Bone and Joint Infection Involving Corynebacterium spp.: From Clinical Features to Pathophysiological Pathways

Introduction: Corynebacteria represent often-neglected etiological agents of post-traumatic and/or post-operative bone and joint infection (BJI). We describe here clinical characteristics and bacteriological determinants of this condition.Methods: A retrospective cohort study described characteristics, outcome and determinants of treatment failure of all patients with proven Corynebacterium spp. BJI (i.e., ≥2 culture-positive gold-standard samples). Available strains were further characterized regarding their antibiotic susceptibilies, abilities to form early (BioFilm Ring Test®) and mature (crystal violet staining method) biofilms and to invade osteoblasts (gentamicin protection assay).Results: The 51 included BJI were mostly chronic (88.2%), orthopedic device-related (74.5%) and polymicrobial (78.4%). After a follow-up of 60.7 weeks (IQR, 30.1–115.1), 20 (39.2%) treatment failures were observed, including 4 Corynebacterium-documented relapses, mostly associated with non-optimal surgical management (OR 7.291; p = 0.039). Internalization rate within MG63 human osteoblasts was higher for strains isolated from delayed (>3 months) BJI (p < 0.001). Infection of murine osteoblasts deleted for the β1-integrin resulted in a drastic reduction in the internalization rate. No difference was observed regarding biofilm formation.Conclusions: Surgical management plays a crucial role in outcome of BJI involving corynebacteria, as often chronic and device-associated infections. Sanctuarisation within osteoblasts, implicating the β1 cellular integrin, may represent a pivotal virulence factor associated with BJI chronicity.

Persistent Infection of Human Mesenchymal Stromal Cells With Bartonella Henselae Exerts a Proangiogenic Effect

Background B henselae is in humans the aetiologic agent of cat-scratch disease and of the vasculoproliferative disorders bacillary angiomatosis and bacillary peliosis. Although endothelial cells are crucial in the pathogenesis other cell types function as reservoir and contribute to pathological angiogenesis. Among them, mesenchymal stromal cells (MSCs) can sense pathogens and mount an appropriate cytokine/chemokine response through different Pattern Recognition Receptors (PRRs). MSCs exert direct antimicrobial effector function but may also shelter bacteria such as M. tuberculosis. Methods Adipose-derived MSCs were infected with B. henselae and analyzed for bacterial persistence by gentamicin protection assay, immunohistochemistry and immunofluorescence. Involvement of PRRs in bacterial infection was evaluated through gene and protein expression analysis. The effect of infection on MSC proliferation, apoptosis and release of soluble factors was assessed. The role of infected-MSC conditioned medium in promoting Bartonella infection of endothelial cells and angiogenesis was demonstrated using respectively gentamicin protection assay and different pro-angiogenic assays including spheroid, wound healing and morphogenesis. Results B. henselae can readily infect MSCs and survive in perinuclear bound vacuoles for up to 8 days. Bartonella infection stimulates MSC proliferation and upregulation of EGFR and of the two pattern recognition receptors (PRRs) TLR2 and NOD1. Specific inhibition of EGFR reduces bacterial internalization and treatment with anti-TLR2 neutralizing antibody or EGFR/NOD1 inhibitors significantly downmodulates CXCL8 production. Secretome analysis shows that, in addition to CXCL8, infected MSCs secrete higher levels of the proangiogenic factors VEGF, FGF-7, MMP-9, PIGF, serpin E1, TSP-1, uPA, IL-6, CCL5 and PDGF-D. Importantly, supernatants from B. henselae-infected MSCs increase the susceptibility of ECs to B. henselae infection while enhancing EC proangiogenic potential. Conclusions Altogether, these findings indicate that MSCs constitute a novel niche for B. henselae, which favors the persistence of vascular proliferative disorders.

Ciprofloxacin interferes with Salmonella Typhimurium intracellular survival and host virulence through repression of Salmonella pathogenicity island-2 (SPI-2) genes expression

ABSTRACT Current study aims to characterize the influence of sub-minimum inhibitory concentration (sub-MIC) of ciprofloxacin on Salmonella intracellular survival and host virulence. Herein, Salmonella resistance patterns to various antibiotics were in agreement with those reported in previous studies. Moreover, intracellular survival of both ciprofloxacin-sensitive and -resistant Salmonella was markedly reduced upon treatment with sub-MIC of ciprofloxacin as determined by gentamicin protection assay. These findings were further confirmed using immunostaining indicating an inhibitory effect of sub-MIC of ciprofloxacin on Salmonella intracellular survival. RT-qPCR revealed that expression of genes encoding Salmonella type three secretion system (TTSS) decreased upon bacterial exposure to sub-MIC of ciprofloxacin. Furthermore, bacterial exposure to sub-MIC of ciprofloxacin significantly reduced expression of both sifA and sifB, which are important for Salmonella filaments formation within the host. Treatment of Salmonella with sub-MIC of ciprofloxacin reduced bacterial capacity to kill mice infection models. A lower mortality rate was observed in mice injected with Salmonella treated with sub-MIC of ciprofloxacin as compared with mice inoculated with untreated bacteria. Collectively, current findings indicate that, in addition to its bactericidal potential, sub-MIC of ciprofloxacin could inhibit Salmonella intracellular survival, virulence genes expression as well as host pathogenesis, providing another mechanism for ciprofloxacin in limiting Salmonella host infection.

Campylobacter fetus is Internalized by Bovine Endometrial Epithelial Cells

Campylobacter fetus is an important venereal pathogen of cattle that causes infertility and abortions. It is transmitted during mating, and it travels from the vagina to the uterus; therefore, an important cell type that interacts with C.fetus are endometrial epithelial cells. Several virulence factors have been identified in the genome of C.fetus, such as adhesins, secretion systems, and antiphagocytic layers, but their expression is unknown. The ability of C.fetus to invade human epithelial cells has been demonstrated, but the ability of this microorganism to infect bovine endometrial epithelial cells has not been demonstrated. Bovine endometrial epithelial cells were isolated and challenged with C.fetus. The presence of C.fetus inside the endometrial epithelial cells was confirmed by the confocal immunofluorescence. C.fetus was not internalized when actin polymerization was disturbed, suggesting cytoskeleton participation in an internalization mechanism. To evaluate the intracellular survival of C.fetus, a gentamicin protection assay was performed. Although C.fetus was able to invade epithelial cells, the results showed that it did not have the capacity to survive in the intracellular environment. This study reports for the first time, the ability of C.fetus to invade bovine endometrial epithelial cells, and actin participation in this phenomenon.

Alternative Enzyme Protection Assay To Overcome the Drawbacks of the Gentamicin Protection Assay for Measuring Entry and Intracellular Survival of Staphylococci

ABSTRACTPrecise enumeration of living intracellular bacteria is the key step to estimate the invasion potential of pathogens and host immune responses to understand the mechanism and kinetics of bacterial pathogenesis. Therefore, quantitative assessment of host-pathogen interactions is essential for development of novel antibacterial therapeutics for infectious disease. The gentamicin protection assay (GPA) is the most widely used method for these estimations by counting the CFU of intracellular living pathogens. Here, we assess the longstanding drawbacks of the GPA by employing an antistaphylococcal endopeptidase as a bactericidal agent to kill extracellularStaphylococcus aureus. We found that the difference between the two methods for the recovery of intracellular CFU ofS. aureuswas about 5 times. We prove that the accurate number of intracellular CFU could not be precisely determined by the GPA due to the internalization of gentamicin into host cells during extracellular bacterial killing. We further demonstrate that lysostaphin-mediated extracellular bacterial clearance has advantages for measuring the kinetics of bacterial internalization on a minute time scale due to the fast and tunable activity and the inability of protein to permeate the host cell membrane. From these results, we propose that accurate quantification of intracellular bacteria and measurement of internalization kinetics can be achieved by employing enzyme-mediated killing of extracellular bacteria (enzyme protection assay [EPA]) rather than the host-permeative drug gentamicin, which is known to alter host physiology.

Inflammasome Activation by ATP Enhances Citrobacter rodentium Clearance through ROS Generation

Background: Nod-like receptor family, pyrin domain containing 3 (NLRP3) is an important cytosolic sensor of cellular stress and infection. Once activated, NLRP3 forms a multiprotein complex (inflammasome) that triggers the maturation and secretion of interleukin (IL)-1β and IL-18. We aimed to define the consequences of NLRP3 induction, utilizing exogenous adenosine triphosphate (ATP) as an inflammasome activator, to determine if inflammasome activation increases macrophage killing of Citrobacter rodentium and define mechanisms. Methods: Bacterial survival was measured using a gentamicin protection assay. Inflammasome activation or inhibition in mouse J774A.1 macrophages were assessed by measuring IL-1β; cytokines and reactive oxygen species (ROS) were measured by ELISA and DCFDA, respectively. Results: Activation of the inflammasome increased bacterial killing by macrophages and its inhibition attenuated this effect with no impact on phagocytosis or cell death. Furthermore, inflammasome activation suppressed pro-inflammatory cytokines during infection, possibly due to more effective bacterial killing. While the infection increased ROS production, this effect was reduced by inflammasome inhibitors, indicating that ROS is inflammasome-dependent. ROS inhibitors increased bacterial survival in the presence of ATP, suggesting that inflammasome-induced bacterial killing is mediated, at least in part, by ROS activity. Conclusion: Improving inflammasome activity during infection may increase bacterial clearance by macrophages and reduce subsequent microbe-induced inflammation.

Fibronectin Binding Proteins SpsD and SpsL Both Support Invasion of Canine Epithelial Cells by Staphylococcus pseudintermedius

In this study, we investigated the cell wall-anchored fibronectin-binding proteins SpsD and SpsL from the canine commensal and pathogenStaphylococcus pseudintermediusfor their role in promoting bacterial invasion of canine progenitor epidermal keratinocytes (CPEK). Invasion was examined by the gentamicin protection assay and fluorescence microscopy. An ΔspsD ΔspsLmutant of strain ED99 had a dramatically reduced capacity to invade CPEK monolayers, while no difference in the invasion level was observed with single mutants.Lactococcus lactistransformed with plasmids expressing SpsD and SpsL promoted invasion, showing that both proteins are important. Soluble fibronectin was required for invasion, and an RGD-containing peptide or antibodies recognizing the integrin α5β1markedly reduced invasion, suggesting an important role for the integrin in this process. Src kinase inhibitors effectively blocked internalization, suggesting a functional role for the kinase in invasion. In order to identify the minimal fibronectin-binding region of SpsD and SpsL involved in the internalization process, recombinant fragments of both proteins were produced. The SpsD520–846and SpsL538–823regions harboring the major fibronectin-binding sites inhibitedS. pseudintermediusinternalization. Finally, the effects of staphylococcal invasion on the integrity of different cell lines were examined. Because SpsD and SpsL are critical factors for adhesion and invasion, blocking these processes could provide a strategy for future approaches to treating infections.