A Novel Apparatus to Standardise the Polishing Protocol to Achieve Different Roughness of Titanium and Zirconia Disc Surfaces.

PURPOSE: This study aimed to propose and evaluate standardised polishing protocols for in vitro experiment using a custom-made apparatus under controlled force to create consistent surface roughness on titanium and zirconia disks. METHODS: A total of 160 discs were manufactured with a diameter of 10mm, 80 titanium (Ti), and 80 zirconium oxide (Zr). Specimens were categorised into 2 groups: controlled force (CF) and without controlled force (WCF). Specimens in the CF group were polished with a custom apparatus incorporating a tension gauge on the Ti and Zr disc surfaces to achieve consistent roughness. The WCF was polished without the use of a tension gauge. Each group had 4 subgroups (10 disks in each): control/machined (C) with no polishing, rough (R), smooth (S) and very smooth (VS). The subgroups were processed using a sequence of diamond-impregnated polishing burs and polishing paste. RESULTS: CF group showed consistent surface roughness and a gradual decrease in surface roughness. Control (Ra=6.5±0.03µm) in Ti and (Ra=5.4±0.04µm) in Zr; R (Ra=3.5± 0.06µm) in Ti and (Ra=3.2± 0.07 µm) in Zr; S (Ra=1.5±0.04µm) in Ti and (Ra=1.1±006µm) in Zr; VS (Ra=0.05± 0.002µm) in Ti and (Ra=0.02±0.005µm) in Zr. There were significant differences for R, S, and SV under CF and WCF in Ti and Zr surfaces. CONCLUSION: The specimens polished under control force produced significantly more uniform surface roughness than those polished without controlled force and were produced with a higher degree of consistency.

Medication-related osteonecrosis of the jaw: A disease of significant importance for older patients.

BACKGROUND: Medication-related osteonecrosis of the jaw (MRONJ) is clinically defined as a non-healing jawbone ulcerative-necrotic lesion appearing after dental therapy or minor trauma in patients treated previously with anti-resorptive, anti-angiogenic or immunomodulators. Older patients with osteoporosis and cancer receive these pharmacological agents regularly. As these patients are long-term survivors, efficient treatment is of paramount importance for their quality of life. METHODS: Literature searches via PubMed were conducted to identify relevant MRONJ studies. Basic information on MRONJ classification, clinical features, and pathosphysiology is presented herein as well as various clinical studies dealing with MRONJ in patients with osteoporosis and cancer. Lastly, we discuss current managment of patients and new trends in treatment of MRONJ. RESULTS: Although close follow-up and local hygiene have been advocated by some authors, severe forms of MRONJ are not responsive to conservative therapy. At present, there is no "gold standard" therapy for this condition. However, as the physiopathological basis of MRONJ is represented by the anti-angiogenic action of various pharmacological agents, new methods to increase and promote local angiogenesis and vascularization have recently been successfully tested in vitro, limited preclinical studies, and in a pilot clinical study. CONCLUSIONS: It appears that the best method implies application on the lesion of endothelial progenitor cells as well as pro-angiogenic factors such as Vascular Endothelial Growth Factor (VEGF) and other related molecules. More recently, scaffolds in which these factors have been incorporated have shown positive results in limited trials. However, these studies must be replicated to include a large number of cases before any official therapeutic protocol is adopted.

The Effect of Titanium Surface Topography on Adherent Macrophage Integrin and Cytokine Expression.

Immunomodulatory biomaterials have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling, as opposed to persistent inflammation and scar tissue formation. This study examined the effects of titanium surface modification on integrin expression and concurrent cytokine secretion by adherent macrophages in vitro in an attempt to delineate the molecular events involved in biomaterial-mediated immunomodulation. Non-polarised (M0) and inflammatory polarised (M1) macrophages were cultured on a relatively smooth (machined) titanium surface and two proprietary modified rough titanium surfaces (blasted and fluoride-modified) for 24 h. The physiochemical characteristics of the titanium surfaces were assessed by microscopy and profilometry, while macrophage integrin expression and cytokine secretion were determined using PCR and ELISA, respectively. After 24 h adhesion onto titanium, integrin α1 expression was downregulated in both M0 and M1 cells on all titanium surfaces. Expression of integrins α2, αM, ß1 and ß2 increased in M0 cells cultured on the machined surface only, whereas in M1 cells, expression of integrins α2, αM and ß1 all increased with culture on both the machined and rough titanium surfaces. These results correlated with a cytokine secretory response whereby levels of IL-1ß, IL-31 and TNF-α increased significantly in M1 cells cultured on the titanium surfaces. These results show that adherent inflammatory macrophages interact with titanium in a surface-dependent manner such that increased levels of inflammatory cytokines IL-1ß, TNF-α and IL-31 secreted by M1 cells were associated with higher expression of integrins α2, αM and ß1.

Titanium Implant Surface Effects on Adherent Macrophage Phenotype: A Systematic Review.

Immunomodulatory biomaterials have the potential to stimulate an immune response able to promote constructive and functional tissue remodeling responses as opposed to persistent inflammation and scar tissue formation. As such, the controlled activation of macrophages and modulation of their phenotype through implant surface modification has emerged as a key therapeutic strategy. METHODS: Online databases were searched for in vitro studies between January 1991 and June 2020 which examined the effect of titanium implant surface topography on the adherent macrophage phenotype at either the gene or protein level. RESULTS: Thirty-nine studies were subsequently included for review. Although there was significant heterogeneity between studies, treatment of titanium surfaces increased the surface roughness or hydrophilicity, and hence increased macrophage attachment but decreased cell spreading. Physical coating of the titanium surface also tended to promote the formation of cell clusters. Titanium and titanium-zirconium alloy with a micro- or nano-scale rough topography combined with a hydrophilic surface chemistry were the most effective surfaces for inducing an anti-inflammatory phenotype in adherent macrophages, as indicated by significant changes in cytokine gene expression and or cytokine secretion profiles. CONCLUSIONS: The published data support the hypothesis that incorporation of specific topographical and physiochemical surface modifications to titanium can modulate the phenotypic response of adherent macrophages.

Streptococcus agalactiae glyceraldehyde-3-phosphate dehydrogenase (GAPDH) elicits multiple cytokines from human cells and has a minor effect on bacterial persistence in the murine female reproductive tract.

Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase (GAPDH), encoded by gapC, is a glycolytic enzyme that is associated with virulence and immune-mediated protection. However, the role of GAPDH in cellular cytokine responses to S. agalactiae, bacterial phagocytosis and colonization of the female reproductive tract, a central host niche, is unknown. We expressed and studied purified recombinant GAPDH (rGAPDH) of S. agalactiae in cytokine elicitation assays with human monocyte-derived macrophage, epithelial cell, and polymorphonuclear leukocyte (PMN) co-culture infection models. We also generated a S. agalactiae mutant that over-expresses GAPDH (oeGAPDH) from gapC using a constitutively active promoter, and analyzed the mutant in murine macrophage antibiotic protection assays and in virulence assays in vivo, using a colonization model that is based on experimental infection of the reproductive tract in female mice. Human cell co-cultures produced interleukin (IL)-1ß, IL-6, macrophage inflammatory protein (MIP)-1, tumor necrosis factor (TNF)-α and IL-10 within 24 h of exposure to rGAPDH. PMNs were required for several of these cytokine responses. However, over-expression of GAPDH in S. agalactiae did not significantly affect measures of phagocytic uptake compared to an empty vector control. In contrast, oeGAPDH-S. agalactiae showed a small but statistically significant attenuation for persistence in the reproductive tract of female mice during the chronic phase of infection (10-28 days post-inoculation), relative to the vector control. We conclude that S. agalactiae GAPDH elicits production of multiple cytokines from human cells, and over-expression of GAPDH renders the bacterium more susceptible to host clearance in the female reproductive tract.One-sentence summary: This study shows Streptococcus agalactiae glyceraldehyde 3-phosphate dehydrogenase, an enzyme that functions in glycolysis, gluconeogenesis and virulence, modifies phagocytosis outcomes, including cytokine synthesis, and affects bacterial persistence in the female reproductive tract.

Fluorescence characteristics of E. faecalis in dentine following treatment with oxidizing endodontic irrigants.

INTRODUCTION: This study aimed to assess changes in the fluorescence characteristics of Enterococcus faecalis in human dentine over a period of 24 h following treatment with endodontic irrigants. METHOD: Sterilised, non-functional extracted third molars were embedded in acrylic resin and uniformly sectioned into 2 mm thick dentine sections. After the removal of smear layer, the dentine sections were inoculated with E. faecalis and cultured for 7 days. The infected dentine sections were subsequently treated with different concentrations of sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2). Bacterial fluorescence readings were assessed at different time points using a calibrated laser device. All data were assessed for normality (Kolmogorov Smirnoff test) and analysed using ANOVA with Bonferroni post-hoc tests. RESULTS: Fluorescence readings were quenched when E. faecalis infected human dentine sections were treated with oxidizing irrigants in vitro. Throughout a 24-hour period, fluorescence recovered in part but did not return to baseline level. CONCLUSION: The fluorescence quenching effect of these oxidizing agents needs to be considered when using laser fluorescence in assessing the quality of root canal debridement or disinfection.

The Antibacterial and Remineralizing Effects of Biomaterials Combined with DMAHDM Nanocomposite: A Systematic Review.

Researchers have developed novel nanocomposites that incorporate additional biomaterials with dimethylaminohexadecyl methacrylate (DMAHDM) in order to reduce secondary caries. The aim of this review was to summarize the current literature and assess the synergistic antibacterial and remineralizing effects that may contribute to the prevention of secondary caries. An electronic search was undertaken in MEDLINE using PubMed, Embase, Scopus, Web of Science and Cochrane databases. The initial search identified 954 papers. After the removal of duplicates and screening the titles and abstracts, 15 articles were eligible for this review. The amalgamation of 2-methacryloyloxyethyl phosphorylcholine (MPC) and silver nanoparticles (AgNPs) with DMAHDM resulted in increased antibacterial potency. The addition of nanoparticles of amorphous calcium phosphate (NACP) and polyamidoamine dendrimers (PAMAM) resulted in improved remineralization potential. Further clinical studies need to be planned to explore the antibacterial and remineralizing properties of these novel composites for clinical success.

Conserved bacterial de novo guanine biosynthesis pathway enables microbial survival and colonization in the environmental niche of the urinary tract.

In bacteria, guaA encodes guanosine monophosphate synthetase that confers an ability to biosynthesize guanine nucleotides de novo. This enables bacterial colonization in different environments and, while guaA is widely distributed among Bacteroidetes and Firmicutes, its contribution to the inhabitation of the human microbiome by commensal bacteria is unclear. We studied Streptococcus as a commensal urogenital tract bacterium and opportunistic pathogen, and explored the role of guaA in bacterial survival and colonization of urine. Analysis of guaA-deficient Streptococcus revealed guanine utilization is essential for bacterial colonization of this niche. The genomic location of guaA in other commensals of the human urogenital tract revealed substantial cross-phyla diversity and organizational structures of guaA that are divergent across phyla. Essentiality of guaA for Streptococcus colonization in the urinary tract establishes that purine biosynthesis is a critical element of the ability of this bacterium to survive and colonize in the host as part of the resident human microbiome.

Peri-implant treatment reduces the salivary levels of Colony stimulator factor-1 and S100A8/A9.

This study evaluated the impact of peri-implant treatment in the salivary levels of Colony stimulator factor -1 (CSF-1), S100A8/A9 and S100A12 in patients having mucositis or peri-implantitis. As a secondary aim, we analysed the correlation between the salivary and peri-implant crevicular fluid (PICF) levels. Forty-seven patient, 27 having mucositis (mean age 63.11 ± 7.78) and 20 having peri-implantitis (61.25 ± 7.01) participated in the study. Clinical parameters, probing pocket depth, clinical attachment level, % of plaque and bleeding on probing were evaluated. Unstimulated whole saliva was collected from all patients, while PICF was collected only from a patient's subgroup (n = 20). Samples were collected before and 3 months after peri-implant treatment. Enzyme-linked immunosorbent assays determined levels of CSF-1, S100A8/A9 and S100A12. Clinical parameters improved and salivary levels of CSF-1 and S100A8/A9, but not S100A12, reduced significantly after treatment in both groups. No significant correlation was found in the salivary and PICF levels of the same molecule. In conclusion, the treatment of peri-implant disease significantly improved the clinical parameters and reduced the salivary levels of CSF-1 and S100A8/A9. The salivary expressions of CSF-1, S100A8/A9 and S100A12 did not correlate with their own expression in PICF.

Novel technique using cold atmospheric plasma coupled with air-polishing for the treatment of titanium discs grown with biofilm: An in-vitro study.

OBJECTIVES: The aim of the present study was to assess the decontamination efficacy and titanium surface alterations of erythritol based air abrasion (AA) and cold atmospheric plasma (CAP) targeting a human complex biofilm. METHODS: Saliva collected from a peri-implantitis patient was used to develop in vitro human biofilm over titanium discs with machined (group A) and moderately rough (group B) surface. The discs were treated with AA, experimental CAP or a combination of both treatments (COM). The amount of biofilm on the discs was measured by crystal violet (CV). Surface features and roughness before and after treatment were assessed by SEM and laser profilometry, respectively. The data were statistically analyzed using Kruskal Wallis followed by Dunn's multiple comparison test after being checked for normality by Shapiro-Wilk test. RESULTS: All the discs in group A performed better to treatments compared to group B. In both groups, CV data showed significantly lower amount of biofilm after AA treatment compared to CAP (p<0.05). Cleaning efficacy revealed relevant decontamination of both the surfaces following AA and COM treatments and almost complete biofilm removal after AA application on group A (99.92%). SEM analysis demonstrated no post-treatment alterations on the discs and laser profilometry did not show statistically significant changes in Sa and Sdr values. SIGNIFICANCE: Decontamination with AA delivering erythritol with or without CAP is highly effective in biofilm removal from different titanium surfaces. All the tested treatments, including CAP showed no noticeable alterations of the titanium discs surface features. Further in vivo studies are necessary to understand the potential of CAP technology in implant surface decontamination.

Restriction of chronic Escherichia coli urinary tract infection depends upon T cell-derived interleukin-17, a deficiency of which predisposes to flagella-driven bacterial persistence.

Urinary tract infections (UTI) frequently progress to chronicity in infected individuals but the mechanisms of pathogenesis underlying chronic UTI are not well understood. We examined the role of interleukin (IL)-17A in UTI because this cytokine promotes innate defense against uropathogenic Escherichia coli (UPEC). Analysis of UPEC persistence and pyelonephritis in mice deficient in IL-17A revealed that UPEC CFT073 caused infection at a rate higher than the multidrug resistant strain EC958. Il17a-/- mice exhibited pyelonephritis with kidney bacterial burdens higher than those of wild-type (WT) mice. Synthesis of IL-17A in the bladder reflected a combination of γδ-T and TH 17 cell responses. Analysis of circulating inflammatory mediators at 24h postinoculation identified predictors of progression to chronicity, including IL-6 and monocyte chemoattractant protein-1 (MCP-1). Histological analysis identified infiltrating populations of neutrophils, NK cells, and γδ T cells in the bladder, whereas neutrophils predominated in the kidney. Analysis of the contribution of flagella to chronicity using hyper-flagellated and fliC-deficient UPEC in WT and Il17a-/- mice revealed that, in a host that is deficient for the production of IL-17A, flagella contribute to bacterial persistence. These findings show a role for IL-17A in defense against chronic UTI and a contribution of flagella to the pathogenesis of infection.

A comprehensive comparison of cell seeding methods using highly porous melt electrowriting scaffolds.

Cell seeding is challenging in the case of additively manufactured 3-dimensional scaffolds, as the open macroscopic pore network impedes the retention of the seeding solution. The present study aimed at comparing several seeding conditions (no fetal bovine serum, 10% or 100% serum) and methods (Static seeding in Tissue Culture Treated plate (CT), Static seeding of the MES in non-Culture Treated plate (nCT), Seeding in nCT plate placed on an orbital shaker at 20 rpm (nCTR), Static seeding of the MES previously incubated with 100% FBS for 1 h to allow for protein adsorption (FBS)) commonly utilised in tissue engineering using highly porous melt electrowritten scaffolds, assessing their seeding efficacy, cell distribution homogeneity and reproducibility. Firstly, we demonstrated that the incubation in 100% serum was superior to the 10% serum pre-incubation and that 1 h only was sufficient to obtain enhanced cell attachment. We further compared this technique to the other methods and demonstrated significant and beneficial impact of the 100% serum pre-incubation, which resulted in enhanced efficacy, homogeneous cell distribution and high reproducibility, leading to accelerated colonisation/maturation of the tissue engineered constructs. We further showed the superior performance of this method using 3D-printed scaffolds also made of different polymers, demonstrating its capacity for up-scaling. Therefore, the pre-incubation of the scaffold in 100% serum is a simple yet highly effective method for enhancing cell adhesion and ensuring seeding reproducibility. This is crucial for tissue engineering applications, especially when cell availability is scarce, and for product standardisation from a translational perspective.

Silver Nanoparticles at Biocompatible Dosage Synergistically Increases Bacterial Susceptibility to Antibiotics.

Antibiotics used to treat bacterial infections can become ineffective over time or result in the emergence of antibiotic resistant pathogens. With the advent of nanotechnology, silver nanoparticles (AgNPs) have gained significant attention as a therapeutic agent due to the well-known antimicrobial properties of silver. However, there are concerns and limited literature on the potential cytotoxicity of nanoparticles at effective antimicrobial concentrations. AgNPs prepared from silver nitrate with glucose reduction were characterized by surface plasmon resonance, dynamic light scattering, zeta potential analysis and transmission electron microscopy. The cytotoxicity of AgNPs towards human gingival fibroblasts over 7 days was determined using cell proliferation assays and confocal microscopy. AgNP MIC and antibacterial effects alone and in combination with 11 antibiotics were determined against a panel of nine microbial species including gram-positive and gram-negative bacterial species. AgNPs concentrations ≤ 1 µg/mL were non-cytotoxic but also showed no antibacterial effects. However, when combined with each of eleven antibiotics, the biocompatible concentration of AgNPs (1 µg/mL) resulted in significant inhibition of bacterial growth for multiple bacterial species that were resistant to either the antibiotics or AgNPs alone. This study presents a promising strategy with further testing in vivo, to develop novel antimicrobial agents and strategies to confront emerging antimicrobial resistance.

Impact of non-surgical periodontal treatment on salivary expression of cytokines related to bone metabolism.

We aimed to evaluate the impact of non-surgical periodontal treatment on the salivary expression of leptin, TNF-α, sclerostin, parathyroid hormone, osteoprotegerin, osteopontin, osteocalcin, IL-6, IL-1ß and fibroblast growth factor 23 in patients with chronic periodontitis after 1 year of follow-up. Fifteen patients with chronic periodontitis (56.0 ± SD 9.6 years) and 15 subjects with gingivitis (39.7 ± SD 4.4 years) were included in the study. Clinical periodontal parameters, such as probing pocket depth (PPD), clinical attachment level (CAL), % of plaque and bleeding on probing (BOP) were evaluated, and non-stimulated whole saliva was collected from all patients before periodontal treatment and after 1 year of follow-up. A bead-based multiplex assay measured cytokines. In the chronic periodontitis group, periodontal treatment significantly improved clinical parameters and reduced the salivary levels of IL-1ß, leptin and TNF-α (p = 0.002, 0.007 and 0.015, respectively). In the gingivitis group, there were also significant improvements in the mean patient %BOP, % Plaque, CAL and PPD. However, there were no significant changes in the cytokine's salivary levels. In conclusion, chronic periodontitis patients showed a significant reduction in the salivary levels of leptin, TNF-α and IL-1ß 1 year after periodontal treatment and a significant improvement in their clinical periodontal parameters suggesting that periodontal treatment alone can downregulate important cytokines associated with bone metabolism.

Optimization of 3D bioprinting of periodontal ligament cells.

Three-dimensional (3D) bioprinting of cells is an emerging area of research but has been not explored yet in the context of periodontal tissue engineering. OBJECTIVE: This study reports on the optimisation of the 3D bioprinting of periodontal ligament cells for potential application in periodontal regeneration. METHODS: We systematically investigated the printability of various concentrations of gelatin methacryloyl (GelMA) hydrogel precursor using a microextrusion based three-dimensional (3D) printer. The influence of different printing parameters such as photoinitiator concentration, UV exposure, pressure and dispensing needle diameter on the viability of periodontal ligament cells encapsulated within the 3D bioprinted construct were subsequently assessed. RESULTS: This systematic evaluation enabled the selection of the most suited printing conditions for achieving high printing resolution, dimensional stability and cell viability for 3D bioprinting of periodontal ligament cells. SIGNIFICANCE: The optimised bioprinting system is the first step towards to the reproducible manufacturing of cell laden, space maintaining scaffolds for the treatment of periodontal lesions.

Type 2 NADH Dehydrogenase Is the Only Point of Entry for Electrons into the Streptococcus agalactiae Respiratory Chain and Is a Potential Drug Target.

The opportunistic pathogen Streptococcus agalactiae is the major cause of meningitis and sepsis in a newborn's first week, as well as a considerable cause of pneumonia, urinary tract infections, and sepsis in immunocompromised adults. This pathogen respires aerobically if heme and quinone are available in the environment, and a functional respiratory chain is required for full virulence. Remarkably, it is shown here that the entire respiratory chain of S. agalactiae consists of only two enzymes, a type 2 NADH dehydrogenase (NDH-2) and a cytochrome bd oxygen reductase. There are no respiratory dehydrogenases other than NDH-2 to feed electrons into the respiratory chain, and there is only one respiratory oxygen reductase to reduce oxygen to water. Although S. agalactiae grows well in vitro by fermentative metabolism, it is shown here that the absence of NDH-2 results in attenuated virulence, as observed by reduced colonization in heart and kidney in a mouse model of systemic infection. The lack of NDH-2 in mammalian mitochondria and its important role for virulence suggest this enzyme may be a potential drug target. For this reason, in this study, S. agalactiae NDH-2 was purified and biochemically characterized, and the isolated enzyme was used to screen for inhibitors from libraries of FDA-approved drugs. Zafirlukast was identified to successfully inhibit both NDH-2 activity and aerobic respiration in intact cells. This compound may be useful as a laboratory tool to inhibit respiration in S. agalactiae and, since it has few side effects, it might be considered a lead compound for therapeutics development.IMPORTANCES. agalactiae is part of the human intestinal microbiota and is present in the vagina of ~30% of healthy women. Although a commensal, it is also the leading cause of septicemia and meningitis in neonates and immunocompromised adults. This organism can aerobically respire, but only using external sources of heme and quinone, required to have a functional electron transport chain. Although bacteria usually have a branched respiratory chain with multiple dehydrogenases and terminal oxygen reductases, here we establish that S. agalactiae utilizes only one type 2 NADH dehydrogenase (NDH-2) and one cytochrome bd oxygen reductase to perform respiration. NADH-dependent respiration plays a critical role in the pathogen in maintaining NADH/NAD+ redox balance in the cell, optimizing ATP production, and tolerating oxygen. In summary, we demonstrate the essential role of NDH-2 in respiration and its contribution to S. agalactiae virulence and propose it as a potential drug target.

A dysbiotic mycobiome dominated by Candida albicans is identified within oral squamous-cell carcinomas.

The aim of this study was to characterize the mycobiome associated with oral squamous-cell carcinoma (OSCC). DNA was extracted from 52 tissue biopsies (cases: 25 OSCC; controls: 27 intra-oral fibro-epithelial polyps [FEP]) and sequenced for the fungal internal transcribed spacer 2 region using Illumina™ 2 x300bp chemistry. Merged reads were classified to species level using a BLASTN-algorithm with UNITE's named species sequences as reference. Downstream analyses were performed using QIIME™ and linear discriminant analysis effect size. A total of 364 species representing 160 genera and two phyla (Ascomycota and Basidiomycota) were identified, with Candida and Malassezia making up 48% and 11% of the average mycobiome, respectively. However, only five species and four genera were detected in ≥50% of the samples. The species richness and diversity were significantly lower in OSCC. Genera Candida, Hannaella, and Gibberella were overrepresented in OSCC; Alternaria and  Trametes  were more abundant in FEP. Species-wise, Candida albicans, Candida etchellsii, and a Hannaella luteola-like species were enriched in OSCC, while aHanseniaspora uvarum-like species, Malassezia restricta, and Aspergillus tamarii were the most significantly abundant in FEP. In conclusion, a dysbiotic mycobiome dominated by C. albicans was found in association with OSCC, a finding worth further investigation.

Complete Genome Sequence of Serotype III Streptococcus agalactiae Sequence Type 17 Strain 874391.

Here we report the complete genome sequence of Streptococcus agalactiae strain 874391. This serotype III isolate is a member of the hypervirulent sequence type 17 (ST-17) lineage that causes a disproportionate number of cases of invasive disease in humans and mammals. A brief historical context of the strain is discussed.

Effect of the Streptococcus agalactiae Virulence Regulator CovR on the Pathogenesis of Urinary Tract Infection.

Background: Streptococcus agalactiae can cause urinary tract infection (UTI). The role of the S. agalactiae global virulence regulator, CovR, in UTI pathogenesis is unknown. Methods: We used murine and human bladder uroepithelial cell models of UTI and S. agalactiae mutants in covR and related factors, including ß-hemolysin/cytolysin (ß-h/c), surface-anchored adhesin HvgA, and capsule to study the role of CovR in UTI. Results: We found that covR-deficient serotype III S. agalactiae 874391 was significantly attenuated for colonization in mice and adhesion to uroepithelial cells. Mice infected with covR-deficient S. agalactiae produced less proinflammatory cytokines than those infected with wild-type 874391. Acute cytotoxicity in uroepithelial cells triggered by covR-deficient but not wild-type 874391 was associated with significant caspase 3 activation. Mechanistically, covR mutation significantly altered the expression of several genes in S. agalactiae 874391 that encode key virulence factors, including ß-h/c and HvgA, but not capsule. Subsequent mutational analyses revealed that HvgA and capsule, but not the ß-h/c, exerted significant effects on colonization of the murine urinary tract in vivo. Conclusions: S. agalactiae CovR promotes bladder infection and inflammation, as well as adhesion to and viability of uroepithelial cells. The pathogenesis of S. agalactiae UTI is complex, multifactorial, and influenced by virulence effects of CovR, HvgA, and capsule.