Cranberry-derived proanthocyanidins induce a differential transcriptomic response within Candida albicans urinary biofilms.
ABSTRACT: Candida albicans is one of the most common causes of hospital-acquired urinary tract infections (UTIs). However, azoles are poorly active against biofilms, echinocandins do not achieve clinically useful urinary concentrations, and amphotericin B exhibits severe toxicities. Thus, novel strategies are needed to prevent Candida UTIs, which are often associated with urinary catheter biofilms. We previously demonstrated that cranberry-derived proanthocyanidins (PACs) prevent C. albicans biofilm formation in an in vitro urinary model. To elucidate functional pathways unique to urinary biofilm development and PAC inhibition, we investigated the transcriptome of C. albicans in artificial urine (AU), with and without PACs. C. albicans biofilm and planktonic cells were cultivated with or without PACs. Genome-wide expression analysis was performed by RNA sequencing. Differentially expressed genes were determined using DESeq2 software; pathway analysis was performed using Cytoscape. Approximately 2,341 of 6,444 total genes were significantly expressed in biofilm relative to planktonic cells. Functional pathway analysis revealed that genes involved in filamentation, adhesion, drug response and transport were up-regulated in urinary biofilms. Genes involved in carbon and nitrogen metabolism and nutrient response were down-regulated. In PAC-treated urinary biofilms compared to untreated control biofilms, 557 of 6,444 genes had significant changes in gene expression. Genes downregulated in PAC-treated biofilms were implicated in iron starvation and adhesion pathways. Although urinary biofilms share key features with biofilms formed in other environments, many genes are uniquely expressed in urinary biofilms. Cranberry-derived PACs interfere with the expression of iron acquisition and adhesion genes within urinary biofilms.
Project description:Cranberry A-type proanthocyanidins (PACs) have been recognized for their inhibitory activity against bacterial adhesion and biofilm-derived infections. However, the precise identification of the specific classes of degree-of-polymerization (DP) conferring PACs bioactivity remains a major challenge owing to the complex chemistry of these flavonoids. In this study, chemically characterized cranberries were used in a multistep separation and structure-determination technique to isolate A-type PAC oligomers of defined DP. The influences of PACs on the 3D architecture of biofilms and Streptococcus mutans-transcriptome responses within biofilms were investigated. Treatment regimens that simulated topical exposures experienced clinically (twice-daily, 60?s each) were used over a saliva-coated hydroxyapatite biofilm model. Biofilm accumulation was impaired, while specific genes involved in the adhesion of bacteria, acid stress tolerance, and glycolysis were affected by the topical treatments (vs the vehicle-control). Genes (rmpC, mepA, sdcBB, and gbpC) associated with sucrose-dependent binding of bacteria were repressed by PACs. PACs of DP 4 and particularly DP 8 to 13 were the most effective in disrupting bacterial adhesion to glucan-coated apatitic surface (>85% inhibition vs vehicle control), and gene expression (eg rmpC). This study identified putative molecular targets of A-type cranberry PACs in S. mutans while demonstrating that PAC oligomers with a specific DP may be effective in disrupting the assembly of cariogenic biofilms.
Project description:BACKGROUND: Bacteria within a biofilm are phenotypically more resistant to antibiotics, desiccation, and the host immune system, making it an important virulence factor for many microbes. Cranberry juice has long been used to prevent infections of the urinary tract, which are often related to biofilm formation. Recent studies have found that the A-type proanthocyanidins from cranberries have anti-biofilm properties against Escherichia coli. METHODS: Using crystal violet biofilm staining, resazurin metabolism assays, and confocal imaging, we examined the ability of A-type proanthocyanidins (PACs) to disrupt the biofilm formation of Pseudomonas aeruginosa. We used mass spectrometry to analyze the proteomic effects of PAC treatment. We also performed synergy assays and in vitro and in vivo infections to determine whether PACs, alone and in combination with gentamicin, could contribute to the killing of P. aeruginosa and the survival of cell lines and G. mellonella. RESULTS: Cranberry PACs reduced P. aeruginosa swarming motility. Cranberry PACs significantly disrupted the biofilm formation of P. aeruginosa. Proteomics analysis revealed significantly different proteins expressed following PAC treatment. In addition, we found that PACs potentiated the antibiotic activity of gentamicin in an in vivo model of infection using G. mellonella. CONCLUSIONS: Results suggest that A-type proanthocyanidins may be a useful therapeutic against the biofilm-mediated infections caused by P. aeruginosa and should be further tested.
Project description:<label>BACKGROUND</label>Urinary tract infections (UTIs) are amongst the most common bacterial infections affecting women. Although antibiotics are the treatment of choice for UTI, cranberry derived products have been used for many years to prevent UTIs, with limited evidence as to their efficacy. Our objective is to assess the efficacy of a cranberry extract capsule standardized in A-type linkage proanthocyanidins (PACs) for the prevention of recurrent urinary tract infection.<label>METHODS</label>We will perform a 1:1 randomized, controlled, double blind clinical trial in women aged 18 years or more who present ≥2 UTIs in 6 months or ≥ 3 UTIs in 12 months. One hundred and forty-eight women will be recruited and randomized in two groups to either receive an optimal dose of cranberry extract quantified and standardized in PACs (2 × 18.5 mg PACs per day) or a control dose (2 × 1 mg PACs per day). The primary outcome for the trial is the mean number of new symptomatic UTIs in women during a 6-month intervention period. Secondary outcomes are: (1) To evaluate the mean number of new symptomatic UTIs with pyuria as demonstrated by a positive leucocyte esterase test; (2) To detect the mean number of new symptomatic culture-confirmed UTIs; (3) To quantify urinary PACs metabolites in women who take a daily dose of 37 mg PACs per day compared to women who take a daily dose of 2 mg per day for 6 months; (4) To characterize women who present recurrent UTI based on known risk factors for recurrent UTI; (5) To describe the side effects of daily intake of cranberry extract containing 37 mg PACs compared to 2 mg PACs. This report provides comprehensive methodological data for this randomized controlled trial.<label>DISCUSSION</label>The results of this trial will inform urologists, gynaecologists, family physicians and other healthcare professionals caring for healthy women with recurrent UTI, as to the benefits of daily use of an optimal dose of cranberry extract for the prevention of recurrent UTI.<label>TRIAL REGISTRATION</label>Clinicaltrials.gov, identifier: NCT02572895 October 8th 2015.
Project description:The exopolysaccharides (EPS) produced by Streptococcus mutans-derived glucosyltransferases (Gtfs) are essential virulence factors associated with the initiation of cariogenic biofilms. EPS forms the core of the biofilm matrix-scaffold, providing mechanical stability while facilitating the creation of localized acidic microenvironments. Cranberry flavonoids, such as A-type proanthocyanidins (PACs) and myricetin, have been shown to inhibit the activity of Gtfs and EPS-mediated bacterial adhesion without killing the organisms. Here, we investigated whether a combination of cranberry flavonoids disrupts EPS accumulation and S. mutans survival using a mixed-species biofilm model under cariogenic conditions. We also assessed the impact of cranberry flavonoids on mechanical stability and the in situ pH at the biofilm-apatite interface. Topical application of an optimized combination of PACs oligomers (100-300 ?M) with myricetin (2 mM) twice daily was used to simulate treatment regimen experienced clinically. Treatments with cranberry flavonoids effectively reduced the insoluble EPS content (>80% reduction vs. vehicle-control; p<0.001), while hindering S. mutans outgrowth within mixed-species biofilms. As a result, the 3D architecture of cranberry-treated biofilms was severely compromised, showing a defective EPS-matrix and failure to develop microcolonies on the saliva-coated hydroxyapatite (sHA) surface. Furthermore, topical applications of cranberry flavonoids significantly weaken the mechanical stability of the biofilms; nearly 90% of the biofilm was removed from sHA surface after exposure to a shear stress of 0.449 N/m2 (vs. 36% removal in vehicle-treated biofilms). Importantly, in situ pH measurements in cranberry-treated biofilms showed significantly higher pH values (5.2 ± 0.1) at the biofilm-apatite interface vs. vehicle-treated biofilms (4.6 ± 0.1). Altogether, the data provide important insights on how cranberry flavonoids treatments modulate virulence properties by disrupting the biochemical and ecological changes associated with cariogenic biofilm development, which could lead to new alternative or adjunctive antibiofilm/anticaries chemotherapeutic formulations.
Project description:Uropathogenic Escherichia coli (UPEC), the most prevalent bacteria isolated in urinary tract infections (UTI), is now frequently resistant to antibiotics used to treat this pathology. The antibacterial properties of cranberry and propolis could reduce the frequency of UTIs and thus the use of antibiotics, helping in the fight against the emergence of antibiotic resistance. Transcriptomic profiles of a clinical UPEC strain exposed to cranberry proanthocyanidins alone (190?µg/mL), propolis alone (102.4?µg/mL) and a combination of both were determined. Cranberry alone, but more so cranberry?+?propolis combined, modified the expression of genes involved in different essential pathways: down-expression of genes involved in adhesion, motility, and biofilm formation, and up-regulation of genes involved in iron metabolism and stress response. Phenotypic assays confirmed the decrease of motility (swarming and swimming) and biofilm formation (early formation and formed biofilm). This study showed for the first time that propolis potentiated the effect of cranberry proanthocyanidins on adhesion, motility, biofilm formation, iron metabolism and stress response of UPEC. Cranberry?+?propolis treatment could represent an interesting new strategy to prevent recurrent UTI.
Project description:BACKGROUND:Bacterial urinary tract infections (UTIs) are common in companion animals. Increasing awareness of biofilm-forming bacteria raises concern regarding the appropriate diagnosis, treatment, and prognosis of UTIs associated with these organisms. HYPOTHESIS/OBJECTIVES:To (1) describe the population of dogs with UTIs associated with biofilm-forming Escherichia coli and (2) determine whether or not clinical differences exist between dogs with biofilm-forming E. coli UTIs and dogs with nonbiofilm-forming E. coli UTIs. We hypothesized that there would be no difference in the population characteristics, but that biofilm-formation would be more prevalent in dogs with chronic, complicated, and asymptomatic UTIs. ANIMALS:Seventy-six client-owned dogs with E. coli UTIs, divided into 2 groups based on the biofilm-forming capability of stored bacterial isolates as assessed by the crystal violet assay. METHODS:Retrospective cross-sectional study. Medical records of the affected dogs were reviewed and their population and infection characteristics were compared. RESULTS:Most (52.6%) E. coli isolates were capable of forming biofilms. Biofilm-forming E. coli had a lower likelihood (P?<?.001) of multidrug resistance than did nonbiofilm-forming E. coli. No statistically significant differences were identified between the population or infection characteristics of the 2 groups of dogs. CONCLUSIONS AND CLINICAL IMPORTANCE:Escherichia coli isolated from canine urinary tracts are frequently capable of forming biofilms. Because no reliable clinical features allowed exclusion of biofilm formation, the potential for biofilm formation should be considered whenever E. coli UTI is diagnosed. The association of antibiotic resistance and biofilm potential may affect treatment of UTIs, but additional investigation is warranted.
Project description:Urinary tract infections (UTIs) are amongst the most common and prevalent infectious diseases worldwide, with uropathogenic Escherichia coli (UPEC) reported as the main causative pathogen. Fluoroquinolone antibiotics are commonly used to treat UTIs but for infections involving UPEC biofilms, which are commonly associated with catheter use and recurrent episodes, ciprofloxacin is often ineffective. Here we report the development of a ciprofloxacin-dinitroxide (CDN) conjugate with potent UPEC biofilm-eradication activity. CDN 11 exhibited a 2-fold increase in potency over the parent antibiotic ciprofloxacin against UPEC biofilms. Moreover, CDN 11 resulted in almost complete UPEC biofilm cell eradication (99.7%) at concentrations as low as 12.5 ?M, and significantly potentiated ciprofloxacin's biofilm-eradication activity against UPEC upon co-administration. The biofilm-eradication activity of CDN 11 highlights the potential of nitroxide functionalized antibiotics as a promising strategy for the treatment of biofilm-related UTIs.
Project description:Urinary tract infections (UTI) are the most common hospital-acquired infections in humans and are caused primarily by uropathogenic Escherichia coli (UPEC). Indwelling urinary catheters become encrusted with UPEC biofilms that are resistant to common antibiotics, resulting in chronic infections. Therefore, it is important to control UPEC biofilms on catheters to reduce the risk for UTIs. This study investigated the efficacy of selenium for inhibiting and inactivating UPEC biofilms on urinary catheters. Urinary catheters were inoculated with UPEC and treated with 0 and 35 mM selenium at 37 °C for 5 days for the biofilm inhibition assay. In addition, catheters with preformed UPEC biofilms were treated with 0, 45, 60, and 85 mM selenium and incubated at 37 °C. Biofilm-associated UPEC counts on catheters were enumerated on days 0, 1, 3, and 5 of incubation. Additionally, the effect of selenium on exopolysacchride (EPS) production and expression of UPEC biofilm-associated genes was evaluated. Selenium at 35 mM concentration was effective in preventing UPEC biofilm formation on catheters compared to controls (p < 0.05). Further, this inhibitory effect was associated with a reduction in EPS production and UPEC gene expression. Moreover, at higher concentrations, selenium was effective in inactivating preformed UPEC biofilms on catheters as early as day 3 of incubation. Results suggest that selenium could be potentially used in the control of UPEC biofilms on urinary catheters.
Project description:OBJECTIVE:Urinary tract infections (UTIs) are the most common infectious diseases, and Escherichia coli is the most common pathogen isolated from patients with UTIs. The products of sfa, afa and foc genes are important for binding of the bacterium to urinary tract epithelium. Our aim was to investigate these genes in E. colis isolated from patients with UTIS. The frequencies of the genes were determined using PCR. Biofilm formation and antibiotic resistance rates were determined using microtiter plate and disk diffusion methods, respectively. The P?<?0.05 was considered statistically significant. RESULTS:The frequencies of sfa, afa and foc were 75.3%, 17.5% and 22.5%, respectively showing a significantly higher prevalence of the sfa gene. The most effective antibiotics against the E. colis were nitrofurantoin and amikacin. The highest microbial resistance rates were also observed against amoxicillin and ampicillin. Furthermore, 12.7%, 6.3%, 74.7% and 6.3% of the isolates showed strong, moderate, weak capacities and no connections to form biofilms, respectively. The expression of the sfa gene was significantly associated with forming strong biofilms. Regarding the variabilities in the characteristics of E. coli strains associated with UTIs, it seems reasonable to adjust diagnostic and therapeutic methods according to the regional microbial characteristics.
Project description:BACKGROUND: Candida albicans can form biofilms on intravenous catheters; this process plays a key role in the pathogenesis of catheter infections. This study evaluated the effect of human serum (HS) on C. albicans biofilm formation and the expression of adhesion-related genes in vitro. A C. albicans laboratory strain (ATCC90028) and three clinical strains were grown for 24 h in RPMI 1640 supplemented with HS or RPMI 1640 alone (as a control). The growth of biofilm cells of four strains was monitored by a Live Cell Movie Analyzer, and by XTT reduction assay. The expression of the adhesion-related genes BCR1, ALS1, ALS3, HWP1 and ECE1 was analyzed by RT-PCR at three time points (60 min, 90 min, and 24 h). RESULTS: In the adhesion phase, C. albicans cells kept a Brownian movement in RPMI medium containing HS until a large number of germ tubes were formed. In the control group, C. albicans cells quickly adhered to the bottom of the reaction plate. Compared with RPMI 1640, medium supplemented with 3-50% HS caused a significant decrease in biofilm development (all p < 0.001). However, the presence of HS had no significant inhibitory effect on the pre-adhered biofilms (all p > 0.05). Biofilm formation was also inhibited by heat-inactivated and proteinase K pre-treated HS. The presence of 50% HS did not significantly affect the planktonic growth of C. albicans (p > 0.05). At three time points, HS inhibited expression of the ALS1 and ALS3 genes and promoted expression of the HWP1 and ECE1 genes. Significant up-regulation of BCR1 was observed only at the 90-min point. CONCLUSIONS: Human serum reduces biofilm formation by inhibiting the adhesion of C. albicans cells. This response may be associated with the down-regulation of adhesion-related genes ALS1, ALS3 and BCR1. The inhibitory serum component is protease-resistant and heat stable.