedastic two-tailed Student's t test). Conclusions. This study suggests that what is classified microbiologically as C.acnes ODRIs comprises: i) true homoclonal infections eliciting an inflammatory response, ii) heteroclonal infections lacking inflammatory response where C.acnes could be an innocent bystander and iii) false positives where no strain achieves true microbiological significance. Our study shows that a stricter threshold of 3 intraoperative positive samples could be more adequate than 2. These results reinforces the need for a more specific definition of C.acnes ODRI

Introduction. Cutibacterium acnes (C. acnes) is now recognized as a clinical entity in periprosthetic joint infections (PJI) of the shoulder and spine. However, the colonization rate of C. acnes in the adult hip is currently unknown. Therefore, the purpose of this study was to investigate the rate of C. acnes colonization from the skin of healthy subjects from various anatomic locations corresponding to direct anterior and lateral/posterolateral surgical approaches. Methods. 90 patients scheduled for hip or knee surgery were recruited for cultured biopsies. Four 3-mm dermal punch biopsies were collected after administration of anesthesia, but prior to delivery of perioperative antibiotics. Pre-biopsy skin prep consisted of a standardized pre-operative 2% chlorhexidine skin cleanse and an additional 70% isopropyl alcohol mechanical skin scrub immediately prior to biopsy collection. Two culture samples 10-cm apart were collected from a location approximating a standard direct anterior skin incision, and two samples 10-cm apart were collected from a location approximating a lateral skin incision (suitable for a posterior, direct-lateral or anterolateral surgical approach). Samples were cultured for two weeks. Results. 22 of the 90 (24%) patients had a positive culture biopsy, fourteen of which (16% of all patients) were positive for C. acnes. Ten (71%) of the culture positive biopsies for C. acnes were obtained from the anterior location with 50% of those obtained from the most proximal sample site. Conclusions. Approximately 16% of the patients in the study demonstrated positive C. acnes colonization about the hip, the majority of which occurred from an anterior location. C. acnes should be considered in the diagnosis of PJI after THA. Given the high rate of skin colonization, particularly as regards the direct anterior approach to the hip, these results have stimulated consideration for different skin preparations for the THA patient. For any tables or figures, please contact the authors directly

Prosthetic Joint Infections (PJIs) are increasing with the use of orthopedic devices on an ageing population. Cutibacterium acnes is a commensal organism that plays an important role in the ecosystem healthy human skin, yet this species is also recognized as a pathogen in foreign body infection: endocarditis, prostatitis and specifically in PJIs. C. acnes is able to escape the immune system. This phenomenon could reflect two bacterial behaviour: the bacterial internalization by host cells and the biofilm formation. In this study, we studied different clinical strains of C. acnes. We noticed that C. acnes isolated from PJIs form 2 fold-more biofilm than the strains isolated from a normal skin in two models (Crystal violet staining and fluorescent microscopy (p=0.04 and p=0.02, respectively, Mann-Whitney test). We did not observe any difference in the internalization rate of those strains by osteoblasts. However, the quantity of biofilm formed by C. acnes before and after the internalization was compared. A significant increase in biofilm formation was observed for the strains isolated from the skin (x2.3±0.07; p=0.008, Mann-Whitney test). However, the hydrophobicity of the skin strains is significantly less important than for the PJIs strains (24.8±13% vs 56.6±12% respectively; p=0.003, Mann-Whitney test) but this did not change after internalization suggesting that there is no cell wall evolution. In conclusion, we studied for the first time the impact of bacterial internalization by osteoblasts on the virulent behaviour of C. acnes, which could explain the hided pathogenicity of this commensal bacterium

Objectives. The purpose of this study was to examine the bactericidal efficacy of hydrogen peroxide (H. 2. O. 2. ) on Cutibacterium acnes (C. acnes). We hypothesize that H. 2. O. 2. reduces the bacterial burden of C. acnes. Methods. The effect of H. 2. O. 2. was assessed by testing bactericidal effect, time course analysis, growth inhibition, and minimum bactericidal concentration. To assess the bactericidal effect, bacteria were treated for 30 minutes with 0%, 1%, 3%, 4%, 6%, 8%, or 10% H. 2. O. 2. in saline or water and compared with 3% topical H. 2. O. 2. solution. For time course analysis, bacteria were treated with water or saline (controls), 3% H. 2. O. 2. in water, 3% H. 2. O. 2. in saline, or 3% topical solution for 5, 10, 15, 20, and 30 minutes. Results were analyzed with a two-way analysis of variance (ANOVA) (p < 0.05). Results. Minimum inhibitory concentration of H. 2. O. 2. after 30 minutes is 1% for H. 2. O. 2. prepared in saline and water. The 3% topical solution was as effective when compared with the 1% H. 2. O. 2. prepared in saline or water. The controls of both saline and water showed no reduction of bacteria. After five minutes of exposure, all mixtures of H. 2. O. 2. reduced the percentage of live bacteria, with the topical solution being most effective (p < 0.0001). Maximum growth inhibition was achieved with topical 3% H. 2. O. 2. . Conclusion. The inexpensive and commercially available topical solution of 3% H. 2. O. 2. demonstrated superior bactericidal effect as observed in the minimum bactericidal inhibitory concentration, time course, and colony-forming unit (CFU) inhibition assays. These results support the use of topical 3% H. 2. O. 2. for five minutes before surgical skin preparation prior to shoulder surgery to achieve eradication of C. acnes for the skin. Cite this article: P. Hernandez, B. Sager, A. Fa, T. Liang, C. Lozano, M. Khazzam. Bactericidal efficacy of hydrogen peroxide on Cutibacterium acnes. Bone Joint Res 2019;8:3–10. DOI: 10.1302/2046-3758.81.BJR-2018-0145.R1

Introduction. Multiple studies have identified Cutibacterium acnes (C.acnes) and other microbes in intervertebral disc tissue using 16S DNA Sequencing and microbial cultures. However, it remains unclear whether these bacteria are native to the discs or result from perioperative contamination. Our study aimed to detect Gram-positive bacteria in non-herniated human disc samples and explore correlations with Toll-like receptors (TLR) 2, TLR4, NLRP3, and Gasdermin D. Methods. Immunohistochemical staining was conducted on 75 human IVD samples for Gram-positive bacteria, S. aureus, C.acnes, TLR2, TLR4, NLRP3, and Gasdermin D. Cell detection and classification were performed using QuPath. NP cells were treated with Lipopolysaccharide (LPS) and Peptidoglycan (PGN) in monolayer and alginate beads for up to 72 hours, followed by secretome analysis using Luminex. Statistical analysis included Kruskal-Wallis, Dunn's multiple comparison test, and Pearson correlation. Results. Immunohistochemical staining revealed Gram-positive bacteria exclusively within cells, with C. acnes positivity ranging from 5–99% and correlating with patient age (r=0.41, p= 0.007). TLR2 positivity ranged from 5–99% and TLR4 from 3–72%, showing a strong correlation (r= 0.62, p= 1.5e-006). Females with mid-degenerative grades exhibited significantly decreased TLR2 expression compared to those without degeneration signs. Treatment with LPS and PGN increased catabolic cyto- and chemokines associated with IVD degeneration. Conclusion. In conclusion, this study confirms Gram-positive bacteria presence in non-herniated human disc samples and highlights their role in triggering a catabolic response in disc cells. No conflicts of interest.  . Sources of funding. This project is part of the Disc4All Training network to advance integrated computational simulations in translational medicine, applies to intervertebral disc degeneration and funded by Horizon 2020 (H2020-MSCA-ITN-ETN-2020 GA: 955735)

Aim. The aim of this study was to develop an in-house multiplex PCR real-time assay on the LightCycler 480 system (Roche, Basel, Switzerland) with the aim of rapid detection of common pathogens in prosthetic joint infections (PJI), followed by validation on clinical samples (sonication fluid and tissue biopsies) routinely collected for PJI diagnosis. Methods. Using the PrimerQuest and CLC WorkBench tool, we designed six primer sets with specific fluorescently labelled TaqMan probes for the nuc gene in different Staphylococcus species (S. aureus, S. epidermidis, S. capitis, S. lugdunensis, S. hominis, S. haemolyticus). In addition, primers previously developed by Renz et al. (2022) for C. acnes were integrated into our assay with internal control of isolation, leading to the development of specific mPCR assay with seven included targets. Analytical sensitivity and specificity were evaluated using reference bacterial strains. To determine the assay's limit of detection (LOD), we conducted serial dilutions of eluates containing known concentrations of bacterial DNA copies/µl. The overall LOD in spiked clinical samples, including sample preparation and DNA isolation on MagnaPure24, was measured through 10-fold serial dilutions (from 10. 9. to 10. -1. CFU/ml) including additional dilutions of 5000, 500, 50 and 5 CFU/ml. Results. The results with LOD in serial dilutions of eluates and spiked clinical samples, together with analytical sensitivity and specificity, are shown in Table 1. Conclusion. The mPCR assay showed excellent analytical sensitivity and specificity, but with considerably lower LOD after sample preparation and further DNA isolation in spiked clinical samples. Although still promising in diagnostics of acute infections, the use of mPCR could be challenging in chronic, low-grade infections with lower microbial burden. Nevertheless, PCR offers significant advantages in terms of speed and can shorten the time to result, especially for C. acnes infections. Additionally, it represents a promising complementary approach in patients with suspected PJI on antibiotic therapy with negative culture results. For any tables or figures, please contact the authors directly

Aims. The outcomes of patients with unexpected positive cultures (UPCs) during revision total hip arthroplasty (THA) and total knee arthroplasty (TKA) remain unknown. The objectives of this study were to establish the prevalence and infection-free implant survival in UPCs during presumed aseptic single-stage revision THA and TKA at mid-term follow-up. Methods. This study included 297 patients undergoing presumed aseptic single-stage revision THA or TKA at a single treatment centre. All patients with at least three UPCs obtained during revision surgery were treated with minimum three months of oral antibiotics following revision surgery. The prevalence of UPCs and causative microorganisms, the recurrence of periprosthetic joint infections (PJIs), and the infection-free implant survival were established at minimum five years’ follow-up (5.1 to 12.3). Results. Of the 297 patients undergoing aseptic revisions, 37 (12.5%) had at least three UPCs obtained during surgery. The UPC cohort included 23 males (62.2%) and 14 females (37.8%), with a mean age of 71.2 years (47 to 82). Comorbidities included smoking (56.8%), hypertension (48.6%), diabetes mellitus (27.0%), and chronic renal impairment (13.5%). The causative microorganisms included Staphylococcus epidermidis (49.6%), Bacillus species (18.9%), Micrococcus species (16.2%), and Cutibacterium acnes (16.2%). None of the study patients with UPCs developed further PJIs or required further surgical intervention during follow-up. Conclusion. The prevalence of UPCs during presumed aseptic revision THA and TKA was 12.5%. The most common causative microorganisms were of low virulence, and included S. epidermidis, Bacillus species, Micrococcus species, and C. acnes. Microorganism-specific antibiotic treatment for minimum three months’ duration of UPCs in presumed aseptic revision arthroplasty was associated with excellent infection-free implant survival at mid-term follow-up. Cite this article: Bone Jt Open 2024;5(10):832–836

Aim. The aim of our study was to identify pathogens involved in septic knee arthritis after ACLR and to describe clinical features, treatment and outcome of infected patients. Methods. We conducted a retrospective observational study including all patients with ACLR infection in 3 orthopedic centers sharing the same infectious disease specialists. Results. During a seven-year period (2011–2017) we identified 74 infected patients among 9858 patients who had ACLR (incidence rate = 0.0075). Fourteen patients had polymicrobial infection. We identified 89 pathogens. Twenty four patients (34.4 %) were infected with S. aureus (27% of all isolates)(only one oxacillin-resistant strain). C. acnes was the second most frequent pathogen, identified in 14 patients (18.9%) (15.7% of all isolates). S. lugdunensis was identified in 9 patients (12.2%) (10.1% of all isolates). S. caprae was as frequent as S. epidermidis identified in 8 patients each (10.8%) (9 % of all isolates for each). No strain of S. lugdunensis and S. caprae was resistant to oxacillin, levofloxacin or rifampicin. Ten patients infected by C. acnes, 8 infected by S. lugdunensis, and 7 infected by S. caprae had an early acute infection. In all cases but one an arthroscopic lavage was performed, in 14 cases two lavages were required and in 4, 3 lavages. All patients infected by a strain susceptible to levofloxacin and rifampicin, including those with C. acnes, S. caprae and S. lugdunensis infection, were treated with an oral combination of levofloxacin and rifampicin, after a couple of days of IV empirical treatment with vancomycin and a broad spectrum beta-lactam. The median duration of treatment was 6 weeks. Seventy one patients were considered cured. Conclusions. To our knowledge this is the largest reported series of infection after ACLR. S. aureus is the main pathogen (27% of all strains). C. acnes, S. lugdunensis and S. caprae accounted for almost 35% of pathogens and 38% of infections. A conservative strategy consisting in arthroscopic lavage(s) and a 6-week treatment with levofloxacin and rifampicin was effective

Aim. To evaluate whether sonication of implant material and subsequent culturing add clinical relevance to culturing of tissue biopsies for improved antibiotic treatment in treatment of bone and joint infection. Method. A retrospective examination of patients’ charts and microbiological analyses in patients who had explanted material (plates, screws, k-wires and prostheses) send for sonication between December 2020 and April 2022. Results. 77/143 (54 %) patients had complete agreement between the cultures from tissue biopsies and sonication fluid. 66/143 (46 %) patients had partial or no agreement between the cultures from tissue biopsies and sonication fluid. Of the 66 patients, 31 (47 %) had a culture positive sonication fluid and tissue biopsies that were positive with one or more bacterial isolates. 26/66 (39 %) patients had a culture positive sonication fluid and tissue biopsies that were negative. 9/66 (14 %) patients had negative sonication fluid and positive tissue biopsies. Of the 26 patients with culture positive sonication fluid and culture negative tissue biopsies, virulent bacteria were found in 5 (19 %) patients, making the diagnosis and treatment of infection straight forward. The remaining 21 (81 %) patients had C. acnes, S. epidermidis and CoNS in the sonication fluid, which made the diagnosis less evident but none the less gave the clinician a relevant treatment option. Conclusion. In this study a high concordance was found between cultures from tissue biopsies and sonication fluid. Additionally, in a small group of patients with culture negative tissue biopsy, the culture of sonication fluid was essential to the identification infections agent. This indicates that culture of sonication fluid is an important diagnostic tool in bone and joint infection, especially in the absence of positive tissue cultures

Aim. Periprosthetic joint infection (PJI) is one of the most devastating complications after joint replacement. It is associated with high morbidity and economic burden when misdiagnosed as an aseptic failure. Among all cases of PJI, up to 25% could yield negative cultures. Conversely, among cases of aseptic failures, up to 30% may actually be undiagnosed PJIs. In PJIs microbiological diagnosis is a key step for successful treatment. Sonication of the removed prosthesis is more sensitive than conventional periprosthetic-tissue culture, especially in patients who received antimicrobial therapy before surgery. This study aimed to compare the diagnostic value of classic sonication fluid cultures (SF-C) and sonication fluid incubation in blood culture bottle (SF-BCB). Method. Between 2016 and 2018 we analysed 160 revision procedures of joint arthroplasties. For each procedure, at least 5 microbiological and multiple histopathological samples were harvested, and explant sonication was performed which was further analysed by SF-C and SF-BCB. For SF-C classical cultivation of sonication fluid was performed. While for SF-BCB, 10 mL of sonication fluid was inoculated into aerobic and anaerobic lytic blood culture bottles. The definite diagnosis of PJI was based on the EBJIS definition. Results. Among 160 revisions, 59 PJIs were identified, 15 patients were treated with the debridement and implant retention, 7 patients with the one-stage and 35 with the two-stage exchange, remaining 2 were partial revisions. The sensitivity of SF-C and SF-BCB were 81.5% and 94.9%, respectively. The mismatch of microbe identification was observed in 5 cases. We observed positive SF-C while negative SF-BCB in 4 cases, among them having 2 positive histology. While 12 patients have negative SF-C and positive SF-BCB, among them 3 have positive and 6 negative histology. Among these 12 patients, typical low-grade microbes were identified in 9 cases (5 cases of C. acnes, 3 cases of S. epidermidis, and 1 case of S. capitis). Conclusions. The weakest point in all PJI diagnostic criteria is their sensitivity. SF-BCB demonstrates higher sensitivity in diagnosing PJI compared to SF-C. Therefore, it appears prudent to incorporate SF-BCB into the diagnostic protocol for all patients exhibiting either low-grade PJI symptoms or experiencing undiagnosed, presumably aseptic failures, where the likelihood of misdiagnosing infection is greatest

Aim. Diagnosing or excluding a chronic prosthetic joint infection (PJI) prior to revision surgery can be a clinical challenge. To enhance accuracy of diagnosis, several biomarkers were introduced in recent years, but most are either expensive or not available as a rapid test. We compared the diagnostic accuracy of leucocyte esterase (€0.20 per sample), calprotectin (€20 per sample) and alpha defensin (€200 per sample). Method. We prospectively evaluated PJI patients with chronic pain with or without prosthetic loosening between 2017 and 2018. Synovial fluid was collected prior to revision surgery. Leucocyte esterase was measured using a reagent strip (2+ considered as positive), and calprotectin and alpha defensin were measured using a lateral flow immunoassay. Intraoperative cultures (5 periprosthetic tissue samples, synovial fluid and sonication fluid) incubated for 9 days, were used as gold standard. At least two positive cultures of low-grade microorganisms with the same antibiogram were required to diagnose PJI. Results. A total of 19 patients were included (knee =11, hip =8). None of the patients were treated with antibiotics prior to revision surgery. A PJI was diagnosed in 8 patients (42.1%). The diagnostic accuracy of leucocyte esterase vs. calprotectin vs. alpha defensin was as follows; sensitivity 50.0% vs. 87.5% vs. 87.5%, specificity 81.8% vs. 90.9% vs. 100%, positive predictive value 60.0% vs. 87.5% vs. 100% and negative predictive value 75.0% vs. 90.9% vs. 91.6%, respectively. Both calprotectin and alpha defensin were false negative in one PJI caused by Cutibacterium acnes. The other two C. acnes PJIs were correctly diagnosed with both tests. Conclusions. Calprotectin is as accurate as alpha defensin in excluding a chronic PJI at 10% of the costs. Future studies with a large number of patients are necessary to analyze its diagnostic accuracy in very low-grade infections, in particularly caused by C. acnes