Alex C. DiBartola

Polymorphisms in fibronectin binding protein A of Staphylococcus aureus are associated with infection of cardiovascular devices

Medical implants, like cardiovascular devices, improve the quality of life for countless individuals but may become infected with bacteria like Staphylococcus aureus. Such infections take the form of a biofilm, a structured community of bacterial cells adherent to the surface of a solid substrate. Every biofilm begins with an attractive force or bond between bacterium and substratum. We used atomic force microscopy to probe experimentally forces between a fibronectin-coated surface (i.e., proxy for an implanted cardiac device) and fibronectin-binding receptors on the surface of individual living bacteria from each of 80 clinical isolates of S. aureus. These isolates originated from humans with infected cardiac devices (CDI; n = 26), uninfected cardiac devices (n = 20), and the anterior nares of asymptomatic subjects (n = 34). CDI isolates exhibited a distinct binding-force signature and had specific single amino acid polymorphisms in fibronectin-binding protein A corresponding to E652D, H782Q, and K786N. In silico molecular dynamics simulations demonstrate that residues D652, Q782, and N786 in fibronectin-binding protein A form extra hydrogen bonds with fibronectin, complementing the higher binding force and energy measured by atomic force microscopy for the CDI isolates. This study is significant, because it links pathogenic bacteria biofilms from the length scale of bonds acting across a nanometer-scale space to the clinical presentation of disease at the human dimension.

Bonds between fibronectin and fibronectin-binding proteins on Staphylococcus aureus and Lactococcus lactis.

Bacterial cell-wall-associated fibronectin binding proteins A and B (FnBPA and FnBPB) form bonds with host fibronectin. This binding reaction is often the initial step in prosthetic device infections. Atomic force microscopy was used to evaluate binding interactions between a fibronectin-coated probe and laboratory-derived Staphylococcus aureus that are (i) defective in both FnBPA and FnBPB (fnbA fnbB double mutant, DU5883), (ii) capable of expressing only FnBPA (fnbA fnbB double mutant complemented with pFNBA4), or (iii) capable of expressing only FnBPB (fnbA fnbB double mutant complemented with pFNBB4). These experiments were repeated using Lactococcus lactis constructs expressing fnbA and fnbB genes from S. aureus. A distinct force signature was observed for those bacteria that expressed FnBPA or FnBPB. Analysis of this force signature with the biomechanical wormlike chain model suggests that parallel bonds form between fibronectin and FnBPs on a bacterium. The strength and covalence of bonds were evaluated via nonlinear regression of force profiles. Binding events were more frequent (p < 0.01) for S. aureus expressing FnBPA or FnBPB than for the S. aureus double mutant. The binding force, frequency, and profile were similar between the FnBPA and FnBPB expressing strains of S. aureus. The absence of both FnBPs from the surface of S. aureus removed its ability to form a detectable bond with fibronectin. By contrast, ectopic expression of FnBPA or FnBPB on the surface of L. lactis conferred fibronectin binding characteristics similar to those of S. aureus. These measurements demonstrate that fibronectin-binding adhesins FnBPA and FnBPB are necessary and sufficient for the binding of S. aureus to prosthetic devices that are coated with host fibronectin.

A Tactile Response in Staphylococcus aureus

It is well established that bacteria are able to respond to temporal gradients (e.g., by chemotaxis). However, it is widely held that prokaryotes are too small to sense spatial gradients. This contradicts the common observation that the vast majority of bacteria live on the surface of a solid substrate (e.g., as a biofilm). Herein we report direct experimental evidence that the nonmotile bacterium Staphylococcus aureus possesses a tactile response, or primitive sense of touch, that allows it to respond to spatial gradients. Attached cells recognize their substrate interface and localize adhesins toward that region. Braille-like avidity maps reflect a cells biochemical sensory response and reveal ultrastructural regions defined by the actual binding activity of specific proteins.

Correlation between histological outcome and surgical cartilage repair technique in the knee: A meta-analysis☆☆☆

BACKGROUND Compare histological outcomes after microfracture (MF), autologous chondrocyte implantation (ACI), and osteochondral autograft transfer (OATS). METHODS Literature review using PubMed MEDLINE, SCOPUS, Cumulative Index for Nursing and Allied Health Literature (CINAHL), and Cochrane Collaboration Library. Inclusion criteria limited to English language studies International Cartilage Repair Society (ICRS) grading criteria for cartilage analysis after ACI (autologous chondrocyte implantation), MF (microfracture), or OATS (osteochondral autografting) repair techniques. RESULTS Thirty-three studies investigating 1511 patients were identified. Thirty evaluated ACI or one of its subtypes, six evaluated MF, and seven evaluated OATS. There was no evidence of publication bias (Beggs p=0.48). No statistically significant correlation was found between percent change in clinical outcome and percent biopsies showing ICRS Excellent scores (R(2)=0.05, p=0.38). Percent change in clinical outcome and percent of biopsies showing only hyaline cartilage were significantly associated (R(2)=0.24, p=0.024). Mean lesion size and histological outcome were not correlated based either on percent ICRS Excellent (R(2)=0.03, p=0.50) or percent hyaline cartilage only (R(2)=0.01, p=0.67). Most common lesion location and histological outcome were not correlated based either on percent ICRS Excellent (R(2)=0.03, p=0.50) or percent hyaline cartilage only (R(2)=0.01, p=0.67). CONCLUSIONS Microfracture has poorer histologic outcomes than other cartilage repair techniques. OATS repairs primarily are comprised of hyaline cartilage, followed closely by cell-based techniques, but no significant difference was found cartilage quality using ICRS grading criteria among OATS, ACI-C, MACI, and ACI-P. LEVEL OF EVIDENCE IV, meta-analysis.

Maximum load to failure of high dose versus low dose gamma irradiation of anterior cruciate ligament allografts: A meta-analysis ☆ ☆☆

BACKGROUND The objective of this study was to systematically evaluate the existing literature to compare the biomechanical effects of low dose and high dose gamma irradiation on commonly used ACL allografts. METHODS A systematic search was performed in PubMed, Cumulative Index for Nursing and Allied Health Literature (CINAHL), Cochrane Reviews, SCOPUS, and SportDiscus. Nine studies were identified that met the following inclusion criteria: 1) controlled laboratory study, 2) investigation of standard allografts for anterior cruciate ligament reconstruction (ACLR), 3) gamma irradiation (dose reported) and a negative control group, and 4) mechanical loading (results reported). RESULTS Nine studies met all inclusion and exclusion criteria. There was a dose-dependent relationship between radiation and decreased mechanical tendon integrity. Low dose radiation (<2.5Mrad [Mrad]) showed graft weakening with an average of 4.3% decrease in load to failure (standardized mean difference [SMD], 0.23; 95% CI 0.216, 0.68; p=0.31), whereas high-dose radiation showed a significantly larger (32.4% average) decrease in load to failure (SMD, 1.79; 95% CI 1.194, 2.38; p<0.001). CONCLUSIONS Gamma irradiation has a negative effect on tendon allograft strength that is dose-dependent, with particularly large effects noted at irradiation doses of ≥2.5Mrad.

Biofilms in orthopedic infections: a review of laboratory methods

Bacterial infection after hardware implantation in orthopedic surgery is a devastating issue as it often necessitates increased hospital costs and stays, multiple revision surgeries, and prolonged use of antibiotics. Because of the nature of hardware implantation into the body, these infections are commonly in the form of attached biofilms. The current literature on a range of methodologies to study clinically explanted infected orthopedic hardware, with potential biofilm, in the laboratory setting is limited. General methods include traditional and advanced culturing techniques, microscopy imaging techniques, and techniques that manipulate genetic material. The future of diagnostic techniques for infected implants, innovative hardware design, and treatment solutions for patients all depend on the successful evaluation and characterization of clinical samples in the laboratory setting. This review provides an overview of current methods to study biofilms associated with orthopedic infections and insight into future directions in the field.

Endovascular Infections Caused by Methicillin-Resistant Staphylococcus aureus Are Linked to Clonal Complex-Specific Alterations in Binding and Invasion Domains of Fibronectin-Binding Protein A as Well as the Occurrence of fnbB

ABSTRACT Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections.

16S rRNA analysis provides evidence of biofilms on all components of three infected periprosthetic knees including permanent braided suture.

Bacterial biofilms are the main etiological agent of periprosthetic joint infections (PJI); however, it is unclear if biofilms colonize one or multiple components. Because biofilms can colonize a variety of surfaces, we hypothesized that biofilms would be present on all components. 16S ribosomal RNA (rRNA) gene sequencing analysis was used to identify bacteria recovered from individual components and non-absorbable suture material recovered from three PJI total knee revision cases. Bray-Curtis non-metric multidimensional scaling analysis revealed no significant differences in similarity when factoring component, material type, or suture versus non-suture material, but did reveal significant differences in organism profile between patients (P < 0.001) and negative controls (P < 0.001). Confocal microscopy and a novel agar encasement culturing method also confirmed biofilm growth on a subset of components. While 16S sequencing suggested that the microbiology was more complex than revealed by culture contaminating, bacterial DNA generates a risk of false positives. This report highlights that biofilm bacteria may colonize all infected prosthetic components including braided suture material, and provides further evidence that clinical culture can fail to sufficiently identify the full pathogen profile in PJI cases.

Interrater and intrarater reliability of arthroscopic measurements of articular cartilage defects in the knee

Background: Cartilage lesions of the knee are difficult to treat. Lesion size is a critical factor in treatment algorithms, and the accurate, reproducible sizing of lesions is important. In this study, we evaluated the interrater and intrarater reliability of, and correlations in relation to, various arthroscopic sizing techniques. Methods: Five lesions were created in each of 10 cadaveric knees (International Cartilage Repair Society grade 3C). Three orthopaedic surgeons used 4 techniques (visualization and use of a 3-mm probe, a simple metal ruler, and a sliding metallic ruler tool) to estimate lesion size. Repeated-measures data were analyzed using a mixed-effect linear model. The differences between observed and gold-standard (plastic mold) values were used as the response. Intraclass and interclass correlation coefficient (ICC) values for intrarater and interrater reliability were computed, as were overall correlation coefficients between measurements and gold standards. Results: The mean lesion size was 2.37 cm2 (range, 0.36 to 6.02 cm2). Rater, lesion location and size, and measurement method all affected the cartilage defect measurements. Surgeons underestimated lesion size, and measurements of larger lesions had a higher percentage of error compared with those of smaller lesions. When compared with plastic molds of lesions, 60.5% of surgeon measurements underestimated lesion size. Overall, the correlation between measurements and gold standards was strongest for the simple metal ruler method and weakest for the visualization method. Conclusions: Several factors may influence arthroscopic estimation of cartilage lesion size: the lesion location, measurement tool, surgeon, and defect size itself. The intrarater and interrater reliability was moderate to good using a 3-mm probe, sliding metallic ruler tool, or simple metal ruler and was fair to moderate using visualization only. Clinical Relevance: There is a need for more accurate methods of determining the size of articular cartilage lesions.

Anterior cruciate ligament reconstruction complicated by Propionibacterium acnes infection: case series

ABSTRACT Anterior cruciate ligament (ACL) reconstruction is one of the most commonly performed orthopaedic procedures. While generally successful, failure resulting in revision surgery is reported to be between 1.9 – 4.9%. However, when not related to traumatic re-injury, failure mechanism is poorly understood. One potential but understudied mechanism of ACL reconstruction failure is infection. We describe three patients with previous ACL reconstructions who later developed infection with Propionibacterium acnes. Two cases presented with knee pain and swelling, and one presented with instability. While only two of the three cases received antibiotic treatment, all three cases were free of knee and infectious symptomatology at most recent follow-up. P. acnes infection may represent one potential mechanism for biologic failure of ACL reconstruction and anaerobic operative cultures may be used to identify its presence.