Uropathogenic Escherichia coli infection: innate immune disorder, bladder damage, and Tailin Fang II.

Background: Uropathogenic Escherichia coli (UPEC) activates innate immune response upon invading the urinary tract, whereas UPEC can also enter bladder epithelial cells (BECs) through interactions with fusiform vesicles on cell surfaces and subsequently escape from the vesicles into the cytoplasm to establish intracellular bacterial communities, finally evading the host immune system and leading to recurrent urinary tract infection (RUTI). Tailin Fang II (TLF-II) is a Chinese herbal formulation composed of botanicals that has been clinically proven to be effective in treating urinary tract infection (UTI). However, the underlying therapeutic mechanisms remain poorly understood. Methods: Network pharmacology analysis of TLF-II was conducted. Female Balb/C mice were transurethrally inoculated with UPEC CFT073 strain to establish the UTI mouse model. Levofloxacin was used as a positive control. Mice were randomly divided into four groups: negative control, UTI, TLF-II, and levofloxacin. Histopathological changes in bladder tissues were assessed by evaluating the bladder organ index and performing hematoxylin-eosin staining. The bacterial load in the bladder tissue and urine sample of mice was quantified. Activation of the TLR4-NF-κB pathway was investigated through immunohistochemistry and western blotting. The urinary levels of interleukin (IL)-1ß and IL-6 and urine leukocyte counts were monitored. We also determined the protein expressions of markers associated with fusiform vesicles, Rab27b and Galectin-3, and levels of the phosphate transporter protein SLC20A1. Subsequently, the co-localization of Rab27b and SLC20A1 with CFT073 was examined using confocal fluorescence microscopy. Results: Data of network pharmacology analysis suggested that TLF-II could against UTI through multiple targets and pathways associated with innate immunity and inflammation. Additionally, TLF-II significantly attenuated UPEC-induced bladder injury and reduced the bladder bacterial load. Meanwhile, TLF-II inhibited the expression of TLR4 and NF-κB on BECs and decreased the urine levels of IL-1ß and IL-6 and urine leukocyte counts. TLF-II reduced SLC20A1 and Galectin-3 expressions and increased Rab27b expression. The co-localization of SLC20A1 and Rab27b with CFT073 was significantly reduced in the TLF-II group. Conclusion: Collectively, innate immunity and bacterial escape from fusiform vesicles play important roles in UPEC-induced bladder infections. Our findings suggest that TLF-II combats UPEC-induced bladder infections by effectively mitigating bladder inflammation and preventing bacterial escape from fusiform vesicles into the cytoplasm. The findings suggest that TLF-II is a promising option for treating UTI and reducing its recurrence.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

Lactobacillus crispatus Limits Bladder Uropathogenic E. coli Infection by Triggering a Host Type I Interferon Response.

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus in vitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.

Antiadhesive natural products against uropathogenic E. coli: What can we learn from cranberry extract?

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts from Cranberry fruits (Vaccinium macrocarpon) are traditionally used against urinary tract infections, mainly due to antiadhesive activity against uropathogenic E. coli (UPEC), but the exact mode of action and compounds, responsible for the activity, are unknown. AIM OF THE STUDY: i. To investigate if cranberry extract acts only by a single component or must be assessed as a multi-active-compound preparation; ii to screen isolated cranberry-related natural products under in vitro conditions to pinpoint natural products with antiadhesive effects against UPEC, followed by in silico calculations (QSAR) to predict potential antiadhesive compounds; iii. investigations by using urine samples from cranberry treated volunteers for evaluation on the bacterial transcriptome and the mannose-binding side of FimH, iv. to investigate if besides Tamm Horsfall Protein induction in the kidney, the extract acts also directly against UPEC. MATERIAL AND METHODS: Antiadhesive activity of 105 compounds was determined by flow cytometric adhesion assay (UPEC UTI89 on T24 bladder cells). Urine samples from 16 volunteers treated with cranberry extract (p.o., 7 days, 900 mg/day) were used for ex vivo testing concerning influence on the bacterial transcriptome (Illumina RNA-seq) and interaction with the mannose binding domain of type-1 fimbriae. RESULTS: i. The antiadhesive effect of cranberry extract cannot be attributed to a single compound or to a single fraction. ii. Unglycosylated flavones and flavonols with bulky substitution of the B ring contribute to the antiadhesive activity. 3'-8″-biflavones and flavolignans (not related to cranberry fruits) were identified as potent antiadhesive compounds against UPEC. iii. QSAR yielded a model with good statistical performance and sufficient internal and external predictive ability. iv. Urine samples from male cranberry-treated volunteers indicated significant interaction with the mannose binding domain of type-1 fimbriae, which correlated with the amount of Tamm-Horsfall Protein in the test samples. v Cranberry extract did not influence the UPEC transcriptome; gene expression of bacterial adhesins (P-, S-fimbrae, curli) was not influenced by the urine samples, while a slight, but non-significant upregulation of type 1 fimbriae was observed. CONCLUSIONS: B-ring substituted flavones and flavonols from cranberry contribute to the antiadhesive activity against UPEC by inhibition of the FimH-mediated interaction with the host cell bladder epithelium.

Evaluation of pooled human urine and synthetic alternatives in a dynamic bladder infection in vitro model simulating oral fosfomycin therapy.

The impact of the bladder environment on fosfomycin activity and treatment response is uncertain. Standard laboratory media does not reflect the biomatrix of urine, where limited nutritional factors are important for growth and antimicrobial kill rates. We compared fosfomycin activity against Enterobacteriaceae in laboratory media, human urine and synthetic alternatives. Sixteen clinical isolates (8-Escherichia coli, 4-Enterobacter cloacae, 4-Klebsiella pneumoniae) were studied with broth microdilution (BMD) susceptibility, static time-kill assays and dynamic testing in a bladder infection model simulating a 3 g oral fosfomycin dose. Mueller-Hinton broth (MHB) with and without 25 mg/L glucose-6-phosphate (G6P), pooled midstream urine (MSU), pooled 24 h urine collection (24 U), artificial urine medium (AUM) and synthetic human urine (SHU) were compared. BMD susceptibility, bacterial growth and response to static fosfomycin concentrations in urine were best matched with SHU and were distinctly different when tested in MHB with G6P. Fosfomycin exposure in the bladder infection model was accurately reproduced (bias 4.7 ± 6.2%). Under all media conditions, 8 isolates (2-E. coli, 2-E. cloacae, 4-K. pneumoniae) re-grew and 4 isolates (4-E. coli) were killed. The remaining isolates (2-E. coli, 2-E. cloacae) re-grew variably in urine and synthetic media. Agar dilution MIC failed to predict re-growth, whereas BMD MIC in media without G6P performed better. Emergence of resistance was restricted in synthetic media. Overall, SHU provided the best substitute for urine for in vitro modelling of antimicrobial treatment of uropathogens, and these data have broader utility for improved preclinical testing of antimicrobials for urinary tract infections.

Crude polysaccharides from the seeds of Vaccaria segetalis prevent the urinary tract infection through the stimulation of kidney innate immunity.

ETHNOPHARMACOLOGICAL RELEVANCE: The seeds of Vaccaria segetalis (Neck.) Garcke is used for the treatment of urinary diseases in Traditional Chinese Medicine according to the Chinese Pharmacopoeia. Crude polysaccharides and the aqueous extract from the seeds of V. segetalis (SVCP) were proved to be effective on treating benign prostatic hyperplasia. AIM OF THE STUDY: The aim of this study was to test the effects of SVCP on urinary tract infection (UTI) induced by uropathogenic Escherichia coli (UPEC) strain CFT073 in the rat model and to investigate the underlying mechanisms. MATERIALS AND METHODS: A rat UTI model was established with the infection of UPEC strain CFT073. After oral administration of SVCP, the urinalysis and histological examination were evaluated. The levels of pro-inflammatory cytokines, procalcitonin (PCT) and polymeric Ig receptor (PIGR) were used to test the effects of SVCP on host immunity. The mRNA level of PapG in CFT073 was used to test the influence of SVCP on virulence factor. The effects of SVCP on the inhibition of bacterial adhesion were evaluated with mice UTI model. RESULTS: In the rat UTI model, the levels of bacterial load, white blood cells (WBC) and red blood cells (RBC) in urine and the pathological injury in the bladder were significantly up-regulated, the expression of PIGR in kidney was down-regulated, no significant change was observed on the pro-inflammatory cytokines in urine. After oral administration of SVCP for 3 days, the levels of bacterial load, WBC and RBC in urine were significantly decreased, the pathological injury in the bladder were remarkably inhibited. The expression of IL-6, IL-8 in urine and PIGR in kidney were significantly up-regulated by SVCP (200 mg/kg). SVCP showed no effect on the concentration of PCT in serum. SVCP failed to down-regulate the mRNA level of PapG in CFT073. In the mice UTI model, pre-treatment of SVCP failed to inhibit the intracellular bacterial load in the bladder. CONCLUSIONS: The therapeutic effects of SVCP on treating UTIs might result from the up-regulation of innate immunity in the kidney. SVCP can be used as an alternative therapeutic agent for UTIs.

Proposed dual antagonist approach for the prevention and treatment of urinary tract infections caused by uropathogenic Escherichia coli.

Urinary tract infections are among the most common infectious diseases worldwide, primarily caused by uropathogenic Escherichia coli (UPEC) strains that harbor type I pili and P pili on the surface. Standard E. coli therapy still entails antibiotic consumption, but urinary tract infections tend to recur at a very high rate. Due to the emergence of antibiotic resistant strains of UPEC, as well as high infection recurrence rates, there is a need for new approaches to efficiently treat and prevent urinary tract infections. Since aforementioned adhesive organelles are the principal virulence factors in UPEC, anti-adhesion strategy seems to be the most promising (and hitherto unexplored) treatment option. Here we propose an antiadhesive dual targeting approach towards FimH and PapG adhesive proteins placed on two key virulence factors for UPEC - type I fimbriae and P pili. Such dual antagonists will contain appropriate pharmacophores (mannose and natural cranberry-containing polyphenol) joined together and will more efficiently block the infection and prevent the progression of the disease in comparison to FimH and PapG as isolated targets. More specifically, polyphenol mannosides (due to the structural similarities with the most potent biaryl inhibitors) can act as high-affinity FimH ligands, while cranberry-associated polyphenol moiety can additionally inhibit the PapG-mediated adhesion. Proposed compound may also contribute to the antioxidant capacity of the human organism. In conclusion, this dual-target hypothesis for the prevention and treatment of UPEC infections represents an important foundation for further research on this topic.

Amaranthus caudatus extract inhibits the invasion of E. coli into uroepithelial cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Amaranthus caudatus is traditionally used to treat infections. Based on its traditional usage, we investigated the effect of A. caudatus on the bladder epithelial cells in the protection of E. coli infection. MATERIALS AND METHODS: The direct antimicrobial effects of A. caudatus on uropathogenic bacteria were investigated using minimum inhibitory concentration (MIC) assay. Bladder epithelial cell lines T24 and 5637 and uropathogenic E. coli strain #12 were used to investigate the effect of A. caudatus. Bacterial adhesion and invasion into bladder cells treated with A. caudatus was analyzed. Expression of uroplakin-1a (UPK1A), ß1 integrin (ITGB1), caveolin-1 (CAV1) and the antimicrobial peptides human ß defensin-2 (DEFB4A) and LL-37 (CAMP) was evaluated using RT-PCR. RESULTS: No direct antibacterial effect on E. coli or any of the tested uropathogenic strains was observed by A. caudatus. However, we demonstrated reduced mRNA expression of uroplakin-1a and caveolin-1, but not ß1 integrin after treatment of uroepithelial cells, mirrored by the decreased adhesion and invasion of E. coli. A. caudatus treatment did not induce increased gene expression of the antimicrobial peptides, LL-37 and human ß-defensin-2. CONCLUSIONS: Our results showed that A. caudatus has a protective role on bladder epithelial cells against uropathogenic E. coli infection by decreasing the bacterial adhesion and invasion, thereby preventing infection.

Antiadhesive hydroalcoholic extract from Apium graveolens fruits prevents bladder and kidney infection against uropathogenic E. coli.

Fruits from Apium graveolens (Celery) are used traditionally in Persian and European medicine for the treatment of uncomplicated urinary tract infections. No data are available on A. graveolens extract on the interplay between uropathogenic E. coli and the eukaryotic host cells and on quorum sensing of the bacteria. The present study aimed to characterize an antiadhesive and anti quorum sensing effect of a characterized A. graveolens extract by specific in vitro assays and to correlate these effects with in vivo data obtained by an animal infection model. Hydroalcoholic extract CSE (EtOH-water, 1:1) from A. graveolens fruits was characterized by UHPLC/+ESI-QTOF-MS and investigated on antiproliferative activity against UPEC (strain NU14) and human T24 bladder cells. Antiadhesive properties of CSE were investigated within two different in vitro adhesion assays. For in vivo studies BALB/c mice were used in an UPEC infection model. The effect of CSE on bacterial load in bladder tissue was monitored within a 4- and 7 days pretreatment (200, 500 mg/kg) of the animals. CSE was dominated by the presence of luteolin-glycosides and related flavons besides furocoumarins. CSE had no cytotoxic effects against UPEC and bladder cells. CSE exerts a dose dependent antiadhesive activity against UPEC strains NU14 and UTI89. CSE inhibited in a concentration-dependent manner bacterial quorum sensing. 4- and 7-day pretreatment of animals with CSE transurethrally infected with UPEC NU14, significantly reduced the bacterial load in bladder tissue. CSE is assessed as an antiadhesive extract for which the traditional use in phytotherapy for UTI is justified.

Evaluation of prevalence, immunogenicity and efficacy of FyuA iron receptor in uropathogenic Escherichia coli isolates as a vaccine target against urinary tract infection.

Uropathogenic Escherichia coli (UPEC) are among the most prevalent agents of urinary tract infections (UTIs). Antibiotic resistance reaches the need for alternative treatment approaches such as vaccination against UTIs. There is no ideal vaccine against UTIs, thus there is a need to evaluate different targets of uropathogens against UTIs. Ferric scavenger receptor FyuA in UPEC has the properties of an ideal vaccine candidate against UTIs. In the present study, the prevalence of FyuA among UPEC isolates, its immunogenicity with and without alum adjuvant, and its efficacy against experimental UTI were assessed. Totally, fyuA gene was present in 77% of the UPEC isolates tested. Alignments of FyuA exhibited a high degree of conservation among different submitted UPEC isolates in GenBank. The bioinformatics studies showed the high confidence value and stability of the FyuA structure. SDS-PAGE and Western blot confirmed the purification of FyuA with high yield by nickel resins. Mice vaccinated subcutaneously with the FyuA induced a significantly higher humoral response (total IgG, IgG1 and IgG2a) than control mice that alum enhanced these responses. The FuyA alone showed the ability to reduce the colonization of UPEC in bladder and kidney of mice as compared to the control group. But the addition of alum to FyuA increased the protection level against UPEC in these organs. Since, FyuA induced significant IgG1 (Th2) and IgG2a (Th1) responses and protected the mice against experimental UTI, it could be a promising target against UPEC infections.

Vitamin D-deficient mice have more invasive urinary tract infection.

Vitamin D deficiency is a common health problem with consequences not limited to bone and calcium hemostasis. Low levels have also been linked to tuberculosis and other respiratory infections as well as autoimmune diseases. We have previously shown that supplementation with vitamin D can induce the antimicrobial peptide cathelicidin during ex vivo infection of human urinary bladder. In rodents, however, cathelicidin expression is not linked to vitamin D and therefore this vitamin D-related effect fighting bacterial invasion is not relevant. To determine if vitamin D had further protective mechanisms during urinary tract infections, we therefore used a mouse model. In vitamin D-deficient mice, we detected more intracellular bacterial communities in the urinary bladder, higher degree of bacterial spread to the upper urinary tract and a skewed cytokine response. Furthermore, we show that the vitamin D receptor was upregulated in the urinary bladder and translocated into the cell nucleus after E. coli infection. This study supports a more general role for vitamin D as a local immune response mediator in the urinary tract.

A high-throughput assay for the measurement of uropathogenic Escherichia coli attachment to urinary bladder cells.

Strains of uropathogenic Escherichia coli (UPEC) are the major causative agent of urinary tract infections (UTI), the most common infectious diseases in the world. Their ability to attach and enter into cells in the urinary tract is a limiting step for their pathogenicity. Many studies are thus focussing on these key mechanisms to propose new therapeutic strategies. To facilitate such studies, we developed a fast and high-throughput assay which makes it possible to monitor the interaction of UPEC with cultured human uroepithelial cells. This assay allows measurement of the in vitro association of fluorescently labelled clinical isolates with bladder epithelial cells using flow cytometry in a microplate format. The assay was sensitive enough to detect variations between isolates expressing different adhesins and virulence factors and the inhibitory effect of proanthocyanidins. Thus we have developed a fast and robust assay which allows us to measure variations in the adhesion properties of UPEC to human bladder cells. This novel assay will be valuable for the study of initial steps of pathogenesis in UTI and for the screening or validation of inhibitory molecules.

Acquired Flucytosine Resistance during Combination Therapy with Caspofungin and Flucytosine for Candida glabrata Cystitis.

Treatment of Candida glabrata cystitis remains a therapeutic challenge, and an antifungal combination using flucytosine is one option. We describe two patients with refractory C. glabrata cystitis who failed flucytosine combined with caspofungin with early-acquired high-level resistance to flucytosine due to nonsense mutations in the FUR1 gene. Rapidly acquired flucytosine resistance with microbiological failure should discourage combination of caspofungin and flucytosine during urinary candidiasis.

Activity of fosfomycin and comparison of several susceptibility testing methods against contemporary urine isolates.

Fosfomycin is recommended as first-line treatment for acute uncomplicated cystitis in women. It has demonstrated in vitro activity against a variety of pathogens; however, a paucity of data are available from the USA. We determined the susceptibility of a collection of urine isolates to fosfomycin and compared multiple methods of susceptibility testing. Consecutive non-duplicate Enterobacteriaceae, enterococci and Pseudomonas aeruginosa isolates were collected from the clinical microbiology laboratory between August 2013 and January 2014. Isolates represented hospitalised or emergency department patients with monomicrobial bacteriuria. Fosfomycin MICs were determined in duplicate, on separate days, by Etest and disk diffusion and results were compared with agar dilution. Nitrofurantoin and ciprofloxacin were used as comparators. MIC results were categorised using Clinical and Laboratory Standards Institute interpretive criteria for Escherichia coli and Enterococcus faecalis. Correlation between the three testing methods was evaluated. Overall susceptibility to fosfomycin was 94.4%, 93.5% and 87.9% by agar dilution, disk diffusion and Etest, respectively. Five fosfomycin-resistant isolates were identified, including two Morganella morganii, one P. aeruginosa, one Proteus mirabilis and one Enterobacter aerogenes. Across all organisms, rates of essential agreement, categorical agreement, minor errors, major errors and very major errors for Etest/disk diffusion compared with agar dilution were 77.3%/NA, 89.5/93.8%, 7.1/5.0%, 3.6/1.3% and 0/0%, respectively. Fosfomycin displayed fairly consistent activity against a majority of isolates collected when using the susceptibility breakpoint of 64 µg/mL. MICs for E. coli were particularly low (≤2 µg/mL). These data lend support to current guidelines that recommend fosfomycin as empirical first-line therapy for uncomplicated UTI.

Anti-Adhesive Activity of Cranberry Phenolic Compounds and Their Microbial-Derived Metabolites against Uropathogenic Escherichia coli in Bladder Epithelial Cell Cultures.

Cranberry consumption has shown prophylactic effects against urinary tract infections (UTI), although the mechanisms involved are not completely understood. In this paper, cranberry phenolic compounds and their potential microbial-derived metabolites (such as simple phenols and benzoic, phenylacetic and phenylpropionic acids) were tested for their capacity to inhibit the adherence of uropathogenic Escherichia coli (UPEC) ATCC®53503™ to T24 epithelial bladder cells. Catechol, benzoic acid, vanillic acid, phenylacetic acid and 3,4-dihydroxyphenylacetic acid showed anti-adhesive activity against UPEC in a concentration-dependent manner from 100-500 µM, whereas procyanidin A2, widely reported as an inhibitor of UPEC adherence on uroepithelium, was only statistically significant (p < 0.05) at 500 µM (51.3% inhibition). The results proved for the first time the anti-adhesive activity of some cranberry-derived phenolic metabolites against UPEC in vitro, suggesting that their presence in the urine could reduce bacterial colonization and progression of UTI.

Azithromycin and ciprofloxacin: a possible synergistic combination against Pseudomonas aeruginosa biofilm-associated urinary tract infections.

Biofilm formation is becoming a predominant feature in nosocomial infections. Since biofilms are increasingly resistant to antibiotics, making monotherapy ineffective, combination therapy appears to be relevant for their eradication. This study assessed the potential of azithromycin (AZM) and ciprofloxacin (CIP) alone and in combination in vitro and in a mouse model of urinary tract infection (UTI) induced with biofilm cells of Pseudomonas aeruginosa. In vitro antibacterial and antibiofilm activities of antibiotics alone and in combination were assessed using the fractional inhibitory concentration index (FICI), time-kill analysis and confocal laser scanning microscopy (CLSM). In vivo efficacy was evaluated in a UTI model by quantitation of bacterial burden in kidney and bladder tissue, renal histopathology, pathology index factors (MDA and NO), and pro-inflammatory (MIP-2 and IL-6) and anti-inflammatory (IL-10) cytokines. MICs of AZM and CIP for strain PAO1 were 256 and 0.5 µg/mL, respectively; MBECs were 4096 and 1024 µg/mL. Synergistic interaction was observed between AZM and CIP both against planktonic and biofilm bacteria (FICI<0.5). The combination was also able to inhibit biofilm formation (at MIC levels) as observed with CLSM. Oral therapy with AZM (500 mg/kg) and CIP (30 mg/kg) combination in mice for 4 days showed accelerated clearance of bacteria from kidney and bladder tissue, improved renal histopathology, decreased levels of MDA and NO, significant decline in MIP-2 and IL-6, and increased IL-10 in the kidney (P<0.0001). We conclude that AZM+CIP therapy holds promise against biofilm-associated UTIs as it confers antibacterial, immunomodulatory and anti-inflammatory effects.

The mechanism of action of BCG therapy for bladder cancer--a current perspective.

Bacillus Calmette-Guérin (BCG) has been used to treat non-muscle-invasive bladder cancer for more than 30 years. It is one of the most successful biotherapies for cancer in use. Despite long clinical experience with BCG, the mechanism of its therapeutic effect is still under investigation. Available evidence suggests that urothelial cells (including bladder cancer cells themselves) and cells of the immune system both have crucial roles in the therapeutic antitumour effect of BCG. The possible involvement of bladder cancer cells includes attachment and internalization of BCG, secretion of cytokines and chemokines, and presentation of BCG and/or cancer cell antigens to cells of the immune system. Immune system cell subsets that have potential roles in BCG therapy include CD4(+) and CD8(+) lymphocytes, natural killer cells, granulocytes, macrophages, and dendritic cells. Bladder cancer cells are killed through direct cytotoxicity by these cells, by secretion of soluble factors such as TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), and, to some degree, by the direct action of BCG. Several gaps still exist in our knowledge that should be addressed in future efforts to understand this biotherapy of cancer.

Modulatory effect of Chandraprabha Vati on antimicrobial peptides and inflammatory markers in kidneys of mice with urinary tract infection.

INTRODUCTION: Chandraprabha Vati (CV) is an Indian polyherbal Siddha drug, traditionally used as an anti-inflammatory agent for arthritis and urinary ailments. This study explores its effect on mice with urinary tract infection. MATERIALS AND METHODS: The in-organic constituents of CV were determined by atomic absorption spectroscopy and phytochemical analysis was carried out. The supplementing dose of CV to infected experimental mice was determined by in vitro antimicrobial assay. Transurethrally infected animals were supplemented with CV extract for 20 days after confirmation of UTI. The animals were euthanized as per the guidelines and the tissues were harvested from the control and infected mice for histopathological examination the antimicrobial peptide Tamm-Horsfall protein (THP) and inflammatory markers (tumor necrosis factor-α and nuclear factor kappa-light-chain-enhancer of activated B cells) to ascertain the modulatory effects of CV. Indicators for oxidative stress and protein levels were also quantified to validate the efficacy of CV. RESULTS: Terpenoids and flavanoids were majorly found to constitute CV along with zinc and iron as in-organic content. Histological and immunohistochemical studies confirmed the pronounced infection in the kidney of the uropathogenic Escherichia coli-infected animals. Supplementation of CV significantly restored the increased levels of the antimicrobial proteins, THP, and inflammatory markers. CONCLUSIONS: This study explored the efficacy of the aqueous extract of CV as an alternative medication for the synthetic analogues administered for UTI. This study also provides information on the possible role of THP as an antimicrobial protein in the kidney in preventing infection due to uropathogenic E coli.

Evaluation of the effects of ozone therapy on Escherichia coli-induced cytitis in rat.

OBJECTIVES: The aim of the study was to investigate the effect of ozone on oxidative/nitrosative stress and bladder injury caused by Escherichia coli in rat bladder. METHODS: Twenty-one Wistar-Albino-type female rats included in the study were divided into three groups of equal number: (1) sham operation (control), (2) E. coli-only (EC), (3) EC + ozone. After ozone therapy for 3 days, urine and tissue samples were obtained for biochemical, microbiological, and histopathological analysis. RESULTS: Tissue malondialdehyde (MDA), myeloperoxidase (MPO), and nitric oxide (NO) level were increased, whereas superoxide dismutase (SOD) and glutathione peroxidase (GPx) activity was decreased in the EC group. MDA, MPO, and NO levels were decreased, whereas SOD, GPx activity was increased in the ozone-treated group. Also, there was no bacterial translocation in this group. CONCLUSION: The results of the present study suggest that ozone may be used as an agent to protect the bladder from oxidative/nitrosative stress occurring in cystitis.