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.

Diverse anti-inflammation and anti-cancer polyketides isolated from the endophytic fungi Alternaria sp. MG1.

Six new polyketides, alternaritins A-D [(±)-1-4] and isoxanalteric acid I (8), and 25 known Alternaria toxins were isolated from the culture of an endophytic fungi Alternaria sp. MG1. 3 is a rare fungal metabolite. 6 is a new natural product, and 5, 7, and 9 are known previously but their absolute configurations have not been determined. Three enantiomers [(±)-1, (±)-7, and (±)-15] were separated via chiral HPLC resolution. The structures of those polyketides (1-9) were elucidated by spectrometric analysis using MS and NMR. The absolute configurations were established using X-ray diffraction analysis and statistical comparative analysis of the experimental ECD and OR data, in conjunction with quantum mechanical calculations. All of the compounds were evaluated for their bioactivities. Known compound 27 exerted the most potent cytotoxic activities against HT-1080 and NCI-H1299 cell lines. The new compounds, 2 and 3, showed moderate inhibition on COX-2, while a pair of isomers, 8 and 9, exhibited medium activity on COX-2 and uropathogenic Escherichia coli.

Phytochemical analysis of medicinal plants of Nepal and their antibacterial and antibiofilm activities against uropathogenic Escherichia coli.

BACKGROUND: A biofilm is an extracellular polymeric substance (EPS) composed of polysaccharides, proteins, nucleic acids, and lipids that impede antibiotics and immune cells, thus providing a shielded environment for bacterial growth. Due to biofilm formation, some microbes can show up to 1000 fold increased resistance towards the antibiotics than the normal planktonic forms. The study was conducted to screen the crude extracts of medicinal plants used in Nepal for their in vitro antibiofilm activities. METHODS: Total phenolic and total flavonoid contents were determined by using a Folin-Ciocalteau reagent and aluminium trichloride method, respectively. Resazurin assay was used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The initial antibiofilm activities and their inhibitory concentration (IC50) values were determined by the microtiter based modified crystal violet staining method. RESULTS: Out of 25 different plant extracts were used for the study, methanolic extracts of 20 plants showed a biofilm inhibition activity against five different strong biofilm producing Escherichia coli strains. Calotropis gigantea exhibited inhibition against all five different E. coli strains with IC50 values ranging from 299.7 ± 20.5 to 427.4 ± 2.7 µg/mL. Apart from that, Eclipta prostrata also showed biofilm formation inhibition, followed by Eupatorium adenophorum, Moringa oleifera, Ocimum tenuifolium, Oxalis lantifolia, Prunus persica, and Urtica parviflora. The extracts of C. gigantea, E. prostrata, Mangifera indica, O. tenuifolium, P. persica, and U. parviflora exhibited a moderate to poor MIC value ranging from 625 to 2500 µg/mL. The highest amount of phenolic content (TPC) was found in Acacia catechu followed by Morus alba, which was 38.9 and 25.1 mg gallic acid equivalents, respectively. The highest amount of flavonoid content was found in A. catechu followed by M. indica, which was 27.1 and 20.8 mg quercetin equivalents, respectively. CONCLUSION: Extracts of C. gigantea, E. prostrata, P. persica, U. parviflora, and O. tenuifolium showed antibacterial as well as antibiofilm activity against pathogenic and strong biofilm producing E. coli. Thus, extracts or the pure compound from these medicinal plants could be used as antibiotics in the future.

Antiadhesive activity of hydroethanolic extract from bean pods of Phaseolus vulgaris (common bean) against uropathogenic E. coli and permeability of its constituents through Caco-2 cells monolayer.

ETHNOPHARMACOLOGICAL RELEVANCE: Phaseaoli pericarpium (bean pods) is a pharmacopeial plant material traditionally used as a diuretic and antidiabetic agents. Diuretic activity of pod extracts was reported first in 1608. Since then Phaseoli pericarpium tea figures in many textbooks as medicinal plant material used by patients. AIM OF THE STUDY: Despite the traditional use of extracts from Phaseolium vulgaris pericarp, limited information is available on bioactivity, chemical composition, and bioavailability of such preparations. The following study aimed to investigate the phytochemical composition, the in vitro permeability of selected extract's constituents over the Caco-2 permeation system, and potential antivirulence activity against uropathogenic Escherichia coli of a hydroalcoholic Phaseoli pericarpium extract (PPX) in vitro to support its traditional use as a remedy used in urinary tract infections. MATERIAL AND METHODS: The chemical composition of the extract PPX [ethanol:water 7:3 (v/v)] investigated by using UHPLC-DAD-MSn and subsequent dereplication. The permeability of compounds present in PPX was evaluated using the Caco-2 monolayer permeation system. The influence of PPX on uropathogenic E. coli (UPEC) strain NU14 proliferation and against the bacterial adhesion to T24 epithelial cells was determined by turbidimetric assay and flow cytometry, respectively. The influence of the extract on the mitochondrial activity of T24 host cells was monitored by MTT assay. RESULTS: LC-MSn investigation and dereplication, indicated PPX extract to be dominated by a variety of flavonoids, with rutin as a major compound, and soyasaponin derivatives. Rutin, selected soyasaponins and fatty acids were shown to permeate the Caco-2 monolayer system, indicating potential bioavailability following oral intake. The extract did not influence the viability of T24 cells after 1.5h incubation at 2 mg/mL and UPEC. PPX significantly reduced the bacterial adhesion of UPEC to human bladder cells in a concentration-dependent manner (0.5-2 mg/mL). Detailed investigations by different incubation protocols indicated that PPX seems to interact with T24 cells, which subsequently leads to reduced recognition and adhesion of UPEC to the host cell membrane. CONCLUSIONS: PPX is characterised by the presence of flavonoids (e.g. rutin) and saponins, from which selected compounds might be bioavailable after oral application, as indicated by the Caco-2 permeation experiments. Rutin and some saponins can be considered as potentially bioavailable after the oral intake. The concentration-dependent inhibition of bacterial adhesion of UPEC to T24 cells justifies the traditional use of Phaseoli pericarpium in the prevention and treatment of urinary tract infections.

Polysaccharides extract from Vaccaria segetalis seeds inhibits kidney infection by regulating cathelicidin expression.

ETHNOPHARMACOLOGICAL RELEVANCE: According to the Chinese Pharmacopoeia, the seeds of Vaccaria segetalis, a traditional medicinal herb, can be used for treating urinary diseases. The polysaccharides extract from V. segetalis seeds (VSP) has been shown to prevent urinary tract infections (UTIs). AIM OF THE STUDY: Investigate the effects of VSP on treating kidney infection induced by uropathogenic Escherichia coli (UPEC) and the underlying mechanisms. MATERIALS AND METHODS: Both in vivo and in vitro infection models were established with the UPEC strain CFT073. After oral administration of VSP, the levels of bacterial load, cathelicidin (CRAMP), Toll-like receptors (TLRs) in the kidney were evaluated. The expression of cathelicidin (LL-37) in human renal cell carcinoma cell line (A498) was tested after the treatment of VSP. RESULTS: In the kidneys of infection models, high-titer bacteria was detected. In the kidney of rat model, the expression of CRAMP was down-regulated, no significant change was observed in the levels of TLRs. After oral administration of VSP, the bacterial load was significantly decreased in rat and mouse models, and the levels of CRAMP and TLRs were significantly up-regulated in rat model. In vitro, the expression of LL-37 was significantly inhibited by CFT073. VSP up-regulated the expression of LL-37 in A498 cells. CONCLUSIONS: The up-regulation of cathelicidin expression may contribute to the therapeutic effects of VSP on kidney infection.

Cranberry Polyphenols and Prevention against Urinary Tract Infections: Relevant Considerations.

Cranberry (Vaccinium macrocarpon) is a distinctive source of polyphenols as flavonoids and phenolic acids that has been described to display beneficial effects against urinary tract infections (UTIs), the second most common type of infections worldwide. UTIs can lead to significant morbidity, especially in healthy females due to high rates of recurrence and antibiotic resistance. Strategies and therapeutic alternatives to antibiotics for prophylaxis and treatment against UTIs are continuously being sought after. Different to cranberry, which have been widely recommended in traditional medicine for UTIs prophylaxis, probiotics have emerged as a new alternative to the use of antibiotics against these infections and are the subject of new research in this area. Besides uropathogenic Escherichia coli (UPEC), the most common bacteria causing uncomplicated UTIs, other etiological agents, such as Klebsiellapneumoniae or Gram-positive bacteria of Enterococcus and Staphylococcus genera, seem to be more widespread than previously appreciated. Considerable current effort is also devoted to the still-unraveled mechanisms that are behind the UTI-protective effects of cranberry, probiotics and their new combined formulations. All these current topics in the understanding of the protective effects of cranberry against UTIs are reviewed in this paper. Further progresses expected in the coming years in these fields are also discussed.

Efficacy of Acacia nilotica aqueous extract in treating biofilm-forming and multidrug resistant uropathogens isolated from patients with UTI syndrome.

Escherichia coli is the dominant bacterial cause of UTI among the uropathogens in both developed and developing countries. This study is to investigate the effect of Acacia nilotica aqueous extract on the survival and biofilm of isolated pathogens to reduce UTIs diseases. A total of 170 urine samples were collected from Luxor general hospital and private medical analysis laboratories in Luxor providence, Egypt. Samples were screened for the incidence of uropathogens by biochemical tests, antibiotics susceptibility, detection of virulence, and antibiotic-resistant genes by multiplex PCR, biofilm formation, and time-killing assay. Escherichia coli is by far the most prevalent causative agent with the percentage of 73.7% followed by Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeuroginosa, and Acinetobacter baumanii. Isolates were multidrug-resistant containing blaTEM, blaSHV, blaCTX, qnrs, and aac(3)-Ia resistant genes. All isolates were sensitive to 15-16.7 mg ml-1 of Acacia nilotica aqueous extract. Time killing assay confirmed the bactericidal effect of the extract over time (20-24 h). A high percentage of 3-Cyclohexane-1-Carboxaldehyde, 2,6,6-trimethyl (23.5%); á-Selinene (15.12%); Oleic Acid (14.52%); Globulol (11.35%) were detected among 19 bioactive phytochemical compounds in the aqueous extract of A. nilotica over the GC-mass spectra analysis. The plant extract reduced significantly the biofilm activity of E. coli, K. pneumoniae, P. mirabilis, and P. aeuroginosa by 62.6, 59. 03, 48.9 and 39.2%, respectively. The challenge to improve the production of A. nilotica phytochemicals is considered a very low price for the return.

Biofilm formation, antimicrobial susceptibility and virulence genes of Uropathogenic Escherichia coli isolated from clinical isolates in Uganda.

INTRODUCTION: Uropathogenic E. coli is the leading cause of Urinary tract infections (UTIs), contributing to 80-90% of all community-acquired and 30-50% of all hospital-acquired UTIs. Biofilm forming Uropathogenic E. coli are associated with persistent and chronic inflammation leading to complicated and or recurrent UTIs. Biofilms provide an environment for poor antibiotic penetration and horizontal transfer of virulence genes which favors the development of Multidrug-resistant organisms (MDRO). Understanding biofilm formation and antimicrobial resistance determinants of Uropathogenic E. coli strains will provide insight into the development of treatment options for biofilm-associated UTIs. The aim of this study was to determine the biofilm forming capability, presence of virulence genes and antimicrobial susceptibility pattern of Uropathogenic E. coli isolates in Uganda. METHODS: This was a cross-sectional study carried in the Clinical Microbiology and Molecular biology laboratories at the Department of Medical Microbiology, Makerere University College of Health Sciences. We randomly selected 200 Uropathogenic E. coli clinical isolates among the stored isolates collected between January 2018 and December 2018 that had significant bacteriuria (> 105 CFU). All isolates were subjected to biofilm detection using the Congo Red Agar method and Antimicrobial susceptibility testing was performed using the Kirby disk diffusion method. The isolates were later subjected PCR for the detection of Urovirulence genes namely; Pap, Fim, Sfa, Afa, Hly and Cnf, using commercially designed primers. RESULTS: In this study, 62.5% (125/200) were positive biofilm formers and 78% (156/200) of these were multi-drug resistant (MDR). The isolates were most resistant to Trimethoprim sulphamethoxazole and Amoxicillin (93%) followed by gentamycin (87%) and the least was imipenem (0.5%). Fim was the most prevalent Urovirulence gene (53.5%) followed by Pap (21%), Sfa (13%), Afa (8%), Cnf (5.5%) and Hyl (0%). CONCLUSIONS: We demonstrate a high prevalence of biofilm-forming Uropathogenic E. coli strains that are highly associated with the MDR phenotype. We recommend routine surveillance of antimicrobial resistance and biofilm formation to understand the antibiotics suitable in the management of biofilm-associated 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.

Identification and Molecular Docking Studies of Bioactive Principles from Alphonsea madraspatana Bedd. against Uropathogens.

AIMS: The present study aims to determine the antimicrobial efficacy of Alphonsea madraspatana leaves extract against selected uropathogens. BACKGROUND: The plant Alphonsea madraspatana is an endangered species, reported to exhibit high antimicrobial activity due to the presence of phenolic compounds. Prevalence of high UTI infection and increased cases of bacterial resistance directed for alternative approach to meet the challenge of drug resistance. OBJECTIVE: Our objective is to determine antimicrobial efficacy of Alphonsea madraspatana leaves extract against selected uropathogens and subsequent in-silico analysis to predict the underlying mechanism. METHODS: Phytochemicals extraction from the dried leaves of Alphonsea madraspatana was performed using solvent gradient technique. All the extracts were subjected to preliminary phytochemical screening using liquid chromatography-mass spectrometry. Antimicrobial activity of the prepared extract was determined against the selected uropathogens using agar diffusion method. Finally, molecular docking study of the selected bio-actives was performed against a representative bacterial resistance enzyme ''DNA Gyrase". RESULTS: Methanolic extract exhibits relatively higher antimicrobial activity against the selected strains with Minimum Inhibitory Concentration (MIC) and minimum bactericidal concentration (MBC) of 1.56 ± 1 ug/mL and 6.25 ± 2 ug/mL, respectively. Phytochemical screening showed the presence of 3 flavonoids compounds such as Luteolin-7-O-glucoside, Kaempferol-3-O- rotinoside-7-O-rhamnoside and Genestein-7-O-glucoside. The results of molecular docking shows Luteolin-7-O-glucoside has best docking scores of -8.5 kcal/mol than other ligand molecules. Experimental simulation in presence of DNA Gyrase inhibitors showed lowest MIC and MBC value for E. Coli, which was found to be 1.56 ±1 ug/mL and 6.25±2 ug/mL respectively, support the docking outcomes. CONCLUSION: Outcomes of this study suggested that the methanolic extract of this plant shows good anti-microbial potential against resistant uropathogens.

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.

Disperse red 15 (DR15) impedes biofilm formation of uropathogenic Escherichia coli.

Catheter associated urinary tract infection (CAUTI) is a highly prevalent hospital-acquired infection that is predominantly caused by uropathogenic Escherichia coli (UPEC). It adheres on catheter surface using type I pili as the initial step of pathogenesis that progresses to form biofilm. In this study, potential inhibitors against FimH adhesin of type I pili were screened computationally that yielded ten compounds. These were further validated in vitro against adhesion and biofilm formation. The compounds, 1-Amino-4-hydroxyanthraquinone (Disperse Red 15 or DR15) and 4-(4'-chloro-4-biphenylylsulfonylamino) benzoic acid (CB1) impaired adhesion and biofilm formation without inhibiting the planktonic growth. Also, both compounds inhibited cell assemblages like autoaggregation and swarming motility by unknown mechanisms. DR15 was further derivatised into N-(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl) undec-10-enamide that self-assembled with linseed oil, which was used as the coating material on urinary Foley catheters. The thin-film coating on the catheter did not leach when incubated in artificial urine and effectively restricted biofilm formation of UPEC. Altogether, the thin-film coating of urinary catheter with DR15 inhibited biofilm formation of UPEC and this application could potentially help to reduce CAUTI incidents in healthcare facilities.

Pectic Oligosaccharides from Cranberry Prevent Quiescence and Persistence in the Uropathogenic Escherichia coli CFT073.

Urinary tract infections (UTIs) caused by Escherichia coli create a large burden on healthcare and frequently lead to recurrent infections. Part of the success of E. coli as an uropathogenic bacterium can be attributed to its ability to form quiescent intracellular reservoirs in bladder cells and its persistence after antibiotic treatment. Cranberry juice and related products have been used for the prevention of UTIs with varying degrees of success. In this study, a group of cranberry pectic oligosaccharides (cPOS) were found to both inhibit quiescence and reduce the population of persister cells formed by the uropathogenic strain, CFT073. This is the first report detailing constituents of cranberry with the ability to modulate these important physiological aspects of uropathogenic E. coli. Further studies investigating cranberry should be keen to include oligosaccharides as part of the 'active' cocktail of chemical compounds.

The anti-virulence effect of cranberry active compound proanthocyanins (PACs) on expression of genes in the third-generation cephalosporin-resistant Escherichia coli CTX-M-15 associated with urinary tract infection.

Background: Urinary tract infections (UTIs) are one of the most common infections found in humans, with uropathogenic Escherichia coli (UPEC) being the most common cause. Prevention of UTI is a major global concern due to its recurrent nature, medical cost, and most importantly, the increased antimicrobial resistance among UPEC. The resistance in UPEC is mainly due to the Extended-Spectrum ß-lactamases (ESBL), particularly the E. coli CTXM-15 type which is known for its rapid dissemination worldwide. Treatment options for E. coli CTXM-15 have become limited over recent years because of their multi-drug resistance, hence anti-virulent strategies based on herbal remedies, have considered as a viable option. The cranberry product, Cysticlean® capsules, contain 240 mg of proanthocyanins (PACs), which have been shown to significantly inhibit E. coli adherence, both in vitro and ex vivo, to uroepithelial cells. Method: In this study, the cephalosporin-resistant E. coli isolate NCTC 1553 (E. coli CTXM-15) was analysed by qRT-PCR (quantitative Reverse Transcriptase -Polymerase Chain Reaction) for the expression of virulence factors after treatment with Cysticlean®. qRT-PCR was carried out to detect virulence determinants encoding for toxins SAT, and USP, the iron acquisition system ChuA, the protectins SoxS, KPSM, TraT and RecA, the antibiotic resistance gene CTX-M (encode ß-lactamases), and the transporters IdfB and HcaT. Results: Cysticlean® significantly reduced the expression of all ten selected genes encoding for virulence factors and ß-lactamases. Conclusion: Cranberry product Cysticlean® could represent a practicable alternative option for the prevention of recurrent UTI caused by multi-drug resistant E. coli CTXM-15, as the product acts on multiple bacterial targets.

Polymethoxylated flavones from Orthosiphon stamineus leaves as antiadhesive compounds against uropathogenic E. coli.

Aqueous and acetone extracts of O. stamineus leaves reduce the adhesion of uropathogenic E. coli (UPEC, strain UTI89) to T24 bladder cells significantly (IC25 ~ 524 mg/mL, resp. 40 µg/mL). The acteonic extract had no cytotoxic effects against UPEC in concentrations that inhibited the bacterial adhesion. The extract significantly reduced the gene expression of fimH, fimC, fimD, csgA and focG, which are strongly involved in the formation of bacterial adhesins. The antiadhesive effect was due to the presence of polymethoxylated flavones, enriched in the acetonic extract. Five flavones have been isolated by fast centrifugal partition chromatography, followed by preparative HPLC. Eupatorin, ladanein, salvigenin, sinensetin, 5,6,7,4'-tetramethoxyflavone and 5-hydroxy-6,7,3',4'-tetramethoxyflavone were identified as the main polymethoxylated flavones. With the exception of eupatorin, all of these flavones reduced the bacterial adhesion in a concentration depending manner, indicating that B-ring hydroxylation and methoxylation seems to have a major impact on the antiadhesive activity. In addition, this was confirmed by investigation of the flavones chrysoeriol and diosmetin, which had only very weak antiadhesive activity. From these data, Orthosiphon extracts can be assessed to have a pronounced antiadhesive activity against UPEC, based on a variety of polymethoxylated flavones.

In-Vitro Biofilm Formation and Antimicrobial Resistance of Escherichia coli in Diabetic and Nondiabetic Patients.

BACKGROUND: Diabetic patients are more susceptible to urinary tract infection compared to nondiabetic patients, Escherichia coli being the most common uropathogen causing UTI. Unreasonable and incorrect antibiotic prescription for UTI in these patients may induce the development of antibiotic-resistant urinary pathogens resulting in delayed recovery and longer hospitalization. In addition to these, biofilm forming capacity of the pathogen may worsen the problem. The main aim of this cross-sectional study (conducted from March to September 2015) is to detect the biofilm forming capacity of UTI causing micro-organisms and compare the antibiotic resistance pattern of Escherichia coli, the most common cause of UTI, which will help the physician in choosing the best antibiotic. METHOD: Total of 1,099 clean-catch mid stream urine (CCMSU) was processed by standard microbiological technique; 182 were from the diabetic group and 917 nondiabetic. Following identification, all isolates were subjected to antibiotic susceptibility testing using modified Kirby-Bauer disc diffusion method. In-vitro biofilm forming capacity of the isolates were detected by Microtitre plate method. The data were analyzed using SPSS software 16. RESULT: Urinary tract infection was found to be significantly higher in diabetic patients (42.9%) compared to nondiabetic patients (17.4%) with Escherichia coli as the most common uropathogen in both diabetic and nondiabetic groups. Similarly, UTI was more common in elderly population (29.5%). Imipenem, nitrofurantoin and amikacin were found to be the most effective drug for uropathogenic E. coli in both diabetic and nondiabetic patients, whereas amoxicillin, ciprofloxacin, and cotrimoxazole were least effective. Of the total bacterial isolates, 43.3% showed positive results for in-vitro biofilm production by the Microtitre plate method. A significantly higher resistance rate was observed among biofilm producing E. coli for quinolones, cotrimoxazole, and third generation cephalosporin ceftriaxone. Most of the biofilm producers (79.5%) were found to be MDR (p-value 0.015). CONCLUSION: Elderly populations with diabetes are at a higher risk of UTI. Higher biofilm production and resistance to in-use antimicrobial agents in this study render its inefficacy for empirical treatment and point out the importance of biofilm screening to ensure the effective management of infection.

Orthosipon stamineus extract exerts inhibition of bacterial adhesion and chaperon-usher system of uropathogenic Escherichia coli-a transcriptomic study.

Specific recognition and bacterial adhesion to host cells by uropathogenic Escherichia coli (UPEC) are the first steps towards infection of epithelial tissue of the human urogenital system. Therefore, targeting of UPEC virulence factors, relevant for adhesion, is a promising approach for prevention of recurrent urinary tract infections (UTI). A fully characterized plant-derived aqueous extract from the leaves of Orthosiphon stamineus (OWE), a plant traditionally used in clinical practice in Europe and Asia for UTI, has been shown to exert strong antiadhesive effects under in vitro and in vivo conditions. For improved understanding of the underlying mechanisms, transcriptome analysis of OWE-treated UPEC strain UTI89 by Illumina sequencing and cross-validation of these data by qPCR indicated significant downregulation of bacterial adhesins (curli, type 1-, F1C-, and P fimbriae) and of the chaperone-mediated protein folding/unfolding and pilus assembly process; in contrast, flagellar and motility-related genes were upregulated. We conclude that OWE transforms the sessile lifestyle of bacteria into a motile one and therefore disables bacterial attachment to the host cell. Additionally, the extract inhibited gene expression of multiple iron-acquisition systems (ent, fep, feo, fhu, chu, sit, ybt). The present study explains the antiadhesive and anti-infective effect of the plant extract by pinpointing specific biochemical and molecular targets.

The anti-biofilm and anti-virulence activities of trans-resveratrol and oxyresveratrol against uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the primary causative agent of urinary tract infections, which are one of the most common infectious disease types in humans. UPEC infections involve bacterial cell adhesion to bladder epithelial cells, and UPEC can also form biofilms on indwelling catheters that are often tolerant to common antibiotics. In this study, the anti-biofilm activities of t-stilbene, stilbestrol, t-resveratrol, oxyresveratrol, ε-viniferin, suffruticosol A, and vitisin A were investigated against UPEC. t-Resveratrol, oxyresveratrol, and ε-viniferin, suffruticosol A, and vitisin A significantly inhibited UPEC biofilm formation at subinhibitory concentrations (10-50 µg ml-1). These findings were supported by observations that t-resveratrol and oxyresveratrol reduced fimbriae production and the swarming motility in UPEC. Furthermore, t-resveratrol and oxyresveratrol markedly diminished the hemagglutinating ability of UPEC, and enhanced UPEC killing by human whole blood. The findings show that t-resveratrol, oxyresveratrol, and resveratrol oligomers warrant further attention as antivirulence strategies against persistent UPEC infections.

Evaluation of the potential of colicins to prevent extraluminal contamination of urinary catheters by Escherichia coli.

The feasibility of using colicins to create an antimicrobial lubricant to prevent extraluminal catheter contamination during urinary catheter insertion was assessed. Levels of resistance of uropathogenic Escherichia coli to antibiotics and colicins were compared. The results showed that antibiotics and colicins possess similar frequencies of resistance to a single drug, whereas colicins exhibit significantly lower levels of multidrug resistance (22%) than antibiotics (42%). Colicins and antibiotics showed complementary inhibitory activity, with each targeting different subsets of pathogenic isolates. The collateral impact of these two antimicrobials on genera that are members of the fecal/vaginal/urinary microbiome was assessed, with colicins showing significantly less collateral damage than antibiotics. Using a novel colicin, SR4, minimum inhibitory concentrations (MICs) for a panel of 30 uropathogenic isolates were determined and showed that SR4 achieved the same antimicrobial efficacy as gentamicin using 20-30% less drug. An SR4-impregnated catheter lubricant was created and its ability to prevent extraluminal urinary catheter contamination in vitro was demonstrated. These data indicate that a colicin-impregnated lubricant may provide a viable prophylactic option for preventing catheter-associated urinary tract infections.

Antiadhesive phthalides from Apium graveolens fruits against uropathogenic E. coli.

ETHNOPHARMACOLOGICAL RELEVANCE: Fruits of Apium graveolens (celery) are used traditionally in Persian and European medicine for the treatment of uncomplicated urinary tract infections. AIM OF THE STUDY: The study aimed at identifying potential antiadhesive compounds from celery extracts to provide strategies for improved standardization of the herbal material. MATERIALS AND METHODS: Decoction, hydroalcoholic and acetone extracts were prepared from celery fruits. Bioassay-guided fractionation was performed by Fast Centrifugal Partition Chromatography and preparative HPLC, followed by LC-MS and NMR investigations for structure elucidation. The antiadhesive activity of extracts, fractions and purified compounds was assessed by flow cytometry, evaluating the adhesion of fluorescent-labelled uropathogenic bacteria (UPEC NU14) to T24 bladder cells; mannose served as positive control. Influence of the extract on gene expression of selected adhesins and fitness genes was monitored by qPCR. RESULTS: Concentration-dependent antiadhesive activity was found for the hydroalcoholic and even more for the acetone extract AE (IC50 85 µg/mL) from celery fruits. Bioassay-guided fractionation revealed the presence of the phthalides senkyunolide (1, inactive) and sedanenolide (2, IC50 790 µM). 2 is assessed as the main antiadhesive compound, which accounts for 4.0% in the water extract, for 18% in the hydroethanolic extract and for 71% in AE. Additionally a similar phthalide, Z-ligustilide (5), was shown to exert an IC50 of 611 µM. Furthermore, AE caused a significant upregulation of fimH and sfaG in free floating, non-attached UPEC and significantly down-regulated these genes in adherent bacteria. CONCLUSIONS: Phthalides were identified as the main active compounds in polar and semi-polar extracts, which exert strong antiadhesive activity against uropathogenic E. coli. The current findings support the traditional use in phytotherapy for urinary tract infections and provide a base for standardization of the herbal material.