The antimicrobial fibupeptide lugdunin forms water-filled channel structures in lipid membranes.

Recently, a novel cyclo-heptapeptide composed of alternating D,L-amino acids and a unique thiazolidine heterocycle, called lugdunin, was discovered, which is produced by the nasal and skin commensal Staphylococcus lugdunensis. Lugdunin displays potent antimicrobial activity against a broad spectrum of Gram-positive bacteria, including challenging-to-treat methicillin-resistant Staphylococcus aureus (MRSA). Lugdunin specifically inhibits target bacteria by dissipating their membrane potential. However, the precise mode of action of this new class of fibupeptides remains largely elusive. Here, we disclose the mechanism by which lugdunin rapidly destabilizes the bacterial membrane potential using an in vitro approach. The peptide strongly partitions into lipid compositions resembling Gram-positive bacterial membranes but less in those harboring the eukaryotic membrane component cholesterol. Upon insertion, lugdunin forms hydrogen-bonded antiparallel ß-sheets by the formation of peptide nanotubes, as demonstrated by ATR-FTIR spectroscopy and molecular dynamics simulations. These hydrophilic nanotubes filled with a water wire facilitate not only the translocation of protons but also of monovalent cations as demonstrated by voltage-clamp experiments on black lipid membranes. Collectively, our results provide evidence that the natural fibupeptide lugdunin acts as a peptidic channel that is spontaneously formed by an intricate stacking mechanism, leading to the dissipation of a bacterial cell's membrane potential.

Molecular characterization of lugdunin inactivation mechanisms and their association with Staphylococcus lugdunensis genetic types.

BACKGROUND AND PURPOSE: Our previous studies showed that lugdunin activities are associated with Staphylococcus lugdunensis genotypes, and most isolates do not exhibit lugdunin activity. As a continuation of our previous analysis, we focused on the reasons for defects in lugdunin production in S. lugdunensis clinical isolates. METHODS: A comparative analysis of 36 S. lugdunensis whole genome sequencing data revealed three major mutation types, unknown deletion mechanism that caused most of lug operon genes lost, mobile genetic element (MGE) insertion, and nonsense mutations, which potentially damaged lugdunin production. A total of 152 S. lugdunensis clinical isolates belonging to lugdunin nonproducers were further examined for the above three mutation types. PCR products were sequenced to examine these variations. RESULTS: Forty-six of the 152 isolates were CRISPR-Cas IIC isolates, including 26 ST27, 14 ST4, and 6 ST29 isolates; further investigation confirmed that all of their lug operons had lost almost all lug operon genes except lugM. An IS256 insertion in lugA was identified in 16 isolates, and most isolates (15 over 16) belonged to ST3. In addition, three nonsense mutations caused by single nucleotide substitutions (an adenine deletion in lugB at the 361th and 1219th nucleotides and an adenine deletion in lugC at the 1612nd nucleotide) that were frequently observed among 36 S. lugdunensis whole genome sequencing data were further observed in our clinical isolates. These three nonsense mutations were frequently found in most of CRISPR-Cas IIIA strains, especially in ST6 isolates. CONCLUSION: Our findings suggest that the mechanisms affecting lugdunin production are associated with S. lugdunensis molecular types.

Characterization of haloacid dehalogenase superfamily acid phosphatase from Staphylococcus lugdunensis.

The haloacid dehalogenase superfamily implicated in bacterial pathogenesis comprises different enzymes having roles in many metabolic pathways. Staphylococcus lugdunensis, a Gram-positive bacterium, is an opportunistic human pathogen causing infections in the central nervous system, urinary tract, bones, peritoneum, systemic conditions and cutaneous infection. The haloacid dehalogenase superfamily proteins play a significant role in the pathogenicity of certain bacteria, facilitating invasion, survival, and proliferation within host cells. The genome of S. lugdunensis encodes more than ten proteins belonging to this superfamily. However, none of them have been characterized. The present work reports the characterization of one of the haloacid dehalogenase superfamily proteins (SLHAD1) from Staphylococcus lugdunensis. The functional analysis revealed that SLHAD1 is a metal-dependent acid phosphatase, which catalyzes the dephosphorylation of phosphorylated metabolites of cellular pathways, including glycolysis, gluconeogenesis, nucleotides, and thiamine metabolism. Based on the substrate specificity and genomic analysis, the physiological function of SLHAD1 in thiamine metabolism has been tentatively assigned. The crystal structure of SLHAD1, lacking 49 residues at the C-terminal, was determined at 1.7 Å resolution with a homodimer in the asymmetric unit. It was observed that SLHAD1 exhibited time-dependent cleavage at a specific point, occurring through a self-initiated process. A combination of bioinformatics, biochemical, biophysical, and structural studies explored unique features of SLHAD1. Overall, the study revealed a detailed characterization of a critical enzyme of the human pathogen Staphylococcus lugdunensis, associated with several life-threatening infections.

Searching for Virulence Factors among Staphylococcus lugdunensis Isolates from Orthopedic Infections: Correlation of ß-hemolysin, hemolysin III, and slush Genes with Hemolytic Activity and Synergistic Hemolytic Activity.

Staphylococcus lugdunensis is an emerging high-virulent pathogen. Here, the presence and expression of virulence genes (icaA, fbl, vwbl, fbpA, slush A, B and C, and genes of the putative ß-hemolysin and hemolysin III) and the ability to induce synergistic hemolytic activity and hemolysis after 24, 48 and 72 h were investigated in a collection of twenty-two S. lugdunensis clinical isolates. The collection of isolates, mainly from implant orthopedic infections, had previously been grouped by ribotyping/dendrogram analysis and studied for biofilm matrices, biomasses and antibiotic resistances. Two isolates, constituting a unique small ribogroup sharing the same cluster, exhibited an amplicon size of the slush operon (S. lugdunensis synergistic hemolysin) which was shorter than the expected 977 bp. This outcome can predict the genetic lineage of the S. lugdunensis strains. One isolate (cra1342) presented two deletions: one of 90 bp in slush A and the other of 91 bp in slush B. Another isolate (N860314) showed a single 193 bp deletion, which encompassed part of the slush B terminal sequence and most of slush C. The isolate N860314 was devoid of hemolytic activity after 24 h, and the first consideration was that the deleted region deals with the coding of the active enzymatic site of the slush hemolysin. On the other hand, cra1342 and N860314 isolates with different slush deletions and with hemolytic activity after 24 and 48 h, respectively, could have replaced the hemolytic phenotype through other processes.

Impact of clonal lineages on susceptibility of Staphylococcus lugdunensis to chlorhexidine digluconate and chloride benzalkonium.

BACKGROUND: Little is known about susceptibility of Staphylococcus lugdunensis to antiseptics. The objective of this study was to evaluate, at the molecular and phenotypic level, the susceptibility of 49 clinical S. lugdunensis strains (belonging to the seven clonal complexes [CCs] defined by multilocus sequence typing) to two antiseptics frequently used in healthcare settings (chlorhexidine digluconate [CHX] and chloride benzalkonium [BAC]). RESULTS: The minimum inhibitory concentrations (MICs), by broth microdilution method, varied for BAC from 0.25 mg/L to 8 mg/L (MIC50 = 1 mg/L, MIC90 = 2 mg/L) and for CHX from 0.5 mg/L to 2 mg/L (MIC50 = 1 mg/L, MIC90 = 2 mg/L). The BAC and CHX minimum bactericidal concentrations (MBCs) varied from 2 mg/L to 8 mg/L (MBC50 = 4 mg/L, MBC90 = 8 mg/L) and from 2 mg/L to 4 mg/L (MBC50 and MBC90 = 4 mg/L), respectively. A reduced susceptibility to CHX (MIC = 2 mg/L) was observed for 12.2% of the strains and that to BAC (MIC ≥ 4 mg/L) for 4.1%. The norA resistance gene was detected in all the 49 isolates, whereas the qacA gene was rarely encountered (two strains; 4.1%). The qacC, qacG, qacH, and qacJ genes were not detected. The two strains harboring the qacA gene had reduced susceptibility to both antiseptics and belonged to CC3. CONCLUSION: The norA gene was detected in all the strains, suggesting that it could belong to the core genome of S. lugdunensis. S. lugdunensis is highly susceptible to both antiseptics tested. Reduced susceptibility to BAC and CHX was a rare phenomenon. Of note, a tendency to higher MICs of BAC was detected for CC3 isolates. These results should be confirmed on a larger collection of strains.

fbl-typing and Antimicrobial Resistance Analysis of Staphylococcus lugdunensis.

BACKGROUND: A broad variety of infections, ranging from skin infections to infective endocarditis can be caused by Staphylococcus lugdunensis. Bacterial virulence is often related to virulence genes, so we sought to investigate the relationship between virulence genes and the pathogenicity of S. lugdunensis and to explore an appropriate typing method to distinguish different pathogenic phenotypes of S. lugdunensis. METHODS: We describe the distribution of several virulence genes in different infection types in an attempt to find the relationship between virulence genes and pathogenicity. Subsequently, we make the Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) dendrogram and fbl-typing were performed using BioNumerics software, tried to compare the correlation between different methods and the different infectious diseases, and antimicrobial resistance of the strains, in order to obtain the epidemic characteristics and antimicrobial resistance information of S. lugdunensis based on a molecular approach. RESULTS: The results of virulence genes showed that the seven virulence genes we have described existed in most strains, and there was no significant correlation between virulence gene distribution and infection type. Compared with the MALDI-TOF MS dendrogram, we found that fbl-typing could better correspond to the pathogenic phenotype, with better recognition and reproducibility. In the phylogenetic tree constructed in the fbl R-region, we found a tendency for some infection types to be distributed in clusters, new type 3 was the most dominant fbl-type, followed by fbl47b. Bone and joint infection isolates and ear infection isolates were significantly clustered together, in addition, all the oxacillin-resistant isolates were concentrated in fbl-type fbl45j and fbl47b. CONCLUSIONS: In this study, we found no significant correlation between virulence genes from S. lugdunensis isolates and the site of infection. The fbl-typing has the characteristics of convenient operation, low cost, high repeatability, and is preferable to indicate the pathogenic phenotype. Based on fbl-typing, we described the epidemiological characteristics of S. lugdunensis in a hospital and supplemented the data for fbl-typing. We recommend that fbl-typing method be extended and supplemented.

Staphylococcus lugdunensis, a Not So Infrequent Pathogen in Orthopedic Surgery.

Abstract Background: Staphylococcus lugdunensis is a coagulase-negative staphylococci that is considered normal skin microbiota. It has been described as a cause of soft tissue infections but is not a common micro-organism as the cause of orthopedic surgery-related infections. This study describes the characteristics, treatment, and results of musculoskeletal infection by Staphylococcus lugdunensis treated in our institution. Methods: We performed a descriptive, retrospective observational study. Clinical records of all musculoskeletal infections treated in our department between 2012 and 2020 were reviewed. We selected those patients with a positive monomicrobial culture for Staphylococcus lugdunensis. Risk factors for infection, patient's medical records, previous surgery performed, time from surgery to infection, culture antibiogram, antibiotic and surgical treatment for the infection, and recovery rate were registered for the analysis. Results: Of the 1,482 patients with musculoskeletal infections diagnosed in our institution, 22 had a monomicrobial positive culture of Staphylococcus lugdunensis after an orthopedic surgery representing 1.5% of all orthopedic infections. Ten patients underwent arthroplasty, six underwent fractures synthesis, three had foot surgeries, two had anterior cruciate ligament reconstructions, and one had spine surgery. All patients needed surgery and antibiotic treatment with a mean of two surgeries. The most used antibiotic scheme was levofloxacin-rifampicin. The mean follow-up was 36 months. Ninety-six percent of the patients achieved a complete clinical and analytical recovery. Conclusions: Although musculoskeletal infections caused by Staphylococcus lugdunensis are not common, we have observed a statistically significant incidence increase of Staphylococcus lugdunensis in recent years. If managed with appropriately aggressive surgical treatment and correct antibiotic therapy, good results can be obtained.

Characteristics and outcomes in adult patients with Staphylococcus lugdunensis bacteremia compared to patients with Staphylococcus epidermidis and Staphylococcus aureus bacteremia: a retrospective study in a 16-year period at the university hospital, Japan.

BACKGROUND: Staphyococcus lugudnensis (S. lugdunensis) is one of coagulase-negative Staphylococcus species with a potential to cause invasive infections. Few studies have evaluated the characteristics and outcomes of patients with S. lugdunensis bacteremia (SLB) compared with those of patients with Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus) bacteremia. METHODS: We performed a single-center retrospective case-control study of patients aged ≥ 18 who had SLB with at least two sets of positive blood cultures at the Kyoto University Hospital, Japan, from January 2005 to June 2022. Patients who had S. epidermidis bacteremia (SEB) with at least two sets of positive blood cultures and those who had S. aureus bacteremia (SAB) with at least one set of positive blood cultures were randomly selected in a 1:5:5 (SLB:SEB:SAB) ratio. RESULTS: A total of 22 patients with SLB, 110 patients with SEB, and 110 patients with SAB were included. The proportions of infective endocarditis (IE) and metastatic infections were statistically higher in the SLB group than in the SEB group (14% vs. 2%, p < 0.01 and 18% vs. 5%, p 0.02, respectively) and were not significantly different between the SLB and SAB groups (14% vs. 5%, p 0.16 and 18% vs. 16%, p 0.78, respectively). The seven-day mortality was higher in the SLB group than in the SEB group (9% vs. 1%, p 0.02) and similar between the SLB and SAB groups (9% vs. 7%, p 0.77). CONCLUSIONS: The clinical course and outcome of SLB were worse than those of SEB and similar to those of SAB. Appropriate evaluation and treatment for SAB may be warranted in patients with SLB.

Novel biochemical aspects of lugdulysin, a Staphylococcus lugdunensis metalloprotease that inhibits formation and disrupts protein biofilm of methicillin-resistant Staphylococcus aureus.

Staphylococcus lugdunensis produces lugdulysin, a metalloprotease that may contribute to its virulence. This study aimed to evaluate the biochemical aspects of lugdulysin and investigate its effect on Staphylococcus aureus biofilms. The protease was isolated and characterized for its optimal pH and temperature, hydrolysis kinetics, and influence of metal cofactor supplementation. The protein structure was determined via homology modeling. The effect on S. aureus biofilms was assessed by the micromethod technique. The protease optimal pH and temperature were 7.0 and 37 °C, respectively. EDTA inhibited protease activity, confirming it as a metalloprotease. Lugdulysin activity was not recovered by divalent ion supplementation post-inhibition, and supplementation with divalent ions did not change enzymatic activity. The isolated enzyme was stable for up to 3 h. Lugdulysin significantly inhibited the formation and disrupted preestablished protein-matrix MRSA biofilm. This preliminary study indicates that lugdulysin has a potential role as a competition mechanism and/or modulation of staphylococcal biofilm.

Biofilm formation is associated with poor outcome in prosthetic joint infections caused by Staphylococcus lugdunensis.

BACKGROUND: Staphylococcus lugdunensis has been described as a pathogen of increasing importance in prosthetic joint infections (PJI). Our aim was to describe the clinical presentation of PJI caused by S. lugdunensis, and to correlate the biofilm-forming ability of the bacterial isolates to clinical outcome. METHOD: S. lugdunensis isolates from PJI episodes during 2015-2019 were included and analysed for biofilm formation using a microtiter plate assay. Medical records from the corresponding patients were reviewed. RESULTS: We identified 36 patients with PJI caused by S. lugdunensis during the study period. Early postoperative PJIs were most frequent (n = 20, 56%). Surgical intervention was performed in a majority of the patients (n = 33, 92%), and the dominating type of antibiotic treatment was a combination of rifampicin and ciprofloxacin (n = 27, 75%). The treatment success-rate was 81% (n = 29). All isolates causing PJI were able to form biofilm in vitro. Biofilm formation was significantly stronger in isolates causing relapsing vs non-relapsing PJI (mean OD550 3.1 ± 0.23 vs 1.14 ± 0.73 p = .001) and strong biofilm formation was also associated with late acute hematogenic PJI (mean OD550 1.8 ± 0.93 vs. 0.93 ± 0.81, p = .01). CONCLUSION: Strong biofilm production in S. lugdunensis isolates was associated with relapse in PJI.

Characterization of oxacillin-resistant Staphylococcus lugdunensis isolated from sterile body fluids in a medical center in Taiwan: A 12-year longitudinal epidemiological study.

BACKGROUND: In this study, our objective was to characterize Staphylococcus lugdunensis isolated from sterile body fluids (SBFs) in a medical center in Taiwan between 2009 and 2020. METHODS: We used MALDI-TOF MS, disk diffusion testing, agar dilution assay, SCCmec typing, and antibiotic resistance gene screening to identify and investigate the characteristics of oxacillin-resistant S. lugdunensis (ORSL). RESULTS: A total of 438 S. lugdunensis isolates were collected and 146 (33.3%) isolates were identified as ORSL. SCCmec type V was dominant (65.7%) in our ORSL isolates, followed by SCCmec type II (18.5%), and type IV (8.9%). After 2013, a slight increase in SCCmec types IV and V was revealed. Moreover, all ORSL isolates with type II and untypable SCCmec were highly resistant to oxacillin (MIC >32 µg/mL), compared to ORSL that had SCCmec types IV, V, and VT. All 146 ORSL isolates were resistant to penicillin and susceptible to teicoplanin and vancomycin. High resistance rates of ORSL to clindamycin (43.2%), erythromycin (43.2%), gentamicin (78.1%) and tetracycline (46.6%) was observed. Moreover, only two (1.4%) and six (4.1%) ORSL isolates were resistant to trimethoprim/sulfamethoxazole and ciprofloxacin, respectively. The erythromycin-resistant ORSL isolates mostly exhibited constitutive MLSB resistant phenotype (61/63, 96.8%) and contained either ermC alone (27/63, 42.9%) or a combination of ermC with ermA (28/63, 44.4%). CONCLUSION: Our present study showed a stable rate of ORSL from SBFs during 2009-2020. Moreover, teicoplanin, vancomycin, trimethoprim/sulfamethoxazole, and ciprofloxacin were shown to be highly efficient for the treatment of ORSL in vitro.

Staphylococcus lugdunensis bacteremia: clinical implications of single set positive blood cultures.

Staphylococcus lugdunensis is exceptionally virulent among the coagulase-negative Staphylococcus species, but the clinical significance of single-positive bacteremia of S. lugdunensis remains uncertain. We investigated S. lugdunensis bacteremia cases over 10 years. Of the 49 cases included, 12 had multiple-positive blood cultures and 37 had single-positive blood cultures. Antimicrobial therapy was given to over 80% of both groups, whereas the duration of therapy was significantly longer in the multiple-positive group. The overall 30-day and 90-day mortality rates were 13.3% and 18.2%, and 36.7% and 18.2% for single and multiple-positive patients, respectively. Five single-positive patients without therapy did not have severe infection, presumed source of infection, or culture positivity within 20 hours, but all defervesced within 2 days and were alive at 30 days. While the clinical spectrum of single-positive S. lugdunensis bacteremia is broad, antimicrobial therapy may be withheld without adverse clinical consequences in a subset of low-risk patients.

Narrowband ultraviolet B response in cutaneous T-cell lymphoma is characterized by increased bacterial diversity and reduced Staphylococcus aureus and Staphylococcus lugdunensis.

Skin microbiota have been linked to disease activity in cutaneous T-cell lymphoma (CTCL). As the skin microbiome has been shown to change after exposure to narrowband ultraviolet B (nbUVB) phototherapy, a common treatment modality used for CTCL, we performed a longitudinal analysis of the skin microbiome in CTCL patients treated with nbUVB. 16S V4 rRNA gene amplicon sequencing for genus-level taxonomic resolution, tuf2 amplicon next generation sequencing for staphylococcal speciation, and bioinformatics were performed on DNA extracted from skin swabs taken from lesional and non-lesional skin of 25 CTCL patients receiving nbUVB and 15 CTCL patients not receiving nbUVB from the same geographical region. Disease responsiveness to nbUVB was determined using the modified Severity Weighted Assessment Tool: 14 (56%) patients responded to nbUVB while 11 (44%) patients had progressive disease. Microbial α-diversity increased in nbUVB-responders after phototherapy. The relative abundance of Staphylococcus, Corynebacterium, Acinetobacter, Streptococcus, and Anaerococcus differentiated nbUVB responders and non-responders after treatment (q<0.05). Microbial signatures of nbUVB-treated patients demonstrated significant post-exposure depletion of S. aureus (q=0.024) and S. lugdunensis (q=0.004) relative abundances. Before nbUVB, responder lesional skin harboured higher levels of S. capitis (q=0.028) and S. warneri (q=0.026) than non-responder lesional skin. S. capitis relative abundance increased in the lesional skin of responders (q=0.05) after phototherapy; a similar upward trend was observed in non-responders (q=0.09). Post-treatment skin of responders exhibited significantly reduced S. aureus (q=0.008) and significantly increased S. hominis (q=0.006), S. pettenkoferi (q=0.021), and S. warneri (q=0.029) relative abundances compared to that of no-nbUVB patients. Staphylococcus species abundance was more similar between non-responders and no-nbUVB patients than between responders and no-nbUVB patients. In sum, the skin microbiome of CTCL patients who respond to nbUVB is different from that of non-responders and untreated patients, and is characterized by shifts in S. aureus and S. lugdunensis. Non-responsiveness to phototherapy may reflect more aggressive disease at baseline.

Clinical and microbiological characteristics of Staphylococcus lugdunensis.

PURPOSE OF REVIEW: This review provides an update on recent findings about the clinical and microbiological characteristics of Staphylococcus lugdunensis . RECENT FINDINGS: European Committee on Antimicrobial Susceptibility Testing (EUCAST) and Clinical and Laboratory Standards Institute (CLSI) differ in their methodology and breakpoints for the detection of penicillin and oxacillin resistance in S. lugdunensis . The EUCAST method for beta-lactamase detection recommends a 1-unit penicillin disk and has demonstrated superior performance compared to the 10-unit penicillin disk recommended by CLSI. A similar outcome has been previously reported in Staphylococcus aureus. In addition, there is emerging oxacillin resistance in some geographical areas. Of particular concern is that oxacillin resistance in mecA positive isolates may not be reliably detected by current cefoxitin breakpoints. SUMMARY: Coagulase negative staphylococci are now recognised as a heterogenous group of organisms that do not microbiologically or clinically behave the same way. The spectrum of clinical disease is species dependent and is particularly true for S. lugdunensis , which causes an array of clinical infections like that of S. aureus. Further studies are needed to assess the performance of phenotypic tests to detect resistance, to ensure that appropriate antimicrobial therapy is delivered to patients.

Staphylococcus lugdunensis prosthetic joint infection: A multicentric cohort study.

OBJECTIVES: To describe Staphylococcus lugdunensis prosthetic joint infection (PJI) management and outcome. METHODS: Adults with proven S. lugdunensis PJI were included in a multicentric retrospective cohort. Determinants for failure were assessed by logistic regression and treatment failure-free survival curve analysis (Kaplan-Meier). RESULTS: One hundred and eleven patients were included (median age 72.4 [IQR, 62.7-79.4] years), with a knee (n = 71, 64.0%) or hip (n = 39, 35.1%) PJI considered as chronic in 77 (69.4%) cases. Surgical management consisted in debridement, antibiotic with implant retention (DAIR; n = 60, 54.1%), two-stage (n = 28, 25.2%) or one-stage (n = 15, 13.5%) exchange. Total duration of antimicrobial therapy was 13.1 (IQR, 11.8-16.9) weeks. After a median follow-up of 99.9 (IQR, 53.9-178.1) weeks, 22 (19.8%) S. lugdunensis-related treatment failures were observed. Independent determinants for outcome were diabetes (OR, 3.741; p = 0.036), sinus tract (OR, 3.846; p = 0.032), DAIR (OR, 3.749; p = 0.039) and rifampin-based regimen (OR, 0.319; p = 0.043). Twenty-four (40.0%) of the 60 DAIR-treated patients experienced treatment failure, with hip location (OR, 3.273; p = 0.048), delay from prosthesis implantation (OR, 1.012 per month; p = 0.019), pre-surgical CRP level >115 mg/L (OR, 4.800; p = 0.039) and mobile component exchange (OR, 0.302; p = 0.069) constituting additional determinants of outcome. CONCLUSIONS: Staphylococcus lugdunensis PJI are difficult-to-treat infections, with pivotal roles of an optimal surgical management.

A 92 protein inflammation panel performed on sonicate fluid differentiates periprosthetic joint infection from non-infectious causes of arthroplasty failure.

Periprosthetic joint infection (PJI) is a major complication of total joint arthroplasty, typically necessitating surgical intervention and prolonged antimicrobial therapy. Currently, there is no perfect assay for PJI diagnosis. Proteomic profiling of sonicate fluid has the potential to differentiate PJI from non-infectious arthroplasty failure (NIAF) and possibly clinical subsets of PJI and/or NIAF. In this study, 200 sonicate fluid samples, including 90 from subjects with NIAF (23 aseptic loosening, 35 instability, 10 stiffness, five osteolysis, and 17 other) and 110 from subjects with PJI (40 Staphylococcus aureus, 40 Staphylococcus epidermidis, 10 Staphylococcus lugdunensis, 10 Streptococcus agalactiae, and 10 Enterococcus faecalis) were analyzed by proximity extension assay using the 92 protein Inflammation Panel from Olink Proteomics. Thirty-seven of the 92 proteins examined, including CCL20, OSM, EN-RAGE, IL8, and IL6, were differentially expressed in PJI versus NIAF sonicate fluid samples, with none of the 92 proteins differentially expressed between staphylococcal versus non-staphylococcal PJI, nor between the different types of NIAF studied. IL-17A and CCL11 were differentially expressed between PJI caused by different bacterial species, with IL-17A detected at higher levels in S. aureus compared to S. epidermidis and S. lugdunensis PJI, and CCL11 detected at higher levels in S. epidermidis compared to S. aureus and S. agalactiae PJI. Receiver operative characteristic curve analysis identified individual proteins and combinations of proteins that could differentiate PJI from NIAF. Overall, proteomic profiling using this small protein panel was able to differentiate between PJI and NIAF sonicate samples and provide a better understanding of the immune response during arthroplasty failure.