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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.

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 analysed 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)-1β, IL-6, macrophage inflammatory protein (MIP)-1, tumour 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 effects bacterial persistence in the female reproductive 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.

Effectiveness of root canal disinfection greatly determines the success of endodontic treatment. The purpose of this study was to evaluate the bacterial killing efficacy of chlorhexidine (CHX) activated with a Gemni 980 nm diode laser. Bacterial kill at the apical one third of the endodontically prepared roots were assessed using the 3D fluorescent microscope (SZX16) after staining the tooth with BacLight SYTO 9 (Live/Dead). The percentage of bacterial kill was calculated by measuring the total amount of green pixels (live bacteria) and red pixels (dead bacteria) using the Image color summarizer image color statistics and clustering program (Circos—Circular Genome Visualization/Martin Krzywinski). The 3D fluorescent ex‐vivo tooth model allowed real time measurement of the percentage of bacterial kill at specified time points by capturing the bacterial kill on the external surface of the root, hence indicating the amount of penetration of the irrigation solution through the radicular dentine. Compared to CHX alone, both Endoactivator and diode laser treatment groups showed significantly better bacterial kill following a 72‐hour study period (P > .05). However, of clinical importance is the larger number of bacteria killed with laser assisted irrigation immediately following treatment when compared to the use of CHX alone.image

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.

Melioidosis, caused by the bacterium Burkholderia pseudomallei , is an often severe infection that regularly involves respiratory disease following inhalation exposure. Intranasal (i.n.) inoculation of mice represents an experimental approach used to study the contributions of bacterial capsular polysaccharide I (CPS I) to virulence during acute disease. We used aerosol delivery of B. pseudomallei to establish respiratory infection in mice and studied CPS I in the context of innate immune responses. CPS I improved B. pseudomallei survival in vivo and triggered multiple cytokine responses, neutrophil infiltration, and acute inflammatory histopathology in the spleen, liver, nasal-associated lymphoid tissue, and olfactory mucosa (OM). To further explore the role of the OM response to B. pseudomallei infection, we infected human olfactory ensheathing cells (OECs) in vitro and measured bacterial invasion and the cytokine responses induced following infection. Human OECs killed >90% of the B. pseudomallei in a CPS I-independent manner and exhibited an antibacterial cytokine response comprising granulocyte colony-stimulating factor, tumor necrosis factor alpha, and several regulatory cytokines. In-depth genome-wide transcriptomic profiling of the OEC response by RNA-Seq revealed a network of signaling pathways activated in OECs following infection involving a novel group of 378 genes that encode biological pathways controlling cellular movement, inflammation, immunological disease, and molecular transport. This represents the first antimicrobial program to be described in human OECs and establishes the extensive transcriptional defense network accessible in these cells. Collectively, these findings show a role for CPS I in B. pseudomallei survival in vivo following inhalation infection and the antibacterial signaling network that exists in human OM and OECs.

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.

Results from microbiome studies on oral cancer have been inconsistent, probably because they focused on compositional analysis, which does not account for functional redundancy among oral bacteria. Based on functional prediction, a recent study revealed enrichment of inflammatory bacterial attributes in oral squamous cell carcinoma (OSCC). Given the high relevance of this finding to carcinogenesis, we aimed here to corroborate them in a case-control study involving 25 OSCC cases and 27 fibroepithelial polyp (FEP) controls from Sri Lanka. DNA extracted from fresh biopsies was sequenced for the V1 to V3 region with Illumina’s 2 × 300–bp chemistry. High-quality nonchimeric merged reads were classified to the species level with a prioritized BLASTN-based algorithm. Downstream compositional analysis was performed with QIIME (Quantitative Insights into Microbial Ecology) and linear discriminant analysis effect size, while PICRUSt (Phylogenetic Investigation of Communities by Reconstruction of Unobserved States) was utilized for bacteriome functional prediction. The OSCC tissues tended to have lower species richness and diversity. Genera Capnocytophaga, Pseudomonas, and Atopobium were overrepresented in OSCC, while Lautropia, Staphylococcus, and Propionibacterium were the most abundant in FEP. At the species level, Campylobacter concisus, Prevotella salivae, Prevotella loeschii, and Fusobacterium oral taxon 204 were enriched in OSCC, while Streptococcus mitis, Streptococcus oral taxon 070, Lautropia mirabilis, and Rothia dentocariosa among others were more abundant in FEP. Functionally, proinflammatory bacterial attributes, including lipopolysaccharide biosynthesis and peptidases, were enriched in the OSCC tissues. Thus, while the results in terms of species composition significantly differed from the original study, they were consistent at the functional level, substantiating evidence for the inflammatory nature of the bacteriome associated with OSCC.

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.

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.

There is increasing interest in the possible role of the oral microbiome in oral squamous cell carcinoma (OSSC). However, studies on the possible association between the oral bacteriome and OSCC remain inconclusive, while the potential role of the “mycobiome” in oral carcinogenesis has never been explored. I hereby present results from our recent studies on the bacteriome and mycobiome associated with OSCC based on analysis of samples from Yemeni and Sri Lankan cohorts. Tissue biopsies were obtained from the cases while deep buccal swabs or fibro-epithelial polyps were collected from the controls. Sequencing of the bacterial V1-V3 region of the 16S rRNA gene and the fungal ITS2 region using Illumina’s 2x300 bp chemistry was employed to study the bacteriome and mycobiome, respectively. Merged reads were classified to species level using a BLASTN-algorithm. Downstream analyses were performed using QIIME, PICRUSt, and LEfSe. Fusobacterium nucleatum subsp. polymorphum, Pseudomonas aeruginosa, Prevotella and Campylobacter spp. were more abundant in OSCC samples while Streptococcus mitis and Rothia spp. were overrepresented in the controls. Functionally, inflammatory bacterial attributes including bacterial mobility, flagellar assembly, bacterial chemotaxis and LPS synthesis were enriched in the tumors. Mycobiome analysis revealed a dysbiotic fungal community dominated by C. albicans in association with OSCC.

Bacteriuria, or the presence of bacteria in urine, is associated with both asymptomatic and symptomatic urinary tract infection and underpins much of the dynamic of microbial colonization of the urinary tract. The prevalence of bacteriuria in dissimilar patient groups such as healthy adults, institutionalized elderly, pregnant women, and immune-compromised patients varies widely. In addition, assessing the importance of 'significant bacteriuria' in infected individuals represents a diagnostic challenge, partly due to various causal microorganisms, and requires careful consideration of the distinct etiologies of bacteriuria in different populations and circumstances. Recent molecular discoveries have revealed how some bacterial traits can enable organisms to grow in human urine, which, as a fitness adaptation, is likely to influence the progression of bacteriuria in some individuals. In this review, we comprehensively analyze currently available data on the prevalence of causal organisms with a focus on asymptomatic bacteriuria in dissimilar populations. We evaluate recent advances in the molecular detection of bacteriuria from a diagnostic viewpoint and briefly discuss the potential benefits and some of the challenges of these approaches. Overall, this review provides an update on the comparative prevalence and etiology of bacteriuria from both microbiological and clinical perspectives.

Immunotoxins are engineered chimeric proteins that consist of a fragment of a toxin fused to a modified antibody or growth factor capable of targeting specific cells. Furthermore, these proteins can be targeted to receptors that are commonly overexpressed on cancer cells. The majority of immunotoxins function by binding to cells, translocating into the cytosol and inhibiting protein synthesis. In this study, the expression of claudin‑4 (CLDN4) in various cancer cells was analysed as a potential target for immunotoxins. To target CLDN4-expressing cancer cells, the c-terminal CLDN4‑binding domain of Clostridium perfringens enterotoxin (CPE) was fused to the Pseudomonas aeruginosa exotoxin A (ETA) domain to create an immunotoxin (CPE‑ETA'). Subsequently, the capacity of such an immunotoxin in suppressing the proliferation of CLDN4-positive cancer cells was investigated. We report that head and neck squamous carcinoma cells (HN5) have an elevated CLDN4 expression compared to the other cell lines tested. Our findings further demonstrate that CPE‑ETA' is highly potent against MCF-7 breast [50% inhibitory concentration (IC50) 9.8 ng/ml] and HN5 head/neck (IC50 8.8 ng/ml) cancer cell lines, while it has no cytotoxic effects on HeLa cells (CLDN4‑negative). The immunotoxin was subsequently expressed in the tumour colonising oncolytic strain, Clostridium ghonii. Most importantly, the strictly anaerobic Clostridium ghonii was able to overexpress and secrete a functional CPE‑ETA' fusion protein. Our findings open the possibility of the targeted delivery of the immunotoxin locally to tumour sites at a high concentration using strictly anaerobic Clostridium ghonii for the treatment of CLDN4-positive cancer cells.

Background: Studies employing next-generation sequencing (NGS) show that the oral fungal community (mycobiome) is far more complex than hitherto thought. However, the role of the oral mycobiome in health and disease, including oral carcinogenesis, has not been explored. Objective: To characterize the mycobiome associated with oral squamous cell carcinoma (OSCC). Methods: Tissue biopsies [cases: 25 OSCC; controls: 27 intra-oral fibro-epithelial polyp (FEP)] were collected from oral and maxillofacial units in Sri Lanka. Total DNA was extracted and subjected to sequencing of the fungal ITS2 region using Illumina’s 2x300 bp chemistry. High quality, non-chimeric 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 LEfSe. Results: 364 species representing 160 genera and 2 phyla (Ascomycota and Basidiomycota) were identified, with Candida and Malassezia making up 48% and 11% of the average mycobiome, respectively. However, only 5 species and 4 genera were detected in ≥50% of the samples. The species richness and diversity were significantly lower in OSCC. At the genus level, Candida, Hannaella and Gibberella were overrepresented in OSCC while Alternaria and Trametes were more abundant in FEP. Species-wise, C. albicans, C. etchellsii and Hannaella luteola-like species were enriched in OSCC while Malassezia restricta, Aspergillus tamarii, Alternaria alternate, Cladosporium halotolerans, and Hanseniaspora uvarum-like species were the most significantly abundant in FEP. Conclusions: A dysbiotic mycobiome dominated by C. albicans was found in association with OSCC. Whether this dysbiosis plays a role in oral carcinogenesis warrants further investigation.

Streptococcus agalactiae can cause urinary tract infection (UTI) including cystitis and asymptomatic bacteriuria (ABU). The early host-pathogen interactions that occur during S. agalactiae UTI and subsequent mechanisms of disease pathogenesis are poorly defined. Here, we define the early interactions between human bladder urothelial cells, monocyte-derived macrophages, and mouse bladder using uropathogenic S. agalactiae (UPSA) 807 and ABU-causing S. agalactiae (ABSA) 834 strains. UPSA 807 adhered, invaded and killed bladder urothelial cells more efficiently compared to ABSA 834 via mechanisms including low-level caspase-3 activation, and cytolysis, according to lactate dehydrogenase release measures and cell viability. Severe UPSA 807-induced cytotoxicity was mediated entirely by the bacterial β-hemolysin/cytolysin (β-H/C) because an β-H/C-deficient UPSA 807 isogenic mutant, UPSA 807ΔcylE, was not cytotoxic in vitro; the mutant was also significantly attenuated for colonization in the bladder in vivo. Analysis of infection-induced cytokines, including IL-8, IL-1β, IL-6 and TNF-α in vitro and in vivo revealed that cytokine and chemokine responses were dependent on expression of β-H/C that also elicited severe bladder neutrophilia. Thus, virulence of UPSA 807 encompasses adhesion to, invasion of and killing of bladder cells, pro-inflammatory cytokine/chemokine responses that elicit neutrophil infiltration, and β-H/C-mediated subversion of innate immune-mediated bacterial clearance from the bladder.

Urinary tract infections (UTIs) are among the most common bacterial infections in humans. Murine models of human UTI are vital experimental tools that have helped to elucidate UTI pathogenesis and advance knowledge of potential treatment and infection prevention strategies. Fundamentally, several variables are inherent in different murine models, and understanding the limitations of these variables provides an opportunity to understand how models may be best applied to research aimed at mimicking human disease. In this review, we discuss variables inherent in murine UTI model studies and how these affect model usage, data analysis and data interpretation. We examine recent studies that have elucidated UTI host–pathogen interactions from the perspective of gene expression, and review new studies of biofilm and UTI preventative approaches. We also consider potential standards for variables inherent in murine UTI models and discuss how these might expand the utility of models for mimicking human disease and uncovering new aspects of pathogenesis.

The most common causes of urinary tract infections (UTIs) are Gram-negative pathogens such as Escherichia coli ; however, Gram-positive organisms, including Streptococcus agalactiae , or group B streptococcus (GBS), also cause UTI. In GBS infection, UTI progresses to cystitis once the bacteria colonize the bladder, but the host responses triggered in the bladder immediately following infection are largely unknown. Here, we used genome-wide expression profiling to map the bladder transcriptome of GBS UTI in mice infected transurethrally with uropathogenic GBS that was cultured from a 35-year-old women with cystitis. RNA from bladders was applied to Affymetrix Gene-1.0ST microarrays; quantitative reverse transcriptase PCR (qRT-PCR) was used to analyze selected gene responses identified in array data sets. A surprisingly small significant-gene list of 172 genes was identified at 24 h; this compared to 2,507 genes identified in a side-by-side comparison with uropathogenic E. coli (UPEC). No genes exhibited significantly altered expression at 2 h in GBS-infected mice according to arrays despite high bladder bacterial loads at this early time point. The absence of a marked early host response to GBS juxtaposed with broad-based bladder responses activated by UPEC at 2 h. Bioinformatics analyses, including integrative system-level network mapping, revealed multiple activated biological pathways in the GBS bladder transcriptome that regulate leukocyte activation, inflammation, apoptosis, and cytokine-chemokine biosynthesis. These findings define a novel, minimalistic type of bladder host response triggered by GBS UTI, which comprises collective antimicrobial pathways that differ dramatically from those activated by UPEC. Overall, this study emphasizes the unique nature of bladder immune activation mechanisms triggered by distinct uropathogens.

Group B streptococcus (GBS), also known as Streptococcus agalactiae is a Gram-positive, β-hemolytic, chain-forming bacterium and a commensal within the genital tract flora in approximately 25% of healthy adult women (Campbell et al., 2000). The organism is a leading cause of serious infection in newborns, pregnant women, and older persons with chronic medical illness (Baker et al., Edwards&Baker, 2005). In neonates GBS infection most commonly causes pneumonia, meningitis, and sepsis. In addition to maternal cervicovaginal colonization and neonatal infection that can result from vertical transmission of GBS from mothers to their infants, the bacterium can also cause urinary tract infection (UTI). The spectrum of GBS UTI includes asymptomatic bacteriuria (ABU), cystitis, pyelonephritis, urethritis, and urosepsis (Bronsema et al., 1993, Edwards&Baker, 2005, Farley et al., 1993, Lefevre et al., 1991, McKenna et al., 2003, Munoz et al., 1992, Ulett et al., 2009). GBS ABU is particularly common among pregnant women, although those most at risk for cystitis due to GBS appear to be elderly individuals (Edwards&Baker, 2005, Falagas et al., 2006, Muller et al., 2006). In addition to acute and asymptomatic UTI other invasive diseases caused by GBS infection include skin infections, bacteraemia, pneumonia, arthritis, and endocarditis (Liston et al., 1979, Patil & Martin, 2010, Tissi et al., 1997, Trivalle et al., 1998). Thus, GBS is considered unique in terms of its ability to cause a spectrum of diseases in newborns and adult humans and its ability to colonize the genital tract of healthy women in a commensal-type manner...