Genome characterization of a multi-drug resistant Escherichia coli strain, L1PEag1, isolated from commercial cape gooseberry fruits (Physalis peruviana L.).

Introduction: Foodborne infections, which are frequently linked to bacterial contamination, are a serious concern to public health on a global scale. Whether agricultural farming practices help spread genes linked to antibiotic resistance in bacteria associated with humans or animals is a controversial question. Methods: This study applied a long-read Oxford Nanopore MinION-based sequencing to obtain the complete genome sequence of a multi-drug resistant Escherichia coli strain (L1PEag1), isolated from commercial cape gooseberry fruits (Physalis peruviana L.) in Ecuador. Using different genome analysis tools, the serotype, Multi Locus Sequence Typing (MLST), virulence genes, and antimicrobial resistance (AMR) genes of the L1PEag1 isolate were determined. Additionally, in vitro assays were performed to demonstrate functional genes. Results: The complete genome sequence of the L1PEag1 isolate was assembled into a circular chromosome of 4825.722 Kbp and one plasmid of 3.561 Kbp. The L1PEag1 isolate belongs to the B2 phylogroup, sequence type ST1170, and O1:H4 serotype based on in silico genome analysis. The genome contains 4,473 genes, 88 tRNA, 8 5S rRNA, 7 16S rRNA, and 7 23S rRNA. The average GC content is 50.58%. The specific annotation consisted of 4,439 and 3,723 genes annotated with KEEG and COG respectively, 3 intact prophage regions, 23 genomic islands (GIs), and 4 insertion sequences (ISs) of the ISAs1 and IS630 families. The L1PEag1 isolate carries 25 virulence genes, and 4 perfect and 51 strict antibiotic resistant gene (ARG) regions based on VirulenceFinder and RGI annotation. Besides, the in vitro antibiotic profile indicated resistance to kanamycin (K30), azithromycin (AZM15), clindamycin (DA2), novobiocin (NV30), amikacin (AMK30), and other antibiotics. The L1PEag1 isolate was predicted as a human pathogen, matching 464 protein families (0.934 likelihood). Conclusion: Our work emphasizes the necessity of monitoring environmental antibiotic resistance, particularly in commercial settings to contribute to develop early mitigation techniques for dealing with resistance diffusion.

The artificial neural network selects saccharides from natural sources a promise for potential FimH inhibitor to prevent UTI infections.

The major challenge in the development of affordable medicines from natural sources is the unavailability of logical protocols to explain their mechanism of action in biological targets. FimH (Type 1 fimbrin with D-mannose specific adhesion property), a lectin on E. coli cell surface is a promising target to combat the urinary tract infection (UTI). The present study aimed at predicting the inhibitory capacity of saccharides on FimH. As mannosides are considered FimH inhibitors, the readily accessible saccharides from the PubChem collection were utilized. The artificial neural networks (ANN)-based machine learning algorithm Self-organizing map (SOM) has been successfully employed in predicting active molecules as they could discover relationships through self-organization for the ligand-based virtual screening. Docking was used for the structure-based virtual screening and molecular dynamic simulation for validation. The result revealed that the predicted molecules malonyl hexose and mannosyl glucosyl glycerate exhibit exactly similar binding interactions and better docking scores as that of the reference bioassay active, heptyl mannose. The pharmacokinetic profile matches that of the selected bioflavonoids (quercetin malonyl hexose, kaempferol malonyl hexose) and has better values than the control drug bioflavonoid, monoxerutin. Thus, these two molecules can effectively inhibit type 1 fimbrial adhesin, as antibiotics against E. coli and can be explored as a prophylactic against UTIs. Moreover, this investigation can pave the way to the exploration of the potential benefits of plant-based treatments. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-024-00212-5.

It takes two to attach - endo-1,3-ß-d-glucanase as a potential receptor of mannose-independent, FimH-dependent Salmonella Typhimurium binding to spinach leaves.

Currently, fresh, unprocessed food has become a relevant element of the chain of transmission of enteropathogenic infections. To survive on a plant surface and further spread the infections, pathogens like Salmonella have to attach stably to the leaf surface. Adhesion, driven by various virulence factors, including the most abundant fim operon encoding type 1 fimbriae, is usually an initial step of infection, preventing physical removal of the pathogen. Adhesion properties of Salmonella's type 1 fimbriae and its FimH adhesin were investigated intensively in the past. However, there is a lack of knowledge regarding its role in interaction with plant cells. Understanding the mechanisms and structures involved in such interaction may facilitate efforts to decrease the risk of contamination and increase fresh food safety. Here, we applied Salmonella genome site-directed mutagenesis, adhesion assays, protein-protein interactions, and biophysics methods based on surface plasmon resonance to unravel the role of FimH adhesin in interaction with spinach leaves. We show that FimH is at least partially responsible for Salmonella binding to spinach leaves, and this interaction occurs in a mannose-independent manner. Importantly, we identified a potential FimH receptor as endo-1,3-ß-d-Glucanase and found that this interaction is strong and specific, with a dissociation constant in the nanomolar range. This research advances our comprehension of Salmonella's interactions with plant surfaces, offering insights that can aid in minimizing contamination risks and improving the safety of fresh, unprocessed foods.

Design, synthesis, biological evaluation and docking study of some new aryl and heteroaryl thiomannosides as FimH antagonists.

FimH is a mannose-recognizing lectin that is expressed by Escherichia coli guiding its ability to adhere and infect cells. It is involved in pathogenesis of urinary tract infections and Chron's disease. Several X-ray structure-guided ligand design studies were extensively utilized in the discovery and optimization of small molecule aryl mannoside FimH antagonists. These antagonists retain key specific interactions of the mannose scaffolds with the FimH carbohydrate recognition domains. Thiomannosides are attractive and stable scaffolds, and this work reports the synthesis of some of their new aryl and heteroaryl derivatives as FimH antagonists. FimH-competitive binding assays as well as biofilm inhibition of the new compounds (24-32) were determined in comparison with the reference n-heptyl α-d-mannopyranoside (HM). The affinity among these compounds was found to be governed by the structure of the aryl and heteroarylf aglycones. Two compounds 31 and 32 revealed higher activity than HM. Molecular docking and total hydrophobic to topological polar surface area ratio calculations attributed to explain the obtained biological results. Finally, the SAR study suggested that introducing an aryl or heteroaryl aglycone of sufficient hydrophobicity and of proper orientation within the tyrosine binding site considerably enhance binding affinity. The potent and synthetically feasible FimH antagonists described herein hold potential as leads for the development of sensors for detection of E. coli and treatment of its diseases.

Case report: Long-term follow-up of patients who received a FimCH vaccine for prevention of recurrent urinary tract infections caused by antibiotic resistant Enterobacteriaceae: a case report series.

Urinary tract infections (UTI) caused by carbapenem-resistant Enterobacteriaceae (CRE) are considered one of the most urgent health threats to humans according to the Centers for Disease Control (CDC), and the World Health Organization (WHO). A FimCH Vaccine expanded access study is being conducted in patients with a history of antibiotic resistant UTIs who are considered to be at risk for development of CRE UTI. This case series describes the clinical, safety and immunogenicity findings for four participants who received a FimCH four-vaccine series. Participants were followed for 12 months after administration of the fourth vaccine for safety, general health status and UTI occurrence. The study was later amended to allow additional follow-up of up to five years post vaccine administration to assess long-term health status, UTI occurrences and to obtain blood samples for anti-FimH antibody testing. In our population of 4 study participants, the number of symptomatic UTI occurrences caused by gram-negative bacteria in the 12-month period following peak anti-FimH antibody response were approximately 75% lower than the 12-month period preceding study enrollment. These results are consistent with the 30-patient cohort of a Phase 1 study with the same FimCH Vaccine. UTI occurrences increased during the long-term follow-up period for all 4 participants but did not reach the rate observed pre-vaccination. No new safety concerns related to the FimCH Vaccine were identified during long-term follow-up. This case series has clinical importance and public health relevance since it examines and reports on UTI frequency and recurrence following vaccination with the FimCH Vaccine in a high-risk population of patients with recurrent UTI. Additionally, participants described improved well-being following vaccination which was maintained in the long-term follow-up period.

Rate limiting step of the allosteric activation of the bacterial adhesin FimH investigated by molecular dynamics simulations.

The bacterial adhesin FimH is a model for the study of protein allostery because its structure has been resolved in multiple configurations, including the active and the inactive state. FimH consists of a pilin domain (PD) that anchors it to the rest of the fimbria and an allosterically regulated lectin domain (LD) that binds mannose on the surface of infected cells. Under normal conditions, the two domains are docked to each other and LD binds mannose weakly. However, in the presence of tensile force generated by shear the domains separate and conformational changes propagate across LD resulting in a stronger bond to mannose. Recently, the crystallographic structure of a variant of FimH has been resolved, called FimH FocH , where PD contains 10 mutations near the inter-domain interface. Although the X-ray structures of FimH and FimH FocH are almost identical, experimental evidence shows that FimH FocH is activated even in the absence of shear. Here, molecular dynamics simulations combined with the Jarzynski equality were used to investigate the discrepancy between the crystallographic structures and the functional assays. The results indicate that the free energy barrier of the unbinding process between LD and PD is drastically reduced in FimH FocH . Rupture of inter-domain hydrogen bonds involving R166 constitutes a rate limiting step of the domain separation process and occurs more readily in FimH FocH than FimH. In conclusion, the mutations in FimH FocH shift the equilibrium toward an equal occupancy of bound and unbound states for LD and PD by reducing a rate limiting step.

Escherichia coli adhesion protein FimH exacerbates colitis via CD11b+CD103- dendritic cell activation.

Introduction: Immune stimulators are used to improve vaccine efficiency; however, they are accompanied by various side effects. In previous studies, we reported that the Escherichia coli adhesion protein, FimH, induces immune activity; however, we did not examine any side effects in colon inflammation. Methods: FimH was administered orally or intraperitoneally (i.p.) to mice with dextran sulfate sodium (DSS)-induced colitis, and changes in symptoms were observed. Immune cells infiltrated into the colon after the induction of colon inflammation were analyzed using a flow cytometer. Changes in Th1 and Th17 cells that induce colitis were analyzed. Further, mesenteric lymph node (mLN) dendritic cells (DCs) activated by FimH were identified and isolated to examine their ability to induce T-cell immunity. Results: FimH oral and i.p. administration in C57BL/6 mice did not induce inflammation in the colon; however, DSS-induced colitis was exacerbated by oral and i.p. FimH administration. FimH treatment increased immune cell infiltration in the colon compared to that in DSS colitis. Th1 and Th17 cells, which are directly related to colitis, were increased in the colon by FimH; however, FimH did not directly affect the differentiation of these T cells. FimH upregulated the CD11b+CD103- DC activity in the mLNs, which produced the signature cytokines required for Th1 and Th17. In addition, isolated CD11b+CD103- DCs, after stimulation with FimH, directly induced Th1 and Th17 differentiation in a co-culture of CD4 T cells. Conclusion: This study demonstrated the side effects of FimH and indicated that the use of FimH can aggravate the disease in patients with colitis.

Presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli isolates recovered from an apex medical institute in North India.

PURPOSE: To detect the presence of fimH and iss type 1, 2 and 3 genes in uropathogenic Escherichia coli (UPEC) isolates recovered from patients coming to the out patient department (OPD) of our hospital. METHODS: E. coli isolates recovered from patients who had symptoms of urinary tract infection (UTI) were processed for the presence of fimH and iss genes. DNA was extracted using an in house method after which conventional PCR using forward and reverse primers targeting the four genes was carried out. The amplified products were electrophoresed and visualized in a gel documentation imager. Relevant demographic details of the patients were recorded on a pre-designed pro-forma and antimicrobial susceptibility testing of the isolates was done by disc diffusion method. RESULTS: fimH was present in 87.5% of UPEC isolates whereas iss type 1 was seen in 7.3%, type 2 in 4.2% and iss type 3 in 71.9% isolates. Age of the patients ranged from 3 months to 82 â€‹yrs (mean 43.5 SD â€‹± â€‹18.20). UTI was more common in females (60.2%) as compared to males patients (39.8%). Dysuria (66.7%) was the most common symptom in the studied subjects and diabetes mellitus (42.6%) the most common co-morbidity. A total of 56.5% patients gave a history of prior antibiotic intake. The UPEC isolates were resistant to most of the antibiotics tested. However all the isolates were sensitive to polymyxin B and colistin. Fosfomycin resistance was seen in 9.5% of the UPEC isolates harbouring fimH gene. CONCLUSION: This is the first study that highlights the presence of iss type 3 gene in UPEC isolates along with the fimH and iss type 1 and 2 genes. The results of this study can serve as a stepping stone for future in depth research into the significance of the iss genes in causing UTI.

Screening of antigenic epitopes related to the adhesion of the avian Escherichia coli Type 1 Fimbrial Agglutinin Domain.

BACKGROUND: Avian Escherichia coli (E.coli) type 1 fimbriae adhere to avian tracheal epithelial cells through the FimH protein. However, the adhesion-related antigen is still unknown. The purpose of this study was to analyze the antigenicity of the type 1 fimbrial FimH protein of wild-type avian E. coli, screen antigen epitopes, and prepare monoclonal antibodies (mAbs) that can block the adhesion of avian E. coli. RESULTS: In this study, the nucleic acid homologies of MG2 (O11), TS12 (O18), and YR5 (O78) with K12 were 97.7%, 99.6%, and 97.7%, respectively, and the amino acid sequence similarity reached 98.7%, 99.3%, and 98.0%, respectively. The epitopes and hydrophilicities of the FimH proteins of these three strains were similar. The more obvious lectin domain epitopes were located at FimH protein positions 111-124 and 154-162. The mAbs 7C2 and 7D8 against these two epitopes were prepared. An adhesion inhibition test showed that 7C2 and 7D8 blocked bacterial adhesion to avian tracheal epithelial cells. The mAb 7C2 against the 111-124 epitope inhibited O78 strain adhesion by 93%, and the mAb 7D8 against the 154-162 epitope inhibited O78 strain adhesion by 49%, indicating that these two epitopes are closely related to the adhesion of type 1 fimbriae. However, only the 111-124 epitope-recognizing mAb 7C2 inhibited bacterial agglutination of erythrocytes, indicating that host cell receptor binding and erythrocyte agglutination are not mediated by the same spatial locations within the FimH protein. CONCLUSIONS: The results demonstrate that the mAbs 7C2 and 7D8 against FimH protein positions 111-124 and 154-162 could inhibit the adhesion of E.coli to the chicken trachea.

Click chemistry inspired facile one-pot synthesis of mannosyl triazoles and their hemagglutination inhibitory properties: The effect of alkyl chain spacer length between the triazole and phthalimide moieties in the aglycone backbone.

An efficient one-pot synthesis of a new series of mannosyl triazoles has been achieved through CuAAC reaction where the alkyl chain spacer between the phthalimide moiety and the triazole ring in the aglycone backbone is varied from one methylene to six methylene units. The target compounds were evaluated in terms of their inhibitory potency against FimH using hemagglutination inhibition (HAI) assay. It was found that the length of four methylene units was the optimum for the fitting/binding of the compound to FimH as exemplified by compound 11 (HAI = 1.9 µM), which was approximately 200 times more potent than the reference ligand 1(HAI = 385 µM). The successful implementation of one-pot protocol with building blocks 1-7 and the architecture of ligand 11 will be the subject of our future work for developing more potent FimH inhibitors.

Escherichia coli adhesion portion FimH polarizes M2 macrophages to M1 macrophages in tumor microenvironment via toll-like receptor 4.

Background: Macrophages are key effector cells of innate immunity and play a critical role in the immune balance of disease pathogenesis, especially in the tumor microenvironment. In previous studies, we showed that FimH, an Escherichia coli adhesion portion, promoted dendritic cell activation. However, the effect of FimH in macrophage polarization has yet to be fully examined. In this study, we investigated the potential effect of FimH on macrophages, as well as the polarization from M2 to M1 macrophages, contributing to the overall antitumor effect. Methods: Mouse bone marrow derived macrophages and peritoneal macrophages were generated to test the effect of FimH in vitro. The expression of costimulatory molecules and production of cytokines were analyzed. The effect of FimH in the tumor-associated macrophages was examine in the B16F10-tumor bearing C57BL/6. Results: FimH was found to promote M1 macrophage activation. In addition, FimH polarized M2 macrophages, which were induced by interleukin (IL)-4 and IL-13 into M1 macrophages were dependent on toll-like receptor 4 and myeloid differentiation factor 2. Moreover, FimH reprogramed the tumor-associated macrophage (TAM) into M1 macrophages in B16 melanoma tumor-bearing mice and promoted an inflammatory reaction in the tumor microenvironment (TME). Furthermore, FimH promoted M1 macrophage activation, as well as the reversion of M2 macrophages into M1 macrophages in humans. Finally, FimH treatment was found to enhance the anti-cancer immunity of anti-PD-L1 antibody by the induction of M1 polarization from TAM. Conclusion: This study demonstrated the potential effect of FimH on the activation of macrophages, responsible for the repolarization of M2 macrophages into the M1 phenotype via the TLR4 signaling pathway. Moreover, FimH could also reprogram TAM polarization to the M1 status in the TME, as well as enhance the anti-tumor activity of immune checkpoint blockade.

Nature of the Interaction of Alpha-D-Mannose and Escherichia coli Bacteria, and Implications for its Regulatory Classification. A Delphi Panel European Consensus Based on Chemistry and Legal Evidence.

The nature of alpha-D-mannose-natural aldohexose sugar, C-2 glucose epimer, whose intended use is for preventing urinary tract infections-in the interaction with E. coli is addressed in order to drive the issue of its regulatory classification as a medicinal product or medical device. PRISMA systematic review approach was applied; Delphi Panel method was used to target consensus on statements retrieved from evidence. Based on regulatory definitions and research evidence, the mechanism of D-mannose does not involve a metabolic or immunological action while there is uncertainty regarding the pharmacological action. Specific interaction between the product and the bacteria within the body occurs, but its nature is inert: it does not induce a direct response activating or inhibiting body processes. Moreover, the action of D-mannose takes place, even if inside the bladder, outside the epithelium on bacteria that have not yet invaded the urothelial tissue. Therefore, its mechanism of action is not directed to host structures but to structures (bacteria) external to the host's tissues. On the basis of current regulation, the uncertainty as regard a pharmacological action of alpha-D-mannose makes possible its medical device classification: new regulations and legal judgments can add further considerations. From a pharmacological perspective, research is driven versus synthetic mannosides: no further considerations are expected on alpha-D-mannose.

Effect of thymol on antimicrobial susceptibility, and adhesion genes expression of uropathogenic Escherichia coli isolated from pediatric urinary tract infection.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) is a common cause of urinary tract infections (UTI) in children and currently is one of the leading medical problems. Due to the increase in antibiotic resistance rate, herbal medicines with lower side effects were considered. OBJECTIVE: This study aimed to identify the afa, fimH, and sfa genes of UPEC bacteria isolated from pediatric UTI to investigate the effect of the thyme on the expression of fimH gene. STUDY DESIGN: In this cross-sectional study, 160 UPEC were isolated from pediatric UTIs. An antibiotic susceptibility test was performed on six families of antibiotics, including beta-lactams, quinolones, aminoglycosides, carbapenems, sulfonamides, and nitrofurantoin. The micro-broth dilution method was used to determine MIC of thymol. The biofilm production ability of isolated strains was quantified by the microtiter plate method. The PCR technique was used to detectfimH, afa, and sfa adhesion genes, and real-time PCR was used to measure the fimHgene expression. RESULTS: The results of the antibiogram showed that the lowest and highest resistance related to meropenem and imipenem (zero), and 72.5% for cephalothin. MIC showed 80.7% of the isolates were sensitive to thymol. The biofilm production results showed that 3.12%, 53.75%, and 43.12% of the isolates were strong, weak, and no-biofilm (Zero) producers, respectively. After thymol treatment, 26.25% and 73.75% of isolates were weak and no-producer (Zero) biofilms, respectively and there was a significant correlation (P-value = 0.042) compared to the control group. The frequency of fimH, sfa, and afa genes was 53.1%, 49.4%, and 29.4%, respectively. The expression of fimHgene after 48 h thymol treatment decreased significantly (P-value< 0.05). CONCLUSION: Due to the significant effects of thymol in preventing the expression of the adhesion gene (fimH) of UPEC bacteria, our study is a proof-of-concept study evaluating bacterial sensitivity to Thymol and its effect on biofilm production in vitro. Given the demonstrated promising results of Thymol's effectiveness and the increase in bacterial antibiotic resistance, further studies should be undertaken to determine the safety and effectiveness of Thymol use in the clinical treatment of urinary tract infection. We believe that Thymol may prove to be an effective adjunct to the treatment of bacterial urinary tract infections.

Expression of E. coli FimH Enhances Trafficking of an Orally Delivered Lactobacillus acidophilus Vaccine to Immune Inductive Sites via Antigen-Presenting Cells.

The development of lactic acid bacteria as mucosal vaccine vectors requires the identification of robust mucosal adjuvants to increase vaccine effectiveness. The E. coli type I fimbriae adhesion protein FimH is of interest as a mucosal adjuvant as it targets microfold (M) cells enhancing vaccine uptake into Peyer's patches and can activate the innate immune system via Toll-like receptor (TLR) 4 binding. Here, we displayed the N-terminal domain of FimH on the surface of a Lactobacillus acidophilus vaccine vector and evaluated its ability to increase uptake of L. acidophilus into Peyer's patches and activate innate immune responses. FimH was robustly displayed on the L. acidophilus surface but did not increase uptake into the Peyer's patches. FimH did increase trafficking of L. acidophilus to mesenteric lymph nodes by antigen-presenting cells including macrophages and dendritic cells. It also increased transcription of retinaldehyde dehydrogenase and decreased transcription of IL-21 in the Peyer's patches and mesenteric lymph nodes. The N-terminal domain of FimH did not activate TLR4 in vitro, indicating that FimH may stimulate innate immune responses through a not-yet-identified mechanism. These results indicate that E. coli FimH alters the innate immune response to L. acidophilus and should be further studied as an adjuvant for lactic acid bacterial vaccine platforms.

A Label-Free Carbohydrate-Based Electrochemical Sensor to Detect Escherichia coli Pathogenic Bacteria Using D-mannose on a Glassy Carbon Electrode.

Controlling water and food contamination by pathogenic organisms requires quick, simple, and low-cost methods. Using the affinity between mannose and type I fimbriae in the cell wall of Escherichia coli (E. coli) bacteria as evaluation elements compared to the conventional plate counting technique enables a reliable sensing platform for the detection of bacteria. In this study, a simple new sensor was developed based on electrochemical impedance spectroscopy (EIS) for rapid and sensitive detection of E. coli. The biorecogniton layer of the sensor was formed by covalent attachment of p-carboxyphenylamino mannose (PCAM) to gold nanoparticles (AuNPs) electrodeposited on the surface of a glassy carbon electrode (GCE). The resultant structure of PCAM was characterized and confirmed using a Fourier Transform Infrared Spectrometer (FTIR). The developed biosensor demonstrated a linear response with a logarithm of bacterial concentration (R2 = 0.998) in the range of 1.3 × 10 1~1.3 × 106 CFU·mL-1 with the limit of detection of 2 CFU·mL-1 within 60 min. The sensor did not generate any significant signals with two non-target strains, demonstrating the high selectivity of the developed biorecognition chemistry. The selectivity of the sensor and its applicability to analysis of the real samples were investigated in tap water and low-fat milk samples. Overall, the developed sensor showed to be promising for the detection of E. coli pathogens in water and low-fat milk due to its high sensitivity, short detection time, low cost, high specificity, and user-friendliness.

Binary CuO\CoO nanoparticles inhibit biofilm formation and reduce the expression of papC and fimH genes in multidrug-resistant Klebsiella oxytoca.

BACKGROUND AND AIM: Binary copper-cobalt oxide nanoparticles (CuO\CoO NPs) are modern kinds of antimicrobials, which may get a lot of interest in clinical application. This study aimed to detect the effect of the binary CuO\CoO NPs on the expression of papC and fimH genes in multidrug-resistant (MDR) isolates of Klebsiella oxytoca to reduce medication time and improve outcomes. METHODS: Ten isolates of K. oxytoca were collected and identified by different conventional tests besides PCR. Antibiotic sensitivity and biofilm-forming ability were carried out. The harboring of papC and fimH genes was also detected. The effect of binary CuO\CoO nanoparticles on the expression of papC and fimH genes was investigated. RESULTS: Bacterial resistance against cefotaxime and gentamicin was the highest (100%), while the lowest percentage of resistance was to amikacin (30%). Nine of the ten bacterial isolates had the ability to form a biofilm with different capacities. MIC for binary CuO\CoO NPs was 2.5 µg/mL. Gene expression of papC and fimH was 8.5- and 9-fold lower using the NPs. CONCLUSION: Binary CuO\CoO NPs have a potential therapeutic effect against infections triggered by MDR K. oxytoca strains due to the NPs-related downregulation ability on the virulence genes of K. oxytoca.

Reduction of biofilm formation of Escherichia coli by targeting quorum sensing and adhesion genes using the CRISPR/Cas9-HDR approach, and its clinical application on urinary catheter.

BACKGROUND: Escherichia coli is a common cause of biofilm-associated urinary tract infections (UTIs). Biofilm formation in E. coli is responsible for various indwelling medical device-associated infections, including catheter-associated urinary tract infections (CAUTIs). This study aimed to reduce biofilm formation of E. coli ATCC 25922 by knocking out genes involved in quorum sensing (QS) (luxS) and adhesion (fimH and bolA) using the CRISPR/Cas9-HDR approach. METHOD: Single-guide RNAs (sgRNAs) were designed to target luxS, fimH and bolA genes. Donor DNA for homologous recombination was constructed to provide accurate repairs of double-strand breaks (DSBs). A biofilm quantification assay (crystal violet assay) was performed to quantify the biofilm formation of mutant and wild-type strains. Morphological changes in biofilm architecture were confirmed by scanning electron microscopy (SEM). Further application of the biofilm formation of mutant and wild-type strains on urinary catheter was tested. RESULTS: Crystal violet assay showed that the biofilm formation of ΔfimH, ΔluxS, and ΔbolA strains was significantly reduced compared to the wild-type strain (P value<0.001). The percentage of biofilm reduction of mutant strains was as follows: ΔluxS1 77.51 %, ΔfimH1 78.37 %, ΔfimH2 84.17 %, ΔbolA1 78.24 %, and ΔbolA2 75.39 %. Microscopic analysis showed that all mutant strains lack extracellular polymeric substances (EPS) production compared to the wild-type strain, which was embedded in its EPS matrix. The adherence, cell aggregation, and biofilm formation of wild-type strain on urinary catheters were significantly higher compared to ΔfimH, ΔluxS and ΔbolA strains. CONCLUSION: Altogether, our results demonstrated that the knockout of luxS, fimH, and bolA genes reduced EPS matrix production, which is considered the main factor in the development, maturation, and maintenance of the integrity of biofilm. This pathway could be a potential strategy to disrupt E. coli biofilm-associated UTIs. This study suggests that CRISPR/Cas9-HDR system may provide an efficient and site-specific gene editing approach that exhibits a possible antibiofilm strategy through intervention with the QS mechanism and adhesion property to suppress biofilm formation associated with UTI catheter infections.

Multivalent calix[4]arene-based mannosylated dendrons as new FimH ligands and inhibitors.

We report the synthesis and biological characterization of a novel class of multivalent glycoconjugates as hit compounds for the design of new antiadhesive therapies against urogenital tract infections (UTIs) caused by uropathogenic E. coli strains (UPEC). The first step of UTIs is the molecular recognition of high mannose N-glycan expressed on the surface of urothelial cells by the bacterial lectin FimH, allowing the pathogen adhesion required for mammalian cell invasion. The inhibition of FimH-mediated interactions is thus a validated strategy for the treatment of UTIs. To this purpose, we designed and synthesized d-mannose multivalent dendrons supported on a calixarene core introducing a significant structural change from a previously described family of dendrimers bearing the same dendrons units on a flexible pentaerythritol scaffold core. The new molecular architecture increased the inhibitory potency against FimH-mediated adhesion processes by about 16 times, as assessed by yeast agglutination assay. Moreover, the direct molecular interaction of the new compounds with FimH protein was assessed by on-cell NMR experiments acquired in the presence of UPEC cells.

Structural insights into a cooperative switch between one and two FimH bacterial adhesins binding pauci- and high-mannose type N-glycan receptors.

The FimH type-1 fimbrial adhesin allows pathogenic Escherichia coli to adhere to glycoproteins in the epithelial linings of human bladder and intestinal tract, by using multiple fimbriae simultaneously. Pauci- and high-mannose type N-glycans are natural FimH receptors on those glycoproteins. Oligomannose-3 and oligomannose-5 bind with the highest affinity to FimH by using the same Manα1,3Man branch. Oligomannose-6 is generated from oligomannose-5 in the next step of the biogenesis of high-mannose N-glycans, by the transfer of a mannose in α1,2-linkage onto this branch. Using serial crystallography and by measuring the kinetics of binding, we demonstrate that shielding the high-affinity epitope drives the binding of multiple FimH molecules. First, we profiled FimH glycan binding on a microarray containing paucimannosidic N-glycans and in a FimH LEctPROFILE assay. To make the transition to oligomannose-6, we measured the kinetics of FimH binding using paucimannosidic N-glycans, glycoproteins and all four α-dimannosides conjugated to bovine serum albumin. Equimolar mixed interfaces of the dimannosides present in oligomannose-6 and molecular dynamics simulations suggest a positive cooperativity in the bivalent binding of Manα1,3Manα1 and Manα1,6Manα1 dimannosides. The binding of core α1,6-fucosylated oligomannose-3 in cocrystals of FimH is monovalent but interestingly the GlcNAc1-Fuc moiety retains highly flexibility. In cocrystals with oligomannose-6, two FimH bacterial adhesins bind the Manα1,3Manα1 and Manα1,6Manα1 endings of the second trimannose core (A-4'-B). This cooperative switch towards bivalent binding appears sustainable beyond a molar excess of oligomannose-6. Our findings provide important novel structural insights for the design of multivalent FimH antagonists that bind with positive cooperativity.

Sugar-Lectin Interactions for Direct and Selective Detection of Escherichia coli Bacteria Using QCM Biosensor.

Pathogenic Escherichia coli (E. coli) remains a safety concern in the preservation and quality of green leafy vegetables. Sugar-lectin interactions provide a reliable, specific, and effective sensing platform for the detection of bacteria as compared to the tedious conventional plate counting technique. Herein, we present the synthesis of 4-(N-mannosyl) benzoic acid (4-NMBA) and 4-thiophenyl-N-mannose (4-TNM) via a two-step reductive amination for the detection of E. coli using a quartz crystal microbalance (QCM) biosensor. The 4-NMBA was synthesized with mannose and para-aminobenzoic (4-PBA), while the 4-TNM was synthesized with mannose and 4-aminophenyl disulfide (4-AHP) using water and acetic acid in a 1:1 ratio. The resultant structure of mannose derivatives (4-NMBA and 4-TNM) was characterized and confirmed using analytical tools, such as Mass Spectrometer, SEM, and FTIR. The choice of ligands (mannose derivatives) is ascribed to the specific recognition of mannose to the FimH lectin of the type 1 pilus of E. coli. Furthermore, the 4-PBA and 4-AHP conjugated to mannose increase the ligand affinity to FimH lectins. The setup of the QCM biosensor was composed of modification of the crystal surface and the covalent attachment of ligands for the detection of E. coli. The piezoelectric effect (frequency shift of the quartz) was proportional to the change in mass added to the gold crystal surface. Both the 4-NMBA- and 4-TNM-coated QCM sensors had a limit of detection of 3.7 CFU/mL and 6.6 CFU/mL with a sensitivity of 2.56 × 103 ng/mL and 8.99 × 10-5 ng/mL, respectively, within the dynamic range of 103 to 106 CFU/mL. This study demonstrates the application of ligand-coated QCM biosensors as a cost-effective, simple, and label-free technology for monitoring pathogenic bacteria via molecular interactions on crystal surfaces.