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Liquid biopsy is a revolutionary tool that is gaining momentum in the field of cancer research. As a body fluid, urine can be used in non-invasive diagnostics for various types of cancer. We investigated the performance of UAS™ as a preservative for urinary analytes. Firstly, the need for urine preservation was investigated using urine samples from healthy volunteers. Secondly, the performance of UAS™ was assessed for cell-free DNA (cfDNA) and host cell integrity during storage at room temperature (RT) and after freeze-thaw cycling. Finally, UAS™ was used in a clinical setting on samples from breast and prostate cancer patients. In the absence of a preservative, urinary cfDNA was degraded, and bacterial overgrowth occurred at RT. In urine samples stored in UAS™, no microbial growth was seen, and cfDNA and cellular integrity were maintained for up to 14 days at RT. After freeze-thaw cycling, the preservation of host cell integrity and cfDNA showed significant improvements when using UAS™ compared to unpreserved urine samples. Additionally, UAS™ was found to be compatible with several commercially available isolation methods.

Background: Periodontal disease represents a major health concern. The administration of beneficial microbes has been increasing in popularity over efforts to manipulate the microbes using antimicrobial agents. This study determined the ability of Streptococcus salivarius to inhibit IL-6 and IL-8 production by gingival fibroblasts when activated by periodontal pathogens and their effect on the salivary microbiome., Methods: Primary human gingival fibroblasts were challenged with Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum and a combination of all three. IL-6 and IL-8 cytokine release were measured. Using this same model, S. salivarius K12, M18 and different supernatant and whole-cell lysate fractions of S. salivarius K12 were administered to pathogen-induced fibroblasts. A patient study of healthy participants was also conducted to determine the effect S. salivarius K12 had on the native microbiome using 16S next generation sequence analysis., Results: All pathogens tested induced a significant IL-6 and IL-8 response. S. salivarius K12 or M18, did not exhibit an increase in inflammatory cytokines. When either of the probiotic strains were co-administered with a pathogen, there were significant reductions in both IL-6 and IL-8 release. This effect was also observed when gingival fibroblasts were pre-treated with either S. salivarius K12 or M18 and then stimulated with the oral pathogens. Chewing gum containing S. salivarius K12 did not alter the salivary microbiome and did not increase inflammatory markers in the oral cavity., Conclusion: S. salivarius K12 and M18 prevented immune activation induced by periodontal disease pathogens. S. salivarius K12 did not alter the salivary microbiome or induce immune activation when administered as a chewing gum. These results warrant further study to determine if it may be an effective treatment in a model of periodontal disease.

Objectives: Probiotics act as a unique approach to maintaining oral health by supplementing the endogenous oral bacteria with additional naturally occurring beneficial microbes to provide defense against pathogens harmful to teeth and gingiva. The aim of this pilot study was to clinically evaluate the effects of probiotics on plaque accumulation and gingival inflammation in subjects with fixed orthodontics., Methods: The pilot study was comprised of 15 healthy patients, aged 11 to 18 years, undergoing fixed orthodontic treatment. Patients used an all-natural, dissolving lozenge containing six proprietary probiotic strains (Dentaq® Oral and ENT Health Probiotic Complex)for 28 days. Gingival Index (GI) according to Löe-Silness and Plaque Index (PI) according to Quigley-Hein for all teeth were measured at baseline (Day Zero) and at the end of the probiotic regimen (Day 28)., Results: The mean baseline GI and PI scores within each patient decreased by 28.4% and 35.8%, respectively, by Day 28. Patients reported decreased tooth and gingival pain, decreased oral bleeding, and increased motivation to maintain proper oral hygiene over the course of the study., Conclusions: This pilot study provided preliminary support for the use of Dentaq Oral and ENT Health Probiotic Complex as a safe and effective natural health product for the reduction of plaque accumulation and gingival inflammation. The results demonstrate its potential therapeutic value and open the door for larger scale placebo-controlled clinical studies to verify these findings., Competing Interests: The authors do not declare any serious conflicts of interest. K.M.J. is an employee of Integra Medical Inc., but did not participate in clinical data collection. K.M.J. performed data analysis and manuscript drafting.

Oral candidiasis is a disease caused by opportunistic species of Candida that normally reside on human mucosal surfaces. The transition of Candida from budding yeast to filamentous hyphae allows for covalent attachment to oral epithelial cells, followed by biofilm formation, invasion and tissue damage. In this study, combinations of Lactobacillus plantarum SD5870, Lactobacillus helveticus CBS N116411 and Streptococcus salivarius DSM 14685 were assessed for their ability to inhibit the formation of and disrupt Candida albicans biofilms. Co-incubation with probiotic supernatants under hyphae-inducing conditions reduced C. albicans biofilm formation by >75 % in all treatment groups. Likewise, combinations of live probiotics reduced biofilm formation of C. albicans by >67 %. When live probiotics or their supernatants were overlaid on preformed C. albicans biofilms, biofilm size was reduced by >63 and >65 % respectively. Quantitative real-time PCR results indicated that the combined supernatants of SD5870 and CBS N116411 significantly reduced the expression of several C. albicans genes involved in the yeast-hyphae transition: ALS3 (adhesin/invasin) by 70 % (P < 0.0001), EFG1 (hyphae-specific gene activator) by 47 % (P = 0.0061), SAP5 (secreted protease) by 49 % (P < 0.0001) and HWP1 (hyphal wall protein critical to biofilm formation) by >99 % (P < 0.0001). These findings suggest the combination of L. plantarum SD5870, L. helveticus CBS N116411 and S. salivarius DSM 14685 is effective at both preventing the formation of and removing preformed C. albicans biofilms. Our novel results point to the downregulation of several Candida genes critical to the yeast-hyphae transition, biofilm formation, tissue invasion and cellular damage.

Persister cells represent a multidrug-tolerant (MDT), physiologically distinct subpopulation of bacteria. The ability of these organisms to survive lethal antibiotic doses raises concern over their potential role in chronic disease, such as recurrent urinary tract infection (RUTI). Persistence is believed to be conveyed through global metabolic dormancy, which yields organisms unresponsive to external stimuli. However, recent studies have contested this stance. Here, various antibiotics that target different cellular processes were used to dissect the activity of transcription, translation, and peptidoglycan turnover in persister cells. Differential susceptibility patterns were found in type I and type II persisters, and responses differed between Staphylococcus saprophyticus and Escherichia coli uropathogens. Further, SOS-deficient strains were sensitized to ciprofloxacin, suggesting DNA gyrase activity in persisters and indicating the importance of active DNA repair systems for ciprofloxacin tolerance. These results indicate that global dormancy per se cannot sufficiently account for antibiotic tolerance. Rather, the activity of individual cellular processes dictates multidrug tolerance in an antibiotic-specific fashion. Furthermore, the susceptibility patterns of persisters depended on their mechanisms of onset, with subinhibitory antibiotic pretreatments selectively shutting down cognate targets and increasing the persister fraction against the same agent. Interestingly, antibiotics targeting transcription and translation enhanced persistence against multiple agents indirectly related to these processes. Conducting these assays with uropathogenic E. coli isolated from RUTI patients revealed an enriched persister fraction compared to organisms cleared with standard antibiotic therapy. This finding suggests that persister traits are either selected for during prolonged antibiotic treatment or initially contribute to therapy failure.

Unlabelled: What's known on the subject? and What does the study add? Infection, encrustation and ureteral-stent-related symptoms (USRS) including pain, urgency and frequency are all major problems associated with stent use. No current ureteral stent or exogenously applied therapy adequately deals with these problems and antibiotic use is ineffective once a bacterial biofilm forms on the device. Triclosan is a broad spectrum antibacterial agent widely used in numerous healthcare products and has been previously shown to reduce inflammation on the skin and in the oral cavity. This study tested a triclosan-impregnated ureteral stent for its ability to reduce infection, encrustation and USRS. This study shows that while a triclosan-impregnated ureteral stent cannot reduce infection rates alone compared with antibiotic use, the stent can reduce several USRS including pain during indwelling. This study suggests that the triclosan eluting stent may have a role in treating patients, perhaps in combination with standard antibiotic therapy., Objective: To evaluate the capacity of triclosan-loaded ureteral stents to reduce stent-associated bacterial attachment, biofilm formation and encrustation, thereby potentially reducing infection development and other device-related sequelae., Patients and Methods: Twenty subjects requiring short-term stenting (7-15 days) were randomized to receive either a Percuflex Plus(®) non-eluting stent (control) or a Triumph(®) triclosan eluting stent. Control-stented subjects received 3 days of levofloxacin prophylaxis (500 mg once daily) while Triumph(®)-stented subjects did not. All subjects were assessed for positive urine and stent cultures, stent biofilm development and encrustation. Following device removal, each subject completed an analogue-scale symptom assessment questionnaire., Results: Ureteral stenting was performed after nine ureteroscopic and one extracorporeal shock wave lithotripsy procedure in the control group and eight ureteroscopic and two shock wave lithotripsy procedures in the triclosan group. No significant differences were observed for culture, biofilm and encrustation between the two groups. Subjects in the triclosan group reported significant reductions in lower flank pain scores during activity (58.1% reduction, P = 0.017) and urination (42.6%, P = 0.041), abdominal pain during activity (42.1%, P = 0.042) and urethral pain during urination (31.7%, P = 0.049)., Conclusions: In this study, the use of the Triumph(®) triclosan eluting stent had no marked impact on biofilm formation, encrustation or infection development in short-term stented patients. The Triumph(®) device led to significant reductions in several common ureteral-stent-related symptoms, supporting its use in this patient population., (© 2012 BJU INTERNATIONAL.)

Background and Purpose: Staphylococcus saprophyticus is a frequent cause of both uncomplicated and complicated urinary tract infections (UTI) in young females and has recently been established as the most prominent gram-positive uropathogen. Although the effects of subinhibitory concentrations of antimicrobials on numerous other pathogenic bacteria have been studied, little is known regarding how S saprophyticus responds under such conditions., Materials and Methods: In this study, we investigated the effects of subminimum inhibitory concentrations (MIC) of ciprofloxacin (CIP) on S saprophyticus attachment to glass microscope slides, ureteral stent material, and T24 bladder cells, as well as its effects on S saprophyticus-induced proinflammatory cytokine expression in bladder cells., Results: Adherence to glass microscope slides, ureteral stent material, and bladder cell monolayers were all significantly increased in the presence of sub-MIC levels of CIP. While the S saprophyticus challenge of T24 bladder cell monolayers significantly upregulated both interleukin (IL)-6 and IL-8 expression, sub-MIC CIP abrogated these effects, returning their secretion to control levels., Conclusions: Our results demonstrate that exposure to sub-MIC CIP increases S saprophyticus adherence to both abiotic and biotic surfaces including urinary device material and cultured bladder cells. In addition, low levels of this antimicrobial downregulate S saprophyticus-stimulated proinflammatory cytokine secretion in the bladder. These changes may make S saprophyticus more effective at colonizing the urinary tract and highlights the need for clinicians to consider the impact of subinhibitory concentrations of antimicrobials on bacteria when designing treatment strategies to manage UTI.

We describe an efficient high-throughput method for accurate DNA sequencing of entire cDNA clones. Developed as part of our involvement in the Mammalian Gene Collection full-length cDNA sequencing initiative, the method has been used and refined in our laboratory since September 2000. Amenable to large scale projects, we have used the method to generate >7 Mb of accurate sequence from 3695 candidate full-length cDNAs. Sequencing is accomplished through the insertion of Mu transposon into cDNAs, followed by sequencing reactions primed with Mu-specific sequencing primers. Transposon insertion reactions are not performed with individual cDNAs but rather on pools of up to 96 clones. This pooling strategy reduces the number of transposon insertion sequencing libraries that would otherwise be required, reducing the costs and enhancing the efficiency of the transposon library construction procedure. Sequences generated using transposon-specific sequencing primers are assembled to yield the full-length cDNA sequence, with sequence editing and other sequence finishing activities performed as required to resolve sequence ambiguities. Although analysis of the many thousands (22 785) of sequenced Mu transposon insertion events revealed a weak sequence preference for Mu insertion, we observed insertion of the Mu transposon into 1015 of the possible 1024 5mer candidate insertion sites.