Staphylococcus epidermidis serine–aspartate repeat protein G (SdrG) binds to osteoblast integrin alpha V beta 3

Staphylococcus epidermidis is the leading etiologic agent of orthopaedic implant infection. Contamination of the implanted device during insertion allows bacteria gain entry into the sterile bone environment leading to condition known as osteomyelitis. Osteomyelitis is characterised by weakened bones associated with progressive bone loss. The mechanism through which S. epidermidis interacts with bone cells to cause osteomyelitis is poorly understood. We demonstrate here that S. epidermidis can bind to osteoblasts in the absence of matrix proteins. S. epidermidis strains lacking the cell wall protein SdrG had a significantly reduced ability to bind to osteoblasts. Consistent with this, expression of SdrG in Lactococcus lactis resulted in significantly increased binding to the osteoblasts. Protein analysis identified that SdrG contains a potential integrin recognition motif. αVβ3 is a major integrin expressed on osteoblasts and typically recognises RGD motifs in its ligands. Our results demonstrate that S. epidermidis binds to recombinant purified αVβ3, and that a mutant lacking SdrG failed to bind. Blocking αVβ3 on osteoblasts significantly reduced binding to S. epidermidis. These studies are the first to identify a mechanism through which S. epidermidis binds to osteoblasts and potentially offers a mechanism through which implant infection caused by S. epidermidis leads to osteomyelitis.     

Co-Culture of S. epidermidis and Human Osteoblasts on Implant Surfaces: An Advanced In Vitro Model for Implant-Associated Infections

Objectives

Total joint arthroplasty is one of the most frequent and effective surgeries today. However, despite improved surgical techniques, a significant number of implant-associated infections still occur. Suitable in vitro models are needed to test potential approaches to prevent infection. In the present study, we aimed to establish an in vitro co-culture setup of human primary osteoblasts and S. epidermidis to model the onset of implant-associated infections, and to analyze antimicrobial implant surfaces and coatings.

Materials and Methods

For initial surface adhesion, human primary osteoblasts (hOB) were grown for 24 hours on test sample discs made of polystyrene, titanium alloy Ti6Al4V, bone cement PALACOS R^®, and PALACOS R^® loaded with antibiotics. Co-cultures were performed as a single-species infection on the osteoblasts with S. epidermidis (multiplicity of infection of 0.04), and were incubated for 2 and 7 days under aerobic conditions. Planktonic S. epidermidis was quantified by centrifugation and determination of colony-forming units (CFU). The quantification of biofilm-bound S. epidermidis on the test samples was performed by sonication and CFU counting. Quantification of adherent and vital primary osteoblasts on the test samples was performed by trypan-blue staining and counting. Scanning electron microscopy was used for evaluation of topography and composition of the species on the sample surfaces.

Results

After 2 days, we observed approximately 10⁴ CFU/ml biofilm-bound S. epidermidis (10³ CFU/ml initial population) on the antibiotics-loaded bone cement samples in the presence of hOB, while no bacteria were detected without hOB. No biofilm-bound bacteria were detectable after 7 days in either case. Similar levels of planktonic bacteria were observed on day 2 with and without hOB. After 7 days, about 10⁵ CFU/ml planktonic bacteria were present, but only in the absence of hOB. Further, no bacteria were observed within the biofilm, while the number of hOB was decreased to 10% of its initial value compared to 150% in the mono-culture of hOB.

Conclusion

We developed a co-culture setup that serves as a more comprehensive in vitro model for the onset of implant-associated infections and provides a test method for antimicrobial implant materials and coatings. We demonstrate that observations can be made that are unavailable from mono-culture experiments.     

Distinct clonal lineages and within-host diversification shape invasive Staphylococcus epidermidis populations

S. epidermidis is a substantial component of the human skin microbiota, but also one of the major causes of nosocomial infection in the context of implanted medical devices. We here aimed to advance the understanding of S. epidermidis genotypes and phenotypes conducive to infection establishment. Furthermore, we investigate the adaptation of individual clonal lines to the infection lifestyle based on the detailed analysis of individual S. epidermidis populations of 23 patients suffering from prosthetic joint infection. Analysis of invasive and colonizing S. epidermidis provided evidence that invasive S. epidermidis are characterized by infection-supporting phenotypes (e.g. increased biofilm formation, growth in nutrient poor media and antibiotic resistance), as well as specific genetic traits. The discriminating gene loci were almost exclusively assigned to the mobilome. Here, in addition to IS256 and SCCmec, chromosomally integrated phages was identified for the first time. These phenotypic and genotypic features were more likely present in isolates belonging to sequence type (ST) 2. By comparing seven patient-matched nasal and invasive S. epidermidis isolates belonging to identical genetic lineages, infection-associated phenotypic and genotypic changes were documented. Besides increased biofilm production, the invasive isolates were characterized by better growth in nutrient-poor media and reduced hemolysis. By examining several colonies grown in parallel from each infection, evidence for genetic within-host population heterogeneity was obtained. Importantly, subpopulations carrying IS insertions in agrC, mutations in the acetate kinase (AckA) and deletions in the SCCmec element emerged in several infections. In summary, these results shed light on the multifactorial processes of infection adaptation and demonstrate how S. epidermidis is able to flexibly repurpose and edit factors important for colonization to facilitate survival in hostile infection environments.     

An 18 kDa Scaffold Protein Is Critical for Staphylococcus epidermidis Biofilm Formation

Virulence of the nosocomial pathogen Staphylococcus epidermidis is crucially linked to formation of adherent biofilms on artificial surfaces. Biofilm assembly is significantly fostered by production of a bacteria derived extracellular matrix. However, the matrix composition, spatial organization, and relevance of specific molecular interactions for integration of bacterial cells into the multilayered biofilm community are not fully understood. Here we report on the function of novel 18 kDa Small basic protein (Sbp) that was isolated from S. epidermidis biofilm matrix preparations by an affinity chromatographic approach. Sbp accumulates within the biofilm matrix, being preferentially deposited at the biofilm–substratum interface. Analysis of Sbp-negative S. epidermidis mutants demonstrated the importance of Sbp for sustained colonization of abiotic surfaces, but also epithelial cells. In addition, Sbp promotes assembly of S. epidermidis cell aggregates and establishment of multilayered biofilms by influencing polysaccharide intercellular-adhesin (PIA) and accumulation associated protein (Aap) mediated intercellular aggregation. While inactivation of Sbp indirectly resulted in reduced PIA-synthesis and biofilm formation, Sbp serves as an essential ligand during Aap domain-B mediated biofilm accumulation. Our data support the conclusion that Sbp serves as an S. epidermidis biofilm scaffold protein that significantly contributes to key steps of surface colonization. Sbp-negative S. epidermidis mutants showed no attenuated virulence in a mouse catheter infection model. Nevertheless, the high prevalence of sbp in commensal and invasive S. epidermidis populations suggests that Sbp plays a significant role as a co-factor during both multi-factorial commensal colonization and infection of artificial surfaces.     

Monitoring Bacterial Burden,Inflammation and Bone Damage Longitudinally Using Optical and μCT Imaging in an Orthopaedic Implant Infection in Mice

Background

Recent advances in non-invasive optical, radiographic and μCT imaging provide an opportunity to monitor biological processes longitudinally in an anatomical context. One particularly relevant application for combining these modalities is to study orthopaedic implant infections. These infections are characterized by the formation of persistent bacterial biofilms on the implanted materials, causing inflammation, periprosthetic osteolysis, osteomyelitis, and bone damage, resulting in implant loosening and failure.

Methodology/Principal Findings

An orthopaedic implant infection model was used in which a titanium Kirshner-wire was surgically placed in femurs of LysEGFP mice, which possess EGFP-fluorescent neutrophils, and a bioluminescent S. aureus strain (Xen29; 1×10³ CFUs) was inoculated in the knee joint before closure. In vivo bioluminescent, fluorescent, X-ray and μCT imaging were performed on various postoperative days. The bacterial bioluminescent signals of the S. aureus-infected mice peaked on day 19, before decreasing to a basal level of light, which remained measurable for the entire 48 day experiment. Neutrophil EGFP-fluorescent signals of the S. aureus-infected mice were statistically greater than uninfected mice on days 2 and 5, but afterwards the signals for both groups approached background levels of detection. To visualize the three-dimensional location of the bacterial infection and neutrophil infiltration, a diffuse optical tomography reconstruction algorithm was used to co-register the bioluminescent and fluorescent signals with μCT images. To quantify the anatomical bone changes on the μCT images, the outer bone volume of the distal femurs were measured using a semi-automated contour based segmentation process. The outer bone volume increased through day 48, indicating that bone damage continued during the implant infection.

Conclusions/Significance

Bioluminescent and fluorescent optical imaging was combined with X-ray and μCT imaging to provide noninvasive and longitudinal measurements of the dynamic changes in bacterial burden, neutrophil recruitment and bone damage in a mouse orthopaedic implant infection model.     

NADPH Oxidase Deficient Mice Develop Colitis and Bacteremia upon Infection with Normally Avirulent,TTSS-1- and TTSS-2-Deficient Salmonella Typhimurium

Infections, microbe sampling and occasional leakage of commensal microbiota and their products across the intestinal epithelial cell layer represent a permanent challenge to the intestinal immune system. The production of reactive oxygen species by NADPH oxidase is thought to be a key element of defense. Patients suffering from chronic granulomatous disease are deficient in one of the subunits of NADPH oxidase. They display a high incidence of Crohn’s disease-like intestinal inflammation and are hyper-susceptible to infection with fungi and bacteria, including a 10-fold increased risk of Salmonellosis. It is not completely understood which steps of the infection process are affected by the NADPH oxidase deficiency. We employed a mouse model for Salmonella diarrhea to study how NADPH oxidase deficiency (Cybb ^−/−) affects microbe handling by the large intestinal mucosa. In this animal model, wild type S. Typhimurium causes pronounced enteropathy in wild type mice. In contrast, an avirulent S. Typhimurium mutant (S.Tm^avir; invGsseD), which lacks virulence factors boosting trans-epithelial penetration and growth in the lamina propria, cannot cause enteropathy in wild type mice. We found that Cybb ^−/− mice are efficiently infected by S.Tm^avir and develop enteropathy by day 4 post infection. Cell depletion experiments and infections in Cybb ^−/− Myd88 ^−/− mice indicated that the S.Tm^avir-inflicted disease in Cybb ^−/− mice hinges on CD11c⁺CX₃CR1⁺ monocytic phagocytes mediating colonization of the cecal lamina propria and on Myd88-dependent proinflammatory immune responses. Interestingly, in mixed bone marrow chimeras a partial reconstitution of Cybb-proficiency in the bone marrow derived compartment was sufficient to ameliorate disease severity. Our data indicate that NADPH oxidase expression is of key importance for restricting the growth of S.Tm^avir in the mucosal lamina propria. This provides important insights into microbe handling by the large intestinal mucosa and the role of NADPH oxidase in maintaining microbe-host mutualism at this exposed body surface.     

Increased Susceptibility of Humanized NSG Mice to Panton-Valentine Leukocidin and Staphylococcus aureus Skin Infection

Staphylococcus aureus is a leading cause of skin and soft-tissue infections worldwide. Mice are the most commonly used animals for modeling human staphylococcal infections. However a supra-physiologic S. aureus inoculum is required to establish gross murine skin pathology. Moreover, many staphylococcal factors, including Panton-Valentine leukocidin (PVL) elaborated by community-associated methicillin-resistant S. aureus (CA-MRSA), exhibit selective human tropism and cannot be adequately studied in mice. To overcome these deficiencies, we investigated S. aureus infection in non-obese diabetic (NOD)/severe combined immune deficiency (SCID)/IL2rγ^null (NSG) mice engrafted with human CD34⁺ umbilical cord blood cells. These “humanized” NSG mice require one to two log lower inoculum to induce consistent skin lesions compared with control mice, and exhibit larger cutaneous lesions upon infection with PVL⁺ versus isogenic PVL^- S. aureus. Neutrophils appear important for PVL pathology as adoptive transfer of human neutrophils alone to NSG mice was sufficient to induce dermonecrosis following challenge with PVL⁺ S. aureus but not PVL^- S. aureus. PMX53, a human C5aR inhibitor, blocked PVL-induced cellular cytotoxicity in vitro and reduced the size difference of lesions induced by the PVL⁺ and PVL^- S. aureus, but PMX53 also reduced recruitment of neutrophils and exacerbated the infection. Overall, our findings establish humanized mice as an important translational tool for the study of S. aureus infection and provide strong evidence that PVL is a human virulence factor.     

The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325–4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325–4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325–4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325–4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325–4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.     

Bacterial Hypoxic Responses Revealed as Critical Determinants of the Host-Pathogen Outcome by TnSeq Analysis of Staphylococcus aureus Invasive Infection

Staphylococcus aureus is capable of infecting nearly every organ in the human body. In order to infiltrate and thrive in such diverse host tissues, staphylococci must possess remarkable flexibility in both metabolic and virulence programs. To investigate the genetic requirements for bacterial survival during invasive infection, we performed a transposon sequencing (TnSeq) analysis of S. aureus during experimental osteomyelitis. TnSeq identified 65 genes essential for staphylococcal survival in infected bone and an additional 148 mutants with compromised fitness in vivo. Among the loci essential for in vivo survival was SrrAB, a staphylococcal two-component system previously reported to coordinate hypoxic and nitrosative stress responses in vitro. Healthy bone is intrinsically hypoxic, and intravital oxygen monitoring revealed further decreases in skeletal oxygen concentrations upon S. aureus infection. The fitness of an srrAB mutant during osteomyelitis was significantly increased by depletion of neutrophils, suggesting that neutrophils impose hypoxic and/or nitrosative stresses on invading bacteria. To more globally evaluate staphylococcal responses to changing oxygenation, we examined quorum sensing and virulence factor production in staphylococci grown under aerobic or hypoxic conditions. Hypoxic growth resulted in a profound increase in quorum sensing-dependent toxin production, and a concomitant increase in cytotoxicity toward mammalian cells. Moreover, aerobic growth limited quorum sensing and cytotoxicity in an SrrAB-dependent manner, suggesting a mechanism by which S. aureus modulates quorum sensing and toxin production in response to environmental oxygenation. Collectively, our results demonstrate that bacterial hypoxic responses are key determinants of the staphylococcal-host interaction.     

Staphylococcus epidermidis in Orthopedic Device Infections: The Role of Bacterial Internalization in Human Osteoblasts and Biofilm Formation

Background

Staphylococcus epidermidis orthopedic device infections are caused by direct inoculation of commensal flora during surgery and remain rare, although S. epidermidis carriage is likely universal. We wondered whether S. epidermidis orthopedic device infection strains might constitute a sub-population of commensal isolates with specific virulence ability. Biofilm formation and invasion of osteoblasts by S. aureus contribute to bone and joint infection recurrence by protecting bacteria from the host-immune system and most antibiotics. We aimed to determine whether S. epidermidis orthopedic device infection isolates could be distinguished from commensal strains by their ability to invade osteoblasts and form biofilms.

Materials and Methods

Orthopedic device infection S. epidermidis strains (n = 15) were compared to nasal carriage isolates (n = 22). Osteoblast invasion was evaluated in an ex vivo infection model using MG63 osteoblastic cells co-cultured for 2 hours with bacteria. Adhesion of S. epidermidis to osteoblasts was explored by a flow cytometric approach, and internalized bacteria were quantified by plating cell lysates after selective killing of extra-cellular bacteria with gentamicin. Early and mature biofilm formations were evaluated by a crystal violet microtitration plate assay and the Biofilm Ring Test method.

Results

No difference was observed between commensal and infective strains in their ability to invade osteoblasts (internalization rate 308+/−631 and 347+/−431 CFU/well, respectively). This low internalization rate correlated with a low ability to adhere to osteoblasts. No difference was observed for biofilm formation between the two groups.

Conclusion

Osteoblast invasion and biofilm formation levels failed to distinguish S. epidermidis orthopedic device infection strains from commensal isolates. This study provides the first assessment of the interaction between S. epidermidis strains isolated from orthopedic device infections and osteoblasts, and suggests that bone cell invasion is not a major pathophysiological mechanism in S. epidermidis orthopedic device infections, contrary to what is observed for S. aureus.     

Gentamicin palmitate as a new antibiotic formulation for mixing with bone tissue and local release

During surgery with bone grafting, the impaction of bone tissue creates an avascular area where local circulation is disrupted. If infections arise, they may prevent systemically administered antibiotics from reaching the infected bone. In this study we evaluated gentamicin palmitate (GP) mixed with gentamicin sulfate (GS) as a coating for bone chips (BCh). The efficacy of the coated BCh was measured by gentamicin base release tests using B. subtilis, S. epidermidis and S. aureus. Gentamicin base release was evaluated in phosphate-buffered saline for up to 7 days using B. subtilis bioassay. Antimicrobial efficacy was tested with S. aureus and S. epidermidis. A significant difference on the release of gentamicin base between GS and GS + GP was observed. S. epidermidis are significantly more susceptible to GS + GP and GS than S. aureus. BCh can act as gentamicin carriers and showed efficacy against S. aureus and S. epidermidis.     

Cytokine mRNA expression in Peromyscus yucatanicus (Rodentia: Cricetidae) infected by Leishmania (Leishmania) mexicana

Peromyscus yucatanicus, the main reservoir of Leishmania (Leishmania) mexicana in the Yucatan peninsula of Mexico, reproduces clinical and histological pictures of LCL in human as well as subclinical infection. Thus, we used this rodent as a novel experimental model. In this work, we analyzed cytokine mRNA expression in P. yucatanicus infected with L. (L.) mexicana. Animals were inoculated with either 2.5 × 10⁶ or 1 × 10² promastigotes and cytokine expressions were analyzed by real-time RT-PCR in skin at 4 and 12 weeks post-infection (wpi). Independently of the parasite inoculum none of the infected rodents had clinical signs of LCL at 4 wpi and all expressed high IFN-γ mRNA. All P. yucatanicus inoculated with 2.5 × 10⁶ promastigotes developed signs of LCL at 12 wpi while the mice inoculated with 1 × 10² remained subclinical. At that time, both IFN-γ and IL-10 were expressed in P. yucatanicus with clinical and subclinical infections. Expressions of TNF-α and IL-4 were significantly higher in clinical animals (2.5 × 10⁶) compared with subclinical ones (1 × 10²). High TGF-β expression was observed in P. yucatanicus with clinical signs when compared with healthy animals. Results suggested that the clinical course of L. (L.) mexicana infection in P. yucatanicus was associated with a specific local pattern of cytokine production at 12 wpi.     

Molecular and Phenotypic Characterization of Staphylococcus epidermidis Isolates from Healthy Conjunctiva and a Comparative Analysis with Isolates from Ocular Infection

Staphylococcus epidermidis is a common commensal of healthy conjunctiva and it can cause endophthalmitis, however its presence in conjunctivitis, keratitis and blepharitis is unknown. Molecular genotyping of S. epidermidis from healthy conjunctiva could provide information about the origin of the strains that infect the eye. In this paper two collections of S. epidermidis were used: one from ocular infection (n = 62), and another from healthy conjunctiva (n = 45). All isolates were genotyped by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec), detection of the genes icaA, icaD, IS256 and polymorphism type of agr locus. The phenotypic data included biofilm production and antibiotic resistance. The results displayed 61 PFGE types from 107 isolates and they were highly discriminatory. MLST analysis generated a total of 25 STs, of which 11 STs were distributed among the ocular infection isolates and lineage ST2 was the most frequent (48.4%), while 14 STs were present in the healthy conjunctiva isolates and lineage ST5 was the most abundant (24.4%). By means of a principal coordinates analysis (PCoA) and a discriminant analysis (DA) it was found that ocular infection isolates had as discriminant markers agr III or agr II, SCCmec V or SCCmec I, mecA gene, resistance to tobramycin, positive biofilm, and IS256⁺. In contrast to the healthy conjunctiva isolates, the discriminating markers were agr I, and resistance to chloramphenicol, ciprofloxacin, gatifloxacin and oxacillin. The discriminant biomarkers of ocular infection were examined in healthy conjunctiva isolates, and it was found that 3 healthy conjunctiva isolates [two with ST2 and another with ST9] (3/45, 6.66%) had similar genotypic and phenotypic characteristics to ocular infection isolates, therefore a small population from healthy conjunctiva could cause an ocular infection. These data suggest that the healthy conjunctiva isolates do not, in almost all cases, infect the eye due to their large genotypic and phenotypic difference with the ocular infection isolates.     

Biofilm Morphotypes and Population Structure among Staphylococcus epidermidis from Commensal and Clinical Samples

Bacterial species comprise related genotypes that can display divergent phenotypes with important clinical implications. Staphylococcus epidermidis is a common cause of nosocomial infections and, critical to its pathogenesis, is its ability to adhere and form biofilms on surfaces, thereby moderating the effect of the host’s immune response and antibiotics. Commensal S. epidermidis populations are thought to differ from those associated with disease in factors involved in adhesion and biofilm accumulation. We quantified the differences in biofilm formation in 98 S. epidermidis isolates from various sources, and investigated population structure based on ribosomal multilocus typing (rMLST) and the presence/absence of genes involved in adhesion and biofilm formation. All isolates were able to adhere and form biofilms in in vitro growth assays and confocal microscopy allowed classification into 5 biofilm morphotypes based on their thickness, biovolume and roughness. Phylogenetic reconstruction grouped isolates into three separate clades, with the isolates in the main disease associated clade displaying diversity in morphotype. Of the biofilm morphology characteristics, only biofilm thickness had a significant association with clade distribution. The distribution of some known adhesion-associated genes (aap and sesE) among isolates showed a significant association with the species clonal frame. These data challenge the assumption that biofilm-associated genes, such as those on the ica operon, are genetic markers for less invasive S. epidermidis isolates, and suggest that phenotypic characteristics, such as adhesion and biofilm formation, are not fixed by clonal descent but are influenced by the presence of various genes that are mobile among lineages.     

Panton–Valentine Leukocidin Enhances the Severity of Community-Associated Methicillin-Resistant Staphylococcus aureus Rabbit Osteomyelitis

Background

Extensive spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in the United States, and the concomitant increase in severe invasive staphylococcal infections, including osteomyelitis, in healthy children, has led to renewed interest in Panton-Valentine leukocidin (PVL). However, the pathogenetic role of PVL in staphylococcal infections remains controversial, possibly because it depends on the site of infection.

Methodology/Principal Findings

We compared the course of experimental rabbit osteomyelitis due to the PVL-positive CA-MRSA strain USA 300 (LAC) and its PVL-negative isogenic derivative (LACΔpvl), using a low and a high inoculum (8×10⁵ and 4×10⁸ CFU). With the low inoculum, bone infection was less frequent on day 7 (D7) and day 28 (D28) with LACΔpvl than with LAC (respectively 12/19 and 18/19 animals, p = 0.042). With the high inoculum of both strains, all the animals were infected on D7 and the infection persisted on D28 in almost every case. However, tibial bacterial counts and the serum CRP concentration fell significantly between D7 and D28 with LACΔpvl but not with LAC. Respectively 67% and 60% of LAC-infected rabbits had bone deformation and muscle/joint involvement on D7, compared to 0% and 7% of LACΔpvl-infected rabbits (p = 0.001 and p = 0.005 respectively). Between D0 and D28, the anti-PVL antibody titer increased significantly only with the high inoculum of LAC.

Conclusions/Significance

PVL appears to play a role in the persistence and rapid local extension of rabbit osteomyelitis, in keeping with the greater severity of human bone infections due to PVL-positive S. aureus. The possible therapeutic implications of these findings are discussed.     

A Mouse Model of Post-Arthroplasty Staphylococcus aureus Joint Infection to Evaluate In Vivo the Efficacy of Antimicrobial Implant Coatings

Background

Post-arthroplasty infections represent a devastating complication of total joint replacement surgery, resulting in multiple reoperations, prolonged antibiotic use, extended disability and worse clinical outcomes. As the number of arthroplasties in the U.S. will exceed 3.8 million surgeries per year by 2030, the number of post-arthroplasty infections is projected to increase to over 266,000 infections annually. The treatment of these infections will exhaust healthcare resources and dramatically increase medical costs.

Methodology/Principal Findings

To evaluate novel preventative therapeutic strategies against post-arthroplasty infections, a mouse model was developed in which a bioluminescent Staphylococcus aureus strain was inoculated into a knee joint containing an orthopaedic implant and advanced in vivo imaging was used to measure the bacterial burden in real-time. Mice inoculated with 5×10³ and 5×10⁴ CFUs developed increased bacterial counts with marked swelling of the affected leg, consistent with an acute joint infection. In contrast, mice inoculated with 5×10² CFUs developed a low-grade infection, resembling a more chronic infection. Ex vivo bacterial counts highly correlated with in vivo bioluminescence signals and EGFP-neutrophil fluorescence of LysEGFP mice was used to measure the infection-induced inflammation. Furthermore, biofilm formation on the implants was visualized at 7 and 14 postoperative days by variable-pressure scanning electron microscopy (VP-SEM). Using this model, a minocycline/rifampin-impregnated bioresorbable polymer implant coating was effective in reducing the infection, decreasing inflammation and preventing biofilm formation.

Conclusions/Significance

Taken together, this mouse model may represent an alternative pre-clinical screening tool to evaluate novel in vivo therapeutic strategies before studies in larger animals and in human subjects. Furthermore, the antibiotic-polymer implant coating evaluated in this study was clinically effective, suggesting the potential for this strategy as a therapeutic intervention to combat post-arthroplasty infections.     

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification,Distraction Osteogenesis,or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.     

Effect of Quorum Sensing by Staphylococcus epidermidis on the Attraction Response of Female Adult Yellow Fever Mosquitoes,Aedes aegypti aegypti (Linnaeus) (Diptera: Culicidae), to a Blood-Feeding Source

Aedes aegypti, the principal vector of yellow fever and dengue fever, is responsible for more than 30,000 deaths annually. Compounds such as carbon dioxide, amino acids, fatty acids and other volatile organic compounds (VOCs) have been widely studied for their role in attracting Ae. aegypti to hosts. Many VOCs from humans are produced by associated skin microbiota. Staphyloccocus epidermidis, although not the most abundant bacteria according to surveys of relative 16S ribosomal RNA abundance, commonly occurs on human skin. Bacteria demonstrate population level decision-making through quorum sensing. Many quorum sensing molecules, such as indole, volatilize and become part of the host odor plum. To date, no one has directly demonstrated the link between quorum sensing (i.e., decision-making) by bacteria associated with a host as a factor regulating arthropod vector attraction. This study examined this specific question with regards to S. epidermidis and Ae. aegypti. Pairwise tests were conducted to examine the response of female Ae. aegypti to combinations of tryptic soy broth (TSB) and S. epidermidis wildtype and agr- strains. The agr gene expresses an accessory gene regulator for quorum sensing; therefore, removing this gene inhibits quorum sensing of the bacteria. Differential attractiveness of mosquitoes to the wildtype and agr- strains was observed. Both wildtype and the agr- strain of S. epidermidis with TSB were marginally more attractive to Ae. aegypti than the TSB alone. Most interestingly, the blood-feeder treated with wildtype S. epidermidis/TSB attracted 74% of Ae. aegypti compared to the agr- strain of S. epidermidis/TSB (P ≤ 0.0001). This study is the first to suggest a role for interkingdom communication between host symbiotic bacteria and mosquitoes. This may have implications for mosquito decision-making with regards to host detection, location and acceptance. We speculate that mosquitoes “eavesdrop” on the chemical discussions occurring between host-associated microbes to determine suitability for blood feeding. We believe these data suggest that manipulating quorum sensing by bacteria could serve as a novel approach for reducing mosquito attraction to hosts, or possibly enhancing the trapping of adults at favored oviposition sites.     

In vivo efficacy of tobramycin-loaded synthetic calcium phosphate beads in a rabbit model of staphylococcal osteomyelitis

Background

Osteomyelitis is an acute or chronic inflammatory process of the bone following infection with pyogenic organisms like Staphylococcus aureus. Tobramycin (TOB) is a promising aminoglycoside antibiotic used to treat various bacterial infections, including S. aureus. The aim of this study was to investigate the efficacy of tobramycin-loaded calcium phosphate beads (CPB) in a rabbit osteomyelitis model.

Methods

Tobramycin (30 mg/mL) was incorporated into CPB by dipping method and the efficacy of TOB-loaded CPB was studied in a rabbit osteomyelitis model. For juxtaposition, CPB with and without TOB were prepared. Twenty-five New Zealand white rabbits were grouped (n?=?5) as sham (group 1), TOB-loaded CPB without S. aureus (group 2), S. aureus only (group 3), S. aureus?+?CPB (group 4), and S. aureus?+?TOB-loaded CPB (group 5). Groups infected with S. aureus followed by CPB implantation were immediately subjected to surgery at the mid-shaft of the tibia. After 28 days post-surgery, all rabbits were euthanized and the presence or absence of chronic osteomyelitis and the extent of architectural destruction of the bone were assessed by radiology, bacteriology and histological studies.

Results

Tobramycin-loaded CPB group potentially inhibited the growth of S. aureus causing 3.2 to 3.4 log₁₀ reductions in CFU/g of bone tissue compared to the controls. Untreated groups infected with S. aureus showed signs of chronic osteomyelitis with abundant bacterial growth and alterations in bone architecture. The sham group and TOB-loaded CPB group showed no evidence of bacterial growth.

Conclusions

TOB-incorporated into CPB for local bone administration was proven to be more successful in increasing the efficacy of TOB in this rabbit osteomyelitis model and hence could represent a good alternative to other formulations used in the treatment of osteomyelitis.