Emerging roles for ABC transporters as virulence factors in uropathogenic Escherichia coli.

Urinary tract infections (UTI) account for a substantial financial burden globally. Over 75% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which have demonstrated an extraordinarily rapid growth rate in vivo. This rapid growth rate appears paradoxical given that urine and the human urinary tract are relatively nutrient-restricted. Thus, we lack a fundamental understanding of how uropathogens propel growth in the host to fuel pathogenesis. Here, we used large in silico, in vivo, and in vitro screens to better understand the role of UPEC transport mechanisms and their contributions to uropathogenesis. In silico analysis of annotated transport systems indicated that the ATP-binding cassette (ABC) family of transporters was most conserved among uropathogenic bacterial species, suggesting their importance. Consistent with in silico predictions, we determined that the ABC family contributed significantly to fitness and virulence in the urinary tract: these were overrepresented as fitness factors in vivo (37.2%), liquid media (52.3%), and organ agar (66.2%). We characterized 12 transport systems that were most frequently defective in screening experiments by generating in-frame deletions. These mutant constructs were tested in urovirulence phenotypic assays and produced differences in motility and growth rate. However, deletion of multiple transport systems was required to achieve substantial fitness defects in the cochallenge murine model. This is likely due to genetic compensation among transport systems, highlighting the centrality of ABC transporters in these organisms. Therefore, these nutrient uptake systems play a concerted, critical role in pathogenesis and are broadly applicable candidate targets for therapeutic intervention.

Conformational ensembles in Klebsiella pneumoniae FimH impact uropathogenesis.

Klebsiella pneumoniae is an important pathogen causing difficult-to-treat urinary tract infections (UTIs). Over 1.5 million women per year suffer from recurrent UTI, reducing quality of life and causing substantial morbidity and mortality, especially in the hospital setting. Uropathogenic E. coli (UPEC) is the most prevalent cause of UTI. Like UPEC, K. pneumoniae relies on type 1 pili, tipped with the mannose-binding adhesin FimH, to cause cystitis. However, K. pneumoniae FimH is a poor binder of mannose, despite a mannose-binding pocket identical to UPEC FimH. FimH is composed of two domains that are in an equilibrium between tense (low-affinity) and relaxed (high-affinity) conformations. Substantial interdomain interactions in the tense conformation yield a low-affinity, deformed mannose-binding pocket, while domain-domain interactions are broken in the relaxed state, resulting in a high-affinity binding pocket. Using crystallography, we identified the structural basis by which domain-domain interactions direct the conformational equilibrium of K. pneumoniae FimH, which is strongly shifted toward the low-affinity tense state. Removal of the pilin domain restores mannose binding to the lectin domain, thus showing that poor mannose binding by K. pneumoniae FimH is not an inherent feature of the mannose-binding pocket. Phylogenetic analyses of K. pneumoniae genomes found that FimH sequences are highly conserved. However, we surveyed a collection of K. pneumoniae isolates from patients with long-term indwelling catheters and identified isolates that possessed relaxed higher-binding FimH variants, which increased K. pneumoniae fitness in bladder infection models, suggesting that long-term residence within the urinary tract may select for higher-binding FimH variants.

Predictive phage therapy for Escherichia coli urinary tract infections: Cocktail selection for therapy based on machine learning models.

This study supports the development of predictive bacteriophage (phage) therapy: the concept of phage cocktail selection to treat a bacterial infection based on machine learning (ML) models. For this purpose, ML models were trained on thousands of measured interactions between a panel of phage and sequenced bacterial isolates. The concept was applied to Escherichia coli associated with urinary tract infections. This is an important common infection in humans and companion animals from which multidrug-resistant (MDR) bloodstream infections can originate. The global threat of MDR infection has reinvigorated international efforts into alternatives to antibiotics including phage therapy. E. coli exhibit extensive genome-level variation due to horizontal gene transfer via phage and plasmids. Associated with this, phage selection for E. coli is difficult as individual isolates can exhibit considerable variation in phage susceptibility due to differences in factors important to phage infection including phage receptor profiles and resistance mechanisms. The activity of 31 phage was measured on 314 isolates with growth curves in artificial urine. Random Forest models were built for each phage from bacterial genome features, and the more generalist phage, acting on over 20% of the bacterial population, exhibited F1 scores of >0.6 and could be used to predict phage cocktails effective against previously untested strains. The study demonstrates the potential of predictive ML models which integrate bacterial genomics with phage activity datasets allowing their use on data derived from direct sequencing of clinical samples to inform rapid and effective phage therapy.

Beyond schistosomiasis: unraveling co-infections and altered immunity.

Schistosomiasis is a neglected tropical disease caused by the helminth Schistosoma spp. and has the second highest global impact of all parasites. Schistosoma are transmitted through contact with contaminated fresh water predominantly in Africa, Asia, the Middle East, and South America. Due to the widespread prevalence of Schistosoma, co-infection with other infectious agents is common but often poorly described. Herein, we review recent literature describing the impact of Schistosoma co-infection between species and Schistosoma co-infection with blood-borne protozoa, soil-transmitted helminths, various intestinal protozoa, Mycobacterium, Salmonella, various urinary tract infection-causing agents, and viral pathogens. In each case, disease severity and, of particular interest, the immune landscape, are altered as a consequence of co-infection. Understanding the impact of schistosomiasis co-infections will be important when considering treatment strategies and vaccine development moving forward.

Risk factors for recurrent urinary tract infections among women in a large integrated health care organization in the United States.

BACKGROUND: Urinary tract infections (UTIs) occur commonly and often recur. However, recent data on the epidemiology of recurrent UTI (rUTI) are scarce. METHODS: Between 01/01/2016-31/12/2020, index uncomplicated UTIs (uUTI) from office, emergency department (ED), hospital, and virtual care settings were identified from electronic health records of women at Kaiser Permanente Southern California. We defined rUTI as ≥3 UTI within 365 days or ≥2 UTI within 180 days. We determined the proportion of women with cystitis index uUTI who had rUTI and examined factors associated with rUTIs using modified multivariable Poisson regression. RESULTS: Among 374,171 women with cystitis index uUTI, 54,318 (14.5%) had rUTI. A higher proportion of women with rUTI compared to those without rUTI were age 18-27 or ≥78 years at index uUTI (19.7% vs 18.7% and 9.0% vs 6.0%, respectively), were immunocompromised, or had a positive urine culture at index uUTI. In multivariable analyses, characteristics associated with rUTI included younger or older age (48-57 vs 18-27 years aRR=0.83 [95% CI: 0.80-0.85]; ≥78 vs 18-27 years aRR=1.07 [95%CI=1.03-1.11]), Charlson Comorbidity Index (≥3 vs 0, aRR=1.12 [95%CI:1.08-1.17]), and diabetes mellitus (aRR=1.07 [95%CI:1.04-1.10]). More frequent prior year outpatient and ED encounters, oral antibiotic prescriptions, oral contraceptive prescriptions, positive culture at index uUTI, and antibiotic resistant organisms were also associated with increased risk of rUTI. CONCLUSIONS: The high risk of rUTI among women with cystitis is concerning, especially given previous reports of increasing UTI incidence. Current assessment of the epidemiology of rUTI may guide the development of preventive interventions against UTI.

Competitive fitness of asymptomatic bacteriuria E. coli strain 83972 against uropathogens in human urine.

Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. The main causative agent of UTI is uropathogenic Escherichia coli (UPEC). There is an immediate need for novel prophylactic and treatment strategies against UTI because of the increasing incidence of antimicrobial resistance among uropathogens. ABU 83972, an asymptomatic bacteriuria-causing E. coli strain, prevents UTI by suppressing the colonization of UPEC. However, the nature of competition and growth repression of UPEC by ABU 83972 is unclear and is the subject of our investigation. Here, we characterized the growth kinetics of ABU 83972 and uropathogens in human urine and laboratory media. Next, we performed a series of competitive co-culture experiments where ABU 83972 and uropathogens were inoculated at a 1:1 ratio in human urine and in various media, and their relative abundance was determined. In human urine, ABU 83972 outcompeted UPEC and additional uropathogens, reaching up to 90% of the total population after 24 hours of incubation. In contrast, UPEC outcompeted ABU 83972 in LB and M9 minimal media and exhibited superior colonization than ABU 83972 in the mouse urinary bladder. Since engineered living materials (ELMs) can be used to retain an organism of interest in a particular location, we developed ABU 83972-containing ELMs that effectively outcompeted UPEC in human urine. In summary, our work establishes that ABU 83972 outcompetes UPEC in a milieu- and cell-density-dependent manner, highlighting the importance of the metabolites and nutrients found in the human urine as determinants of the competitive fitness of ABU 83972.

A cynomolgus monkey E. coli urinary tract infection model confirms efficacy of new FimH vaccine candidates.

The increase in urinary tract infections (UTI) caused by antibiotic-resistant Escherichia coli requires the development of new therapeutic agents and prophylactic vaccines. To evaluate the efficacy of new lead candidates, we implemented a cynomolgus macaque UTI challenge model that mimics human uncomplicated cystitis in response to transurethral challenge with a multidrug-resistant (MDR) E. coli serotype O25b ST131 isolate. E. coli fimbrial adhesin FimH and O-antigens are separately under clinical evaluation by others as vaccine candidates to prevent UTI and invasive urosepsis disease, respectively. Accordingly, we assessed the protective efficacy of three 50-µg intramuscular doses of a novel recombinant FimH antigen adjuvanted with liposomal QS21/MPLA compared with saline placebo in groups of nine animals. A third group was vaccinated with this FimH formulation in combination with 1 µg each of a four-valent mixture of serotype O1a, O2, O6, and O25b O-antigen CRM197 lattice glycoconjugates. Both vaccines elicited high levels of serum FimH IgG and adhesin blocking antibodies at the time of bacterial challenge and, for the combination group, O-antigen-specific antibodies. Following bacterial challenge, both vaccinated groups showed >200- and >700-fold reduction in bacteriuria at day 2 and day 7 post-infection compared with placebo, respectively. In parallel, both vaccines significantly reduced levels of inflammatory biomarkers IL-8 and myeloperoxidase in the urine at day 2 post-infection relative to placebo. Results provide preclinical proof-of-concept for the prevention of an MDR UTI infection by these new vaccine formulations.

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.

Cefazolin as a predictor of urinary cephalosporin activity in indicated Enterobacterales.

Traditionally, cephalothin susceptibility results were used to predict the susceptibility of additional cephalosporins; however, in 2013-2014, the Clinical and Laboratory Standards Institute (CLSI) revisited this practice and determined that cefazolin is a more accurate proxy than cephalothin for uncomplicated urinary tract infections (uUTIs). Therefore, a cefazolin surrogacy breakpoint was established to predict the susceptibility of seven oral cephalosporins for Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in the context of uUTIs. Clinical microbiology laboratories face several operational challenges when implementing the cefazolin surrogacy breakpoint, which may lead to confusion for the best path forward. Here, we review the historical context and data behind the surrogacy breakpoints, review PK/PD profiles for oral cephalosporins, discuss challenges in deploying the breakpoint, and highlight the limited clinical outcome data in this space.

Peptidoglycan endopeptidase MepM of uropathogenic Escherichia coli contributes to competitive fitness during urinary tract infections.

BACKGROUND: Urinary tract infections (UTIs) are common bacterial infections, primarily caused by uropathogenic Escherichia coli (UPEC), leading to significant health issues and economic burden. Although antibiotics have been effective in treating UPEC infections, the rise of antibiotic-resistant strains hinders their efficacy. Hence, identifying novel bacterial targets for new antimicrobial approaches is crucial. Bacterial factors required for maintaining the full virulence of UPEC are the potential target. MepM, an endopeptidase in E. coli, is involved in the biogenesis of peptidoglycan, a major structure of bacterial envelope. Given that the bacterial envelope confronts the hostile host environment during infections, MepM's function could be crucial for UPEC's virulence. This study aims to explore the role of MepM in UPEC pathogenesis. RESULTS: MepM deficiency significantly impacted UPEC's survival in urine and within macrophages. Moreover, the deficiency hindered the bacillary-to-filamentous shape switch which is known for aiding UPEC in evading phagocytosis during infections. Additionally, UPEC motility was downregulated due to MepM deficiency. As a result, the mepM mutant displayed notably reduced fitness in causing UTIs in the mouse model compared to wild-type UPEC. CONCLUSIONS: This study provides the first evidence of the vital role of peptidoglycan endopeptidase MepM in UPEC's full virulence for causing UTIs. MepM's contribution to UPEC pathogenesis may stem from its critical role in maintaining the ability to resist urine- and immune cell-mediated killing, facilitating the morphological switch, and sustaining motility. Thus, MepM is a promising candidate target for novel antimicrobial strategies.

Diabetes, SGLT-2 Inhibitors, and Urinary Tract Infection: a Review.

PURPOSE OF REVIEW: The aim of this review is to focus on epidemiology, pathogenesis, risk factors, management, and complications of UTI in people with diabetes as well as reviewing the association of SGLT-2 inhibitors with genitourinary infections. RECENT FINDINGS: Individuals diagnosed with T2DM are more prone to experiencing UTIs and recurrent UTIs compared to individuals without T2DM. T2DM is associated with an increased risk of any genitourinary infections (GUI), urinary tract infections (UTIs), and genital infections (GIs) across all age categories. SGLT2 inhibitors are a relatively new class of anti-hyperglycemic agents, and studies suggest that they are associated with an increased risk of genitourinary infections. The management of diabetes and lifestyle modifications with a patient-centric approach are the most recognized methods for preventing critical long-term complications including genitourinary manifestations of diabetes. The available data regarding the association of SGLT-2 inhibitors with genitourinary infections is more comprehensive compared to that with UTIs. Further research is needed to better understand the mechanisms underlining the association between SGLT-2 inhibitors and genital infections and UTIs.

A rare urinary tract infection of multidrug-resistant Chryseobacterium urinae sp. nov. isolated from a diabetic, non-catheterized patient.

Chryseobacterium demonstrates a diverse environmental presence and a significant pathogenic potential across various ecosystems. This clinical case showcases a rare instance of bacterial infection in a 75-year-old male with untreated diabetes and recurrent urinary tract infections (UTIs). The patient presented symptoms of abdominal pain, burning urination, fever, and an elevated eosinophil count. A subsequent urine culture identified a Chryseobacterium-related bacterium as the causative agent, exhibiting sensitivity to piperacillin/tazobactam, trimethoprim/sulfamethoxazole, and nitrofurantoin, which led to successful treatment using oral nitrofurantoin. Analysis of the 16S rRNA gene sequence of APV-1T revealed a close relationship of 98.2% similarity to Chryseobacterium gambrini strain 5-1St1aT (AM232810). Furthermore, comparative genome analysis, incorporating Average Nucleotide Identity (ANI), Digital DNA-DNA Hybridization (dDDH) values, and comprehensive phylogenetic assessments utilizing 16S rRNA gene sequences, core genes, and amino acid sequences of core proteins, highlighted the unique phylogenetic positioning of APV-1T within the Chryseobacterium genus. Distinct carbon utilization and assimilation patterns, along with major fatty acid content, set APV-1T apart from C. gambrini strain 5-1St1aT. These findings, encompassing phenotypic, genotypic, and chemotaxonomic characteristics, strongly support the proposal of a novel species named Chryseobacterium urinae sp. nov., with APV-1T designated as the type strain (= MCC 50690 = JCM 36476). Despite its successful treatment, the strain displayed resistance to multiple antibiotics. Genomic analysis further unveiled core-conserved genes, strain-specific clusters, and genes associated with antibiotic resistance and virulence. This report underscores the vital importance of elucidating susceptibility patterns of rare pathogens like Chryseobacterium, particularly in immunocompromised individuals. It advocates for further analyses to understand the functional significance of identified genes and their implications in treatment and pathogenesis.

Genotypic and phenotypic characterisation of asymptomatic bacteriuria (ABU) isolates displaying bacterial interference against multi-drug resistant uropathogenic E. Coli.

Escherichia coli can colonise the urogenital tract of individuals without causing symptoms of infection, in a condition referred to as asymptomatic bacteriuria (ABU). ABU isolates can protect the host against symptomatic urinary tract infections (UTIs) by bacterial interference against uropathogenic E. coli (UPEC). The aim of this study was to investigate the genotypic and phenotypic characteristics of five ABU isolates from midstream urine samples of adults. Comparative genomic and phenotypic analysis was conducted including an antibiotic resistance profile, pangenome analysis, and a putative virulence profile. Based on the genome analysis, the isolates consisted of one from phylogroup A, three from phylogroup B2, and one from phylogroup D. Two of the isolates, PUTS 58 and SK-106-1, were noted for their lack of antibiotic resistance and virulence genes compared to the prototypic ABU strain E. coli 83,972. This study provides insights into the genotypic and phenotypic profiles of uncharacterised ABU isolates, and how relevant fitness and virulence traits can impact their potential suitability for therapeutic bacterial interference.

Evaluation of MV140 in preventing recurrent urinary tract infections: a multicentre double-blind randomized controlled trial protocol.

OBJECTIVES: To determine, firstly, whether MV140 reduces rates of recurrent urinary tract infections (rUTIs) in patients older than 65 years, measured as the number of urinary tract infections (UTIs) detected over 12 months following the completion of a 3-month treatment course and, additionally, to assess the number of re-admissions to the emergency department, the rate of antibiotic use for UTIs, the safety profile of MV140, and quality of life. MATERIALS AND METHODS: This is a multicentre, double-blind, randomized controlled trial with two arms. Patients will be randomized and allocated to receive either a 3-month course of MV140 or placebo (two sublingual sprays daily). Participants will have 3-monthly consultations with the investigator for 12 months to assess differences in rates of rUTIs between the two groups. Study candidates will be identified and recruited from inpatient and outpatient clinics across Sydney via referral to the investigation team. After obtaining consent, participants will undergo initial study consultations including urine microscopy and culture, uroflowmetry, and bladder scan to assess postvoid residual urine volume. Participants will be randomized and provided with a unique trial number. Electronic medical records will be reviewed to collect relevant information. Participants will be provided with a study diary to record relevant data. RESULTS: Follow-up consultations will be conducted every 3 months for a 12-month duration, during which the study diary will be reviewed. These follow-up consultations will primarily occur via telephone review, however, there will be flexibility for in-person reviews for participants who find telephone consultation prohibitively difficult. CONCLUSION: This is a multicentre, double-blinded, randomised control trial, the first in Australia to assess the safety and efficacy of MV140 Uromune vaccine in prevention of recurrent UTIs. Results have been promissing in the global literatures.

Urosepsis 30-day mortality, morbidity, and their risk factors: SERPENS study, a prospective, observational multi-center study.

PURPOSE: To provide a descriptive report of mortality and morbidity in the first 30 days of diagnosis of urosepsis. Secondary aim is to identify risk factors of unfavourable outcomes. METHODS: Prospective observational multicentre cohort study conducted from September 2014 to November 2018 in European hospitals. Adult patients (≥ 18 years) diagnosed with acute urosepsis according to Sepsis-2 criteria with confirmed microbiological infection were included. Outcomes were classified in one of four health states: death, multiple organ failure, single organ failure, and recovery at day 30 from onset of urosepsis. Descriptive statistics and ordinal logistic regression analysis was performed. RESULTS: Three hundred and fifty four patients were recruited, and 30-day mortality rate was 2.8%, rising to 4.6% for severe sepsis. All patients who died had a SOFA score of ≥ 2 at diagnosis. Upon initial diagnosis, 79% (n = 281) of patients presented with OF. Within 30 days, an additional 5% developed OF, resulting in a total of 84% affected. Charlson score (OR 1.14 CI 1.01-1.28), patients with respiratory failure at baseline (OR 2.35, CI 1.32-4.21), ICU admission within the past 12 months (OR 2.05, CI 1.00-4.19), obstruction causative of urosepsis (OR 1.76, CI 1.02-3.05), urosepsis with multi-drug-resistant(MDR) pathogens (OR 2.01, CI 1.15-3.53), and SOFA baseline score ≥ 2 (OR 2.74, CI 1.49-5.07) are significantly associated with day 30 outcomes (OF and death). CONCLUSIONS: Impact of comorbidities and MDR pathogens on outcomes highlights the existence of a distinct group of patients who are prone to mortality and morbidity. These findings underscore the need for the development of pragmatic classifications to better assess the severity of UTIs and guide management strategies. STUDY REGISTRATION: Clinicaltrials.gov registration number NCT02380170.

Clonal relationship, virulence genes, and antimicrobial resistance of Morganella morganii isolated from community-acquired infections and hospitalized patients: a neglected opportunistic pathogen.

Morganella morganii is a bacterium belonging to the normal intestinal microbiota and the environment; however, in immunocompromised individuals, this bacterium can become an opportunistic pathogen, causing a series of diseases, both in hospitals and in the community, being urinary tract infections more prevalent. Therefore, the objective of this study was to evaluate the prevalence, virulence profile, and resistance to antimicrobials and the clonal relationship of isolates of urinary tract infections (UTI) caused by M. morganii, both in the hospital environment and in the community of the municipality of Londrina-PR, in southern Brazil, in order to better understand the mechanisms for the establishment of the disease caused by this bacterium. Our study showed that M. morganii presents a variety of virulence factors in the studied isolates. Hospital strains showed a higher prevalence for the virulence genes zapA, iutA, and fimH, while community strains showed a higher prevalence for the ireA and iutA genes. Hospital isolates showed greater resistance compared to community isolates, as well as a higher prevalence of multidrug-resistant (MDR) and extended-spectrum beta lactamase (ESBL)-producing isolates. Several M. morganii isolates from both sources showed high genetic similarity. The most prevalent plasmid incompatibility groups detected were FIB and I1, regardless of the isolation source. Thus, M. morganii isolates can accumulate virulence factors and antimicrobial resistance, making them a neglected opportunistic pathogen.

Determining the potential targets of silybin by molecular docking and its antibacterial functions on efflux pumps and porins in uropathogenic E. coli.

BACKGROUND: One of the causes of antibiotic resistance is the reduced accumulation of antibiotics in bacterial cells through pumping out the drugs. Silybin, a key component of the Silybum marianum plant, exhibits various beneficial properties, including anti-bacterial, anti-inflammatory, antioxidant, and hepatoprotective effects. METHODS AND RESULTS: Clinical isolates of E. coli were procured from 17 Shahrivar Children's Hospital in Rasht, Guilan, located in northern Iran. Their susceptibility to six antibiotics was assessed using disc diffusion and broth dilution (MIC) methods. The antibacterial effects of silybin-loaded polymersome nanoparticles (SPNs) were investigated with broth dilution (MIC) and biofilm assays. Molecular docking was utilized to evaluate silybin's (the antibacterial component) binding affinity to efflux pumps, porins, and their regulatory elements. Additionally, qRT-PCR analysis explored the expression patterns of acrA, acrB, tolC, ompC, and ompF genes in both SPNs (sub-MIC) and ciprofloxacin (sub-MIC)-treated and untreated E. coli isolates. The combined use of SPNs and ciprofloxacin exhibited a notable reduction in bacterial growth and biofilm formation, in ciprofloxacin-resistant isolates. The study identified eight overlapping binding sites of the AcrABZ-TolC efflux pump in association with silybin, demonstrating a binding affinity ranging from -7.688 to -10.33 Kcal/mol. Furthermore, the qRT-PCR analysis showed that silybin upregulated AcrAB-TolC efflux pump genes and downregulated ompC and ompF porin genes in combination with ciprofloxacin in transcriptional level in uropathogenic E. coli. CONCLUSIONS: Silybin, a safe herbal compound, exhibits potential in inhibiting antibiotic resistance within bacterial isolates, potentially through the regulation of gene expression and plausible binding to target proteins.

Bacteriuria and phenotypic antimicrobial susceptibility testing in 45 min by point-of-care Sysmex PA-100 System: first clinical evaluation.

PURPOSE: This study compared the results of the new Sysmex PA-100 AST System, a point-of-care analyser, with routine microbiology for the detection of urinary tract infections (UTI) and performance of antimicrobial susceptibility tests (AST) directly from urine. METHODS: Native urine samples from 278 female patients with suspected uncomplicated UTI were tested in the Sysmex PA-100 and with reference methods of routine microbiology: urine culture for bacteriuria and disc diffusion for AST. RESULTS: The analyser delivered bacteriuria results in 15 min and AST results within 45 min. Sensitivity and specificity for detection of microbiologically confirmed bacteriuria were 84.0% (89/106; 95% CI: 75.6-90.4%) and 99.4% (155/156; 95% CI: 96.5-100%), respectively, for bacterial species within the analyser specifications. These are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus, which are common species causing uncomplicated UTI. Overall categorical agreement (OCA) for AST results for the five antimicrobials tested in the Sysmex PA-100 (amoxicillin/clavulanic acid, ciprofloxacin, fosfomycin, nitrofurantoin and trimethoprim) ranged from 85.4% (70/82; 95%CI: 75.9-92.2%) for ciprofloxacin to 96.4% (81/84; 95% CI: 89.9-99.3%) for trimethoprim. The Sysmex PA-100 provided an optimal treatment recommendation in 218/278 cases (78.4%), against 162/278 (58.3%) of clinical decisions. CONCLUSION: This first clinical evaluation of the Sysmex PA-100 in a near-patient setting demonstrated that the analyser delivers phenotypic AST results within 45 min, which could enable rapid initiation of the correct targeted treatment with no further adjustment needed. The Sysmex PA-100 has the potential to significantly reduce ineffective or unnecessary antibiotic prescription in patients with UTI symptoms.

Characterization of bacteriophages infecting multidrug-resistant uropathogenic Escherichia coli strains.

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections, and strains that are resistant to antibiotics are a major problem in treating these infections. Phage therapy is a promising alternative approach that can be used to treat infections caused by polyresistant bacterial strains. In the present study, 16 bacteriophages isolated from sewage and surface water were investigated. Phage host specificity was tested on a collection of 77 UPEC strains. The phages infected 2-44 strains, and 80% of the strains were infected by at least one phage. The susceptible E. coli strains belonged predominantly to the B2 phylogenetic group, including strains of two clones, CC131 and CC73, that have a worldwide distribution. All of the phages belonged to class Caudoviricetes and were identified as members of the families Straboviridae, Autographiviridae, and Drexlerviridae and the genera Kagunavirus, Justusliebigvirus, and Murrayvirus. A phage cocktail composed of six phages - four members of the family Straboviridae and two members of the family Autographiviridae - was prepared, and its antibacterial activity was tested in liquid medium. Complete suppression of bacterial growth was observed after 5-22 hours of cultivation, followed by partial regrowth. At 24 hours postinfection, the cocktail suppressed bacterial growth to 43-92% of control values. Similar results were obtained when testing the activity of the phage cocktail in LB and in artificial urine medium. The results indicate that our phage cocktail has potential to inhibit bacterial growth during infection, and they will therefore be preserved in the national phage bank, serving as valuable resources for therapeutic applications.

Presence of intracellular bacterial communities in uroepithelial cells, a potential reservoir in symptomatic and non-symptomatic people.

BACKGROUND: Urinary tract infection is one of the most common infections in humans, affecting women in more proportion. The bladder was considered sterile, but it has a urinary microbiome. Moreover, intracellular bacteria (IB) were observed in uroepithelial cells from children and women with urinary tract infections (UTIs). Here, we evaluated the presence of IB in urine from healthy people and patients with UTI symptoms. METHODS: Midstream urine was self-collected from 141 donors, 77 females and 64 males; 72 belonged to the asymptomatic group and 69 were symptomatic. IB was characterized by a culture-dependent technique and visualized by confocal microscopy. Urine was also subjected to the classical uroculture and isolated bacteria were identified by MALDI-TOF. RESULTS: One-hundred and fifteen uroculture were positive. A significant association was observed between the presence of symptoms and IB (P = 0.007). Moreover, a significant association between the presence of IB, symptoms and being female was observed (P = 0.03). From the cases with IB, Escherichia coli was the most frequent microorganism identified (34.7%), followed by Stenotrophomonas maltophilia (14.2%), Staphylococcus spp (14.2%), and Enterococcus faecalis (10.7%). Intracellular E. coli was associated with the symptomatic group (P = 0.02). Most of the intracellular Staphylococcus spp. were recovered from the asymptomatic group (P = 0.006). CONCLUSIONS: Intracellular bacteria are present in patients with UTI but also in asymptomatic people. Here, we report for the first time, the presence of S. maltophilia, Staphylococcus spp., and Enterobacter cloacae as intracellular bacteria in uroepithelial cells. These findings open new insights into the comprehension of urinary tract infections, urinary microbiome and future therapies. Uroculture as the gold standard could not be enough for an accurate diagnosis in recurrent or complicated cases.