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The dissemination of antibiotic resistance in Escherichia coli poses a significant threat to public health worldwide. This review provides a comprehensive update on the diverse mechanisms employed by E. coli in developing resistance to antibiotics. We primarily focus on pathotypes of E. coli (e.g., uropathogenic E. coli) and investigate the genetic determinants and molecular pathways that confer resistance, shedding light on both well-characterized and recently discovered mechanisms. The most prevalent mechanism continues to be the acquisition of resistance genes through horizontal gene transfer, facilitated by mobile genetic elements such as plasmids and transposons. We discuss the role of extended-spectrum β-lactamases (ESBLs) and carbapenemases in conferring resistance to β-lactam antibiotics, which remain vital in clinical practice. The review covers the key resistant mechanisms, including: 1) Efflux pumps and porin mutations that mediate resistance to a broad spectrum of antibiotics, including fluoroquinolones and aminoglycosides; 2) adaptive strategies employed by E. coli, including biofilm formation, persister cell formation, and the activation of stress response systems, to withstand antibiotic pressure; and 3) the role of regulatory systems in coordinating resistance mechanisms, providing insights into potential targets for therapeutic interventions. Understanding the intricate network of antibiotic resistance mechanisms in E. coli is crucial for the development of effective strategies to combat this growing public health crisis. By clarifying these mechanisms, we aim to pave the way for the design of innovative therapeutic approaches and the implementation of prudent antibiotic stewardship practices to preserve the efficacy of current antibiotics and ensure a sustainable future for healthcare.

Background: Development of multidrug resistance in Uropathogenic Escherichia coli (UPEC) makes treatment of Urinary Tract Infections (UTIs) a major challenge. This study was conducted to investigate the effect of trans-resveratrol (t-RSV) at a subinhibitory concentration (sub-MIC-t-RSV) on phenotypic and genotypic expression of virulence factors of clinical isolates of UPEC and develop a nanoformulation of t-RSV. Fifty-five clinical UPEC strains were investigated for the presence of virulence factors by phenotypic methods and PCR detection of virulence genes. The effect of sub-MIC-t-RSV was studied on the phenotypic and genotypic expression of virulence factors. t-RSV-loaded nanoemulgel formulation was prepared and characterized. Results: Out of the 55 tested isolates, 50.9% were biofilm producers, 23.6% showed both mannose-sensitive and mannose-resistant hemagglutination, 21.8% were serum-resistant, 18.2% were hemolysin producers, while 36.4% showed cytotoxic effect on HEp-2 cells. A total of 25.5% of the isolates harbor one or more of hly-A, cnf-1 and papC genes, while 54.5% were positive for one or more of fimH, iss and BssS genes. A concentration of 100 µg/mL of t-RSV effectively downregulates the phenotypic and genotypic expression of the virulence factors in positive isolates. A stable t-RSV-nanaoemulgel with droplet size of 180.3 nm and Zetapotential of -46.9 mV was obtained. Conclusion: The study proves the effective role of t-RSV as an antivirulence agent against clinical UPEC isolates in vitro and develops a stable t-RSV-nanoemulgel formulation to be assessed in vivo. The promising antibacterial and antivirulence properties of t-RSV place this natural compound to be a better alternative in the treatment of persistent UTIs. [ABSTRACT FROM AUTHOR]

Multidrug resistance is a paramount impediment to successful treatment of most hospital acquired bacterial infections. A plethora of bacterial genera exhibit differential levels of resistance to the existing antibiotics. Prevalent Uropathogenic Escherichia coli or UPEC conduce high mortality among them. Multi-Drug Resistant bacterial strains utilize precise mechanisms to bypass effects of antibiotics. This is probably due to their familiar genomic origin. In this article drug repositioning method have been utilised to target 23 enzymes of UPEC strains viz. CFT073, 536 and UTI89. 3-D drug binding motifs have been predicted using SPRITE and ASSAM servers that compare amino acid side chain similarities. From the hit results anti-viral drugs have been considered for their uniqueness and specificity. Out of 14 anti-viral drugs 3 anti-HIV drugs viz. Amprenavir, Darunavir and Saquinavir have selected for maximum binding score or drug targetability. Finally, active sites of the enzymes were analyzed using GASS-WEB for eloquent drug interference. Further analyses with the active sites of all the enzymes showed that the three selected anti-HIV drugs were very much potent to inhibit their active sites. Combination or sole application of Amprenavir, Darunavir and Saquinavir to MDR-UPEC infections may leads to cure and inhibition of mortality. [ABSTRACT FROM AUTHOR]

Adherence to both cellular and abiotic surfaces is a crucial step in the interaction of bacterial pathogens and commensals with their hosts. Bacterial surface structures known as fimbriae or pili play a fundamental role in the early colonization stages by providing specificity or tropism. Among the various fimbrial families, the chaperone-usher family has been extensively studied due to its ubiquity, diversity, and abundance. This family is named after the components that facilitate their biogenesis. Type 1 fimbria and P pilus, two chaperone-usher fimbriae associated with urinary tract infections, have been thoroughly investigated and serve as prototypes that have laid the foundations for understanding the biogenesis of this fimbrial family. Additionally, the study of the mechanisms regulating their expression has also been a subject of great interest, revealing that the regulation of the expression of the genes encoding these structures is a complex and diverse process, involving both common global regulators and those specific to each operon. [ABSTRACT FROM AUTHOR]

Abstract Background Development of multidrug resistance in Uropathogenic Escherichia coli (UPEC) makes treatment of Urinary Tract Infections (UTIs) a major challenge. This study was conducted to investigate the effect of trans-resveratrol (t-RSV) at a subinhibitory concentration (sub-MIC-t-RSV) on phenotypic and genotypic expression of virulence factors of clinical isolates of UPEC and develop a nanoformulation of t-RSV. Fifty-five clinical UPEC strains were investigated for the presence of virulence factors by phenotypic methods and PCR detection of virulence genes. The effect of sub-MIC-t-RSV was studied on the phenotypic and genotypic expression of virulence factors. t-RSV-loaded nanoemulgel formulation was prepared and characterized. Results Out of the 55 tested isolates, 50.9% were biofilm producers, 23.6% showed both mannose-sensitive and mannose-resistant hemagglutination, 21.8% were serum-resistant, 18.2% were hemolysin producers, while 36.4% showed cytotoxic effect on HEp-2 cells. A total of 25.5% of the isolates harbor one or more of hly-A, cnf-1 and papC genes, while 54.5% were positive for one or more of fimH, iss and BssS genes. A concentration of 100 µg/mL of t-RSV effectively downregulates the phenotypic and genotypic expression of the virulence factors in positive isolates. A stable t-RSV-nanaoemulgel with droplet size of 180.3 nm and Zetapotential of -46.9 mV was obtained. Conclusion The study proves the effective role of t-RSV as an antivirulence agent against clinical UPEC isolates in vitro and develops a stable t-RSV-nanoemulgel formulation to be assessed in vivo. The promising antibacterial and antivirulence properties of t-RSV place this natural compound to be a better alternative in the treatment of persistent UTIs.

The purpose of this study was to investigate the effect of neutrophils and their antimicrobial factors, hydrogen peroxide and defensin α, on the biofilm biomass, the viability of bacteria in the biofilm and the efficiency of conjugative transfer of the pOX38:Cm plasmid from the E. coli N4i pOX38:Cm strain into different E. coli strains (commensal К12 TG1 and uropathogenic DL82, R32 and R45). The biofilm of the recipient E. coli TG1 with the donor E. coli N4i pOX38:Cm increased when 10⁵ cells/ml of neutrophils were added compared to the control, while the biofilm biomass of the uropathogenic E. coli recipient strains DL82/E. coli R45 with the donor E. coli N4i pOX38:Cm decreased when 10⁶/10⁴–10⁶ cells/ml of neutrophils were added, respectively. The survival of recipient E. coli TG1 cells and transconjugants in the biofilm was, compared to the control, higher when 10⁴, 10⁵, 10⁶ cells/ml of neutrophils were added. The addition of 0.1 mM H₂O₂ increased biofilm formation of E. coli DL82 and E. coli R45, and addition of 0.5 mM H₂O₂ reduced biofilm formation of E. coli DL82, while 0.5 mM or 2.5 mM reduced the E. coli R45 bacterial biofilm biomass in the conjugative mixture. The frequency of the pOX38:Cm conjugative transfer was lower in the presence of 2.5 mM H₂O₂ in the N4i pOX38:Cm × DL82 biofilm, and also in the presence of 0.5 and 2.5 mM H₂O₂ in the N4i pOX38:Cm × R45 biofilm, compared to the control. The frequency of pOX38:Cm conjugation from the donor E. coli N4i pOX38:Cm into E. coli DL82 decreased, when 5 or 25 ng/ml defensin α were added and the conjugation frequency in the mating mixture N4i pOX38:Cm×R45 decreased, when 5 ng/ml were added, while, when 25 ng/ml of defensin α were added it increased.

Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z′ factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Region‐specific immune environments in the epididymis influence the immune responses to uropathogenic Escherichia coli (UPEC) infection, a relevant cause of epididymitis in men. Toll‐like receptors (TLRs) are essential to orchestrate immune responses against bacterial infections. The epididymis displays region‐specific inflammatory responses to bacterial‐derived TLR agonists, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), suggesting that TLR‐associated signaling pathways could influence the magnitude of inflammatory responses in epididymitis. Objectives: To investigate the expression and regulation of key genes associated with TLR4 and TLR2/TLR6 signaling pathways during epididymitis induced by UPEC, LPS, and LTA in mice. Material and methods: Epididymitis was induced in mice using UPEC, ultrapure LPS, or LTA, injected into the interstitial space of the initial segment or the lumen of the vas deferens close to the cauda epididymidis. Samples were harvested after 1, 5, and 10 days for UPEC‐treated animals and 6 and 24 h for LPS‐/LTA‐treated animals. Ex vivo epididymitis was induced by incubating epididymal regions from naive mice with LPS or LTA. RT‐qPCR and Western blot assays were conducted. Results: UPEC infection up‐regulated Tlr2, Tlr4, and Tlr6 transcripts and their associated signaling molecules Cd14, Ticam1, and Traf6 in the cauda epididymidis but not in the initial segment. In these epididymal regions, LPS and LTA differentially modulated Tlr2, Tlr4, Tlr6, Cd14, Myd88, Ticam1, Traf3, and Traf6 expression levels. NFKB and AP1 activation was required for LPS‐ and LTA‐induced up‐regulation of TLR‐associated signaling transcripts in the cauda epididymidis and initial segment, respectively. Conclusion: The dynamic modulation of TLR4 and TLR2/TLR6 signaling pathways gene expression during epididymitis indicates bacterial‐derived antigens elicit an increased tissue sensitivity to combat microbial infection in a spatial manner in the epididymis. Differential activation of TLR‐associated signaling pathways may contribute to fine‐tuning inflammatory responses along the epididymis. [ABSTRACT FROM AUTHOR]

Little is known about the characteristics of uropathogenic Escherichia coli (UPEC) associated with recurrent urinary tract infections (RUTIs). The present study aimed to analyze the phenotypic antimicrobial resistance of recurrent UPEC isolates attributable to either relapse or reinfection. A total of 140 E. coli strains were isolated from 70 outpatients with RUTIs. All isolates were analyzed by random amplified polymorphic DNA-polymerase chain reaction to evaluate genetic similarity between the first and second isolates. We found that 64.2% (45/70) of outpatients had a relapse with the primary infecting E. coli strain and 35.7% (25/70) had reinfection with a new E. coli strain. Compared with reinfecting strains, relapse UPEC isolates exhibited much higher antimicrobial resistance; 89% of these isolates were multidrug-resistant and 46.6% were extended-spectrum β-lactamase producers. Our study provides evidence that RUTIs are mainly driven by the persistence of the original strain in the host (relapses) despite appropriate antibiotic treatments, and only RUTIs attributed to relapses seem to favor multidrug resistance in UPEC isolates. [ABSTRACT FROM AUTHOR]

ABSTRACT Uropathogenic Escherichia coli (UPEC) requires an adaptable physiology to survive the wide range of environments experienced in the host, including gut and urinary tract surfaces. To identify UPEC genes required during intracellular infection, we developed a transposon-directed insertion-site sequencing approach for cellular infection models and searched for genes in a library of ~20,000 UTI89 transposon-insertion mutants that are specifically required at the distinct stages of infection of cultured bladder epithelial cells. Some of the bacterial functional requirements apparent in host bladder cell growth overlapped with those for M9-glycerol, notably nutrient utilization, polysaccharide and macromolecule precursor biosynthesis, and cell envelope stress tolerance. Two genes implicated in the intracellular bladder cell infection stage were confirmed through independent gene deletion studies: neuC (sialic acid capsule biosynthesis) and hisF (histidine biosynthesis). Distinct sets of UPEC genes were also implicated in bacterial dispersal, where UPEC erupts from bladder cells in highly filamentous or motile forms upon exposure to human urine, and during recovery from infection in a rich medium. We confirm that the dedD gene linked to septal peptidoglycan remodeling is required during UPEC dispersal from human bladder cells and may help stabilize cell division or the cell wall during envelope stress created by host cells. Our findings support a view that the host intracellular environment and infection cycle are multi-nutrient limited and create stress that demands an array of biosynthetic, cell envelope integrity, and biofilm-related functions of UPEC.IMPORTANCEUrinary tract infections (UTIs) are one of the most frequent infections worldwide. Uropathogenic Escherichia coli (UPEC), which accounts for ~80% of UTIs, must rapidly adapt to highly variable host environments, such as the gut, bladder sub-surface, and urine. In this study, we searched for UPEC genes required for bacterial growth and survival throughout the cellular infection cycle. Genes required for de novo synthesis of biomolecules and cell envelope integrity appeared to be important, and other genes were also implicated in bacterial dispersal and recovery from infection of cultured bladder cells. With further studies of individual gene function, their potential as therapeutic targets may be realized. This study expands knowledge of the UTI cycle and establishes an approach to genome-wide functional analyses of stage-resolved microbial infections.

Introduction. Uropathogenic Escherichia coli (UPEC) are characterized by the ability to survive and reproduce in the urinary tract due to the presence of specific virulence factors. In routine laboratory practice, the detection of diagnostically significant bacteriuria does not provide an idea of the level of infection of the urinary system (renal parenchyma, bladder), the pathogenic potential of the strain in the progression and chronicity of the infectious process, and the occurrence of life-threatening conditions (urosepsis, meningitis). Objective. To characterize the population structure, genetic diversity and pathogenic potential of E. coli strains isolated from urine. Materials and methods. 194 strains of E. coli isolated from urine were studied. Detection of 17 genes encoding the synthesis of: adhesins (pap, fimH, sfa, focG, afa), toxins (hlyA, cvaC, cnf, cdtB), capsular antigens (kpsMTII, kpsMTIII, kpsMT K1), siderophores (fyuA, iutA), invasins (ibeA), genetic markers of the pathogenicity island (PAI) of UPEC CFT073, the gene (traT) encoding serum resistance capacity and phylogenetic groups were performed by PCR (CXT-1000, BioRad, USA) with published primers (Synthol, Sibenzyme, Evrogen, Russia). To assess the statistical significance of differences, Fisher's exact test was used. Differences were considered significant at a confidence interval of 95% (p 0.05). Results. E. coli strains more often (p 0.05) belonged to the phylogenetic group B2 (57.7%). Pathogenetically significant virulence determinants were identified in 97.9% of strains. Based on the combination of 17 genes, 134 individual virulence genotypes were identified. In 93.3% of strains, a genetic predisposition to the occurrence of recurrent urinary tract infections (UTIs) was revealed, in 6.9% there was a potential for the development of pyelonephritis and recurrent cystitis. Markers of life-threatening complications of UTI were identified in 12% of strains, of which 10.7% were the development of urosepsis and 1.3% were meningitis. Conclusion. Detection of a complex of genes in E. coli strains isolated from urine confirms the etiological significance of the isolate and allows one to assess the pathogenic potential for the development of chronic and severe life-threatening complications.

Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) are a significant global health challenge. The UPEC biofilm lifestyle is believed to play an important role in infection recurrency and treatment resistance, but our understanding of how the extracellular matrix (ECM) components curli and cellulose contribute to biofilm formation and pathogenicity is limited. Here, we study the spatial and temporal development of native UPEC biofilm using agar-based detection methods where the non-toxic, optically active fluorescent tracer EbbaBiolight 680 reports the expression and structural location of curli in real-time. An in vitro screen of the biofilm capacity of common UPEC strains reveals significant strain variability and identifies UPEC No. 12 (UPEC12) as a strong biofilm former at 28 °C and 37 °C. Non-interventional microscopy, including time-lapse and 2-photon, reveal significant horizontal and vertical heterogeneity in the UPEC12 biofilm structure. We identify region-specific expression of curli, with a shift in localization from the bottom of the flat central regions of the biofilm to the upper surface in the topographically dramatic intermediate region. When investigating if the rdar morphotype affects wettability of the biofilm surface, we found that the nano-architecture of curli guided by cellulose, rather than the rdar macrostructures, leads to increased hydrophobicity of the biofilm. By providing new insights at exceptional temporal and spatial resolution, we demonstrate how non-interventional analysis of native biofilms will facilitate the next generation of understanding into the roles of ECM components during growth of UPEC biofilms and their contribution to the pathogenesis of UTI.

The emergence of antimicrobial resistance (AMR) is a principal global health crisis projected to cause 10 million deaths annually worldwide by 2050. While the Gram-negative bacteria Escherichia coli is commonly found as a commensal microbe in the human gut, some strains are dangerously pathogenic, contributing to the highest AMR-associated mortality. Strains of E. coli that can translocate from the gastrointestinal tract to distal sites, called extraintestinal E. coli (ExPEC), are particularly problematic and predominantly afflict women, the elderly, and immunocompromised populations. Despite nearly 40 years of clinical trials, there is still no vaccine against ExPEC. One reason for this is the remarkable diversity in the ExPEC pangenome across pathotypes, clades, and strains, with hundreds of genes associated with pathogenesis including toxins, adhesins, and nutrient acquisition systems. Further, ExPEC is intimately associated with human mucosal surfaces and has evolved creative strategies to avoid the immune system. This review summarizes previous and ongoing preclinical and clinical ExPEC vaccine research efforts to help identify key gaps in knowledge and remaining challenges.

Type I fimbriae are the main adhesive organelles of uropathogenic Escherichia coli (UPEC), consisting of four different subunits. Their component with the most important role in establishing bacterial infections is the FimH adhesin located at the fimbrial tip. This two-domain protein mediates adhesion to host epithelial cells through interaction with terminal mannoses on epithelial glycoproteins. Here, we propose that the amyloidogenic potential of FimH can be exploited for the development of therapeutic agents against Urinary Tract Infections (UTIs). Aggregation-prone regions (APRs) were identified via computational methods, and peptide-analogues corresponding to FimH lectin domain APRs were chemically synthesized and studied with the aid of both biophysical experimental techniques and molecular dynamic simulations. Our findings indicate that these peptide-analogues offer a promising set of antimicrobial candidate molecules since they can either interfere with the folding process of FimH or compete for the mannose-binding pocket.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Introduction: Uropathogenic Escherichia coli (UPEC) is a leading cause of community-acquired and healthcare-associated infections, and antimicrobial resistance in UPEC poses significant challenges to managing these infections. This study aimed to investigate the molecular types of UPEC using enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) and analyze their resistance patterns in a tertiary care setting. Methods: A cross-sectional study was conducted at a tertiary care hospital, where 65 consecutive E. coli isolates from urinary specimens were collected. Isolates were identified biochemically and confirmed by 16S rRNA gene PCR. Antibiotic susceptibility testing was conducted following CLSI guidelines, and molecular typing was performed using ERIC-PCR. ERIC-PCR profiles were analyzed using PAST software version 4.0, generating a dendrogram to visualize similarity among ERIC types. Fisher's exact test was used to determine if specific ERIC types were significantly associated with particular antibiotic resistance profiles. Results: The results showed that 95% of the isolates were resistant to at least two antibiotics, with 92.3% being multidrug resistant (MDR). The highest resistance was observed against ampicillin, while no resistance was seen against colistin and tigecycline. The resistant isolates displayed 36 different antibiograms, indicating a significant degree of resistance variability. ERIC-PCR typing revealed 22 unique clusters at a similarity coefficient of approximately 70%, highlighting the genetic diversity of UPEC isolates in our setting. Conclusion: This study enhances the understanding of UPEC epidemiology in healthcare by revealing the molecular characteristics and resistance profiles of prevalent strains. The high occurrence of MDR UPEC and the absence of a correlation between ERIC types and antibiograms suggest adaptability and increased resistance. These results highlight the necessity for continuous surveillance to inform infection control measures and direct targeted interventions against the spread of MDR UPEC. [ABSTRACT FROM AUTHOR]

Infectious diseases, including urinary tract infections (UTIs), significantly impact global health, diminishing the quality of life and increasing clinical and economic burdens. Concerns arise due to the potential development of antimicrobial resistance (AMR) facilitated by biofilm formation. Nanotechnology, specifically silver and silver nanoparticles (1-100 nm), proves effective against bacterial strains, making them valuable in the biomedical field. Advances in microscopic imaging techniques, such as Confocal Laser Scanning Microscopy (CLSM), enhance our understanding of biofilms. This study aims to assess the effectiveness and optimal concentration of gold and silver nanoparticles in combating biofilms formed by uropathogenic Escherichia coli (UPEC) through CLSM analysis. The research comprises the following stages: rejuvenation of bacterial isolates and inoculum preparation, testing the anti-biofilm activity of gold and silver nanoparticles at concentrations of 25, 50, 75, and 100 ppm against UPEC using CLSM analysis. The findings reveal that both gold and silver nanoparticles exhibit anti-biofilm activity against UPEC at concentrations of 25, 50, 75, and 100 ppm, as confirmed by CLSM analysis. The optimal concentration for anti-biofilm activity of gold and silver nanoparticles against UPEC is identified as 100 ppm. [ABSTRACT FROM AUTHOR]

Background Escherichia coli (E. coli) are the most common cause of urinary tract infection (UTI). Clermont and his coworkers recently classi- fied E. coli strains into eight phylogenetic groups using a new quadruplex PCR method. This study aimed to identify the phylogenetic groups of E. coli based on Quadruplex PCR method to detect any of the phylogenetic groups that mainly cause UTIs in the Iraqi population. Method Isolates of Uropathogenic E. coli (UPEC) and other bacteria are identified by culture media and biochemical test, and then UPEC is subjected to phylogenetic analysis by a quadruplex PCR method. Results Out of 210 urine samples, only 114 (54%) samples have a pos- itive culture for E. coli with a count of 10 5 CFU/mL. The frequency of other bacteria are Enterobacter (24%), Proteus (10%), Pseudomonas (6%), Klebsiella (3%), Citrobacter (2%), and Acinetobacter (1%). In the present study, only seven groups of UPEC are detected and PCR profiles showed most UPEC isolate are related to B2 (42(36.8%)) followed by D (24(21.1%)), A 12((10.5%)), C(13(11.4%)), F (9(8%)), clade1 (8 (7%)), E (3(2.6%)) and unknown (3(2.6%)) but not detected any isolate below to group B1 (0(0%)). In the existing study, most phylogenetic groups were isolated from females and from patients in age groups 2, 3, and 4 (young and adults). Conclusion Only seven phylogenetic groups of UPEC are detected, in- cluding A, B2, C, D, E, F, and clade 1. Phylogenetic group B2 was the most common cause of UTI in the Iraqi population. Moreover, most UPEC phylogenetic groups are isolated from young and adult females.

Uropathogenic Escherichia coli (UPEC) is prevalent in urinary tract infections (UTIs). UPEC’s biofilm production enables it to invade and persist in the uroepithelium, leading to recurrent UTIs. The biofilm formation is associated with antibiotic resistance. To overcome this resistance, non-conventional compounds must be developed as an alternative to conventional antibiotics. Silver nanoparticles (AgNPs) are significant due to their antibacterial activity against diverse organisms. This study was done to investigate the antibacterial and anti-biofilm effects of AgNPs on UPEC. AgNPs were biosynthesized using Pseudomonas aeruginosa ATCC 27853. AgNPs were characterized using visual inspection and scanning electron microscopy. The Agar well diffusion method was employed to assess the antibacterial activity of AgNPs against UPEC isolates. The study utilized the tissue culture plate method to investigate both the biofilm and anti-biofilm properties of AgNPs. Following incubation, Ps.aeruginosa and silver nitrate (AgNO3) mixture exhibited a colour change from pale yellow to dark brown. The mean size of spherical AgNPs observed under a scanning electron microscope was 24.187 ± 8.019 nm. 130 UPECs were obtained. AgNPs exhibited antibacterial activity at a concentration of 20 µg/ml against all tested UPEC strains. Among UPEC strains that produced biofilms, a significant inhibition of 99.89 ± 0.45% was observed at a higher concentration of 512 µg/ml of AgNPs. Ps.aeruginosa produces nitrate reductase enzyme that can potentially convert AgNO3 to AgNPs. The biosynthesized AgNPs exhibit antibacterial and anti-biofilm activity against all tested UPEC strains.

المخلص: أهداف البحث: الجرثومة القولونية المسببة لعدوى البول هي العامل المسبب الأكثر شيوعا لإلتهاب المسالك البولية، حيث تشكل أكثر من 80٪ من حالات التهاب المسالك البولية في جميع أنحاء العالم. تقدم هذه الدراسة بيانات حول الأنماط السائدة، ملامح المقاومة، وعوامل شدة الإمراض التي تساعد على التوطين للجرثومة القولونية المسببة لعدوى البول من مناطق جغرافية مختلفة في الهند. طرق البحث: تم تصنيف الأنماط الجرثومية عن طريق التصقل في لوحات القراءة الصغيرة، تم استخدام الأساليب القياسية لكشف عوامل شدة الإمراض المختلفة، أي الأغشية الحية (باستخدام طريقة زراعة الأنسجة)، إفراز الجزيئات الناقلة للحديد (تم الفحص على وسط الكروم أزورول اس وتم تصنيفها وفقا لطرق كساكي وأرنو)، إفراز الكوليسين (باستخدام تقنية تغطية الأغار)، هدم الجيلاتين (على وسط الجيلاتيناز)، التصاق الخلايا السطحي (باستخدام طريقة تجمع الملح) وملامح مقاومة المضادات الحيوية (باستخدام عشرين مضادا حيويا) والبيتا-لاكتاميز ذات الطيف الواسع تم تقييمها وفقا لمعايير معهد الأمراض السريرية والمختبرية. النتائج: أظهرت سلالات الجرثومة القولونية المسببة لعدوى البول معدلات مقاومة عالية جدا لمعظم المضادات الحيوية المستخدمة بشكل شائع مع أعلى معدلات مقاومة للأمبيسيلين (63.4٪)، وحامض الناليديكسيك (63.4٪)، والسيفوتاكسيم (62.1٪). تم الكشف عن معدلات عالية من مقاومة الأدوية المتعددة (63.36٪)، وإنتاج البيتا-لاكتاميز ذات الطيف الواسع (34.1٪)، والجرثومة القولونية المسببة لعدوى البول المقاومة للكاربابينيم (25.0٪) وكانت من جميع المناطق الجغرافية في الهند. التصاق الخلايا السطحي (61.2٪)، إنتاج الأغشية الحية (62.5٪)، وإنتاج الجزيئات الناقلة للحديد (67.7٪) كانت من أكثر خصائص الخبيثة شيوعا في عزلات الإشريكية القولونية المسببة لعدوى الجهاز البولي. كان تعبير مشترك عن عوامل شدة الإمراض شائع جدا (69.8٪) في سلالات الإشريكية القولونية المسببة لعدوى الجهاز البولي. الاستنتاجات: توجد في الهند سلالات من الجرثومة القولونية المسببة لعدوى البول مقاومة للأدوية المضادة للميكروبات بمعدلات عالية جدا، وهي متنوعة من حيث الأنماط الجرثومية وعوامل شدة الإمراض. Abstract: Objectives: Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection, accounting for more than 80% of cases worldwide. This study presents data on prevalent serotypes, resistance profiles, and colonization-aiding virulence characteristics of UPEC from different geographical regions in India. Methods: UPEC were serotyped through microtiter plate agglutination. Standard techniques were used to detect various virulence characteristics, i.e., biofilm formation (tissue culture plate method), siderophore production (screened on Chrome Azurol S agar and categorized with Csaky's and Arnow's methods), colicin release (agar overlay technique), gelatin hydrolysis (on gelatinase agar), and cell surface hydrophobicity (salt aggregation method). Antibiotic resistance profiles (against 20 antimicrobial agents) and extended-spectrum beta-lactamase (ESBL) were evaluated according to Clinical and Laboratory Standards Institute guidelines. Results: UPEC strains exhibited very high drug resistance rates to most of the commonly used antimicrobial agents; the highest resistance rates were observed for ampicillin (63.4%), nalidixic acid (63.4%), and cefotaxime (62.1%). High rates of multi-drug resistance (63.36%), ESBL-production (34.1%), and carbapenem-resistance (25.0%) were detected in UPEC strains from all geographical regions of India. Hydrophobicity (61.2%), biofilm production (62.5%), and siderophore production (67.7%) were the most common virulence characteristics of UPEC isolates. Co-expression of virulence characteristics was common (69.8%) in UPEC strains. Conclusion: UPEC strains with very high antimicrobial-resistance are in circulation in India, and have diverse serotypes and virulence characteristics.

Introduction: Recurrent urinary tract infections (RUTIs) caused by uropathogenic Escherichia coli are costly public health problems impacting patients' quality of life. Aim: In this work, a comparative genomics analysis of three clinical RUTI strains isolated from bladder biopsy specimens was performed. Materials and methods: One hundred seventy-two whole genomes of urinary tract E. coli strains were selected from the NCBI database. The search for virulence factors, fitness genes, regions of interest, and genetic elements associated with resistance was manually carried out. The phenotypic characterization of antibiotic resistance, haemolysis, motility, and biofilm formation was performed. Moreover, adherence and invasion assays with human bladder HTB-5 cells, and transmission electron microscopy (TEM) were performed. Results: The UTI-1_774U and UTI-3_455U/ST1193 strains were associated with the extraintestinal pathotypes, and the UTI-2_245U/ST295 strain was associated with the intestinal pathotype, according to a phylogenetic analysis of 172 E. coli urinary strains. The three RUTI strains were of clinical, epidemiological, and zoonotic relevance. Several resistance genes were found within the plasmids of these strains, and a multidrug resistance phenotype was revealed. Other virulence genes associated with CFT073 were not identified in the three RUTI strains (genes for type 1 and P fimbriae, haemolysin hlyA, and sat toxin). Quantitative adherence analysis showed that UTI-1_774U was significantly (p < 0.0001) more adherent to human bladder HTB-5 cells. Quantitative invasion analysis showed that UTI-2_245U was significantly more invasive than the control strains. No haemolysis or biofilm activity was detected in the three RUTI strains. The TEM micrographs showed the presence of short and thin fimbriae only in the UTI-2_245U strain. Conclusion: The high variability and genetic diversity of the RUTI strains indicate that are a mosaic of virulence, resistance, and fitness genes that could promote recurrence in susceptible patients. [ABSTRACT FROM AUTHOR]

Alpha hemolysin (HlyA) is a hemolytic and cytotoxic protein secreted by uropathogenic strains of E. coli. The role of glycophorins (GPs) as putative receptors for HlyA binding to red blood cells (RBCs) has been debated. Experiments using anti -GPA/GPB antibodies and a GPA-specific epitope nanobody to block HlyA-GP binding on hRBCs, showed no effect on hemolytic activity. Similarly, the hemolysis induced by HlyA remained unaffected when hRBCs from a GPAnull/GPBnull variant were used. Surface Plasmon Resonance experiments revealed similar values of the dissociation constant between GPA and either HlyA, ProHlyA (inactive protoxin), HlyAΔ914-936 (mutant of HlyA lacking the binding domain to GPA) or human serum albumin, indicating that the binding between the proteins and GPA is not specific. Although far Western blot followed by mass spectroscopy analyses suggested that HlyA interacts with Band 3 and spectrins, hemolytic experiments on spectrin-depleted hRBCs and spherocytes, indicated these proteins do not mediate the hemolytic process. Our results unequivocally demonstrate that neither glycophorins, nor Band 3 and spectrins mediate the cytotoxic activity of HlyA on hRBCs, thereby challenging the HlyA-receptor hypothesis. This finding holds significant relevance for the design of anti-toxin therapeutic strategies, particularly in light of the growing antibiotic resistance exhibited by bacteria. [Display omitted] • Anti- GPA, anti GPA-GPB and nanoantibody iH4 do not affect HlyA hemolytic activity. • GPAnull/GPBnull RBCs are as sensitive to HlyA as control RBCs. • The K D values between GP proteins and HlyA, HlyA Δ914–936 and ProHlyA are similar. • Band 3 is not involved in the lytic mechanism of HlyA. • HlyA binds in the same extent to control and spectrin-depleted ghosts. [ABSTRACT FROM AUTHOR]

Extraintestinal pathogenic Escherichia coli (ExPEC) account for over 80% and 60% of bacterial urinary tract infections (UTIs) in humans and animals, respectively. As shared uropathogenic E. coli (UPEC) strains have been previously reported among humans and pets, our study aimed to characterize E. coli lineages among UTI isolates from dogs and cats and to assess their overlaps with human UPEC lineages. We analysed 315 non-duplicate E. coli isolates from the UT of dogs (198) and cats (117) collected in central Germany in 2019 and 2020 utilizing whole genome sequencing and in silico methods. Phylogroup B2 (77.8%), dog-associated sequence type (ST) 372 (18.1%), and human-associated ST73 (16.6%), were predominant. Other STs included ST12 (8.6%), ST141 (5.1%), ST127 (4.8%), and ST131 (3.5%). Among these, 58.4% were assigned to the ExPEC group and 51.1% to the UPEC group based on their virulence associated gene (VAG) profile (ExPEC, presence of ≥VAGs: papAH and/or papC, sfa/focG, afaD/draBC, kpsMTII, and iutA; UPEC, additionally cnf1 or hlyD). Extended-spectrum cephalosporin (ESC) resistance mediated by extended-spectrum β-lactamases (ESBL) and AmpC-β-lactamase was identified in 1.9% of the isolates, along with one carbapenemase-producing isolate and one isolate carrying a mcr gene. Low occurrence of ESC-resistant or multidrug-resistant (MDR) isolates (2.9%) in the two most frequently detected STs implies that E. coli isolated from UTIs of companion animals are to a lesser extent associated with resistance, but possess virulence-associated genes enabling efficient UT colonization and carriage. Detection of human-related pandemic lineages suggests interspecies transmission and underscores the importance of monitoring companion animals. [ABSTRACT FROM AUTHOR]

Copyright of Revista Latinoamericana de Hipertension is the property of Revista Latinoamericana de Hipertension and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Uropathogenic Escherichia coli (UPEC) is prevalent in urinary tract infections (UTIs). UPEC's biofilm production enables it to invade and persist in the uroepithelium, leading to recurrent UTIs. The biofilm formation is associated with antibiotic resistance. To overcome this resistance, non-conventional compounds must be developed as an alternative to conventional antibiotics. Silver nanoparticles (AgNPs) are significant due to their antibacterial activity against diverse organisms. This study was done to investigate the antibacterial and anti-biofilm effects of AgNPs on UPEC. AgNPs were biosynthesized using Pseudomonas aeruginosa ATCC 27853. AgNPs were characterized using visual inspection and scanning electron microscopy. The Agar well diffusion method was employed to assess the antibacterial activity of AgNPs against UPEC isolates. The study utilized the tissue culture plate method to investigate both the biofilm and anti-biofilm properties of AgNPs. Following incubation, Ps.aeruginosa and silver nitrate (AgNO3) mixture exhibited a colour change from pale yellow to dark brown. The mean size of spherical AgNPs observed under a scanning electron microscope was 24.187 ± 8.019 nm. 130 UPECs were obtained. AgNPs exhibited antibacterial activity at a concentration of 20 µg/ml against all tested UPEC strains. Among UPEC strains that produced biofilms, a significant inhibition of 99.89 ± 0.45% was observed at a higher concentration of 512 µg/ml of AgNPs. Ps.aeruginosa produces nitrate reductase enzyme that can potentially convert AgNO3 to AgNPs. The biosynthesized AgNPs exhibit antibacterial and anti-biofilm activity against all tested UPEC strains. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Taibah University Medical Sciences is the property of Elsevier B.V. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Uropathogenic Escherichia coli (UPEC) strains are among the leading causes of urinary tract infections (UTIs) worldwide. They can colonize the urinary tract and form biofilms that allow bacteria to survive and persist, causing relapses of infections and life-threatening sequelae. Here, we analyzed biofilm production, antimicrobial susceptibility, virulence factors, and phylogenetic groups in 74 E. coli isolated from diagnosed patients with UTIs to describe their microbiological features and ascertain their relationship with biofilm capabilities. High levels of ceftazidime resistance are present in hospital-acquired UTIs. Isolates of multidrug resistance strains (p = 0.0017) and the yfcV gene (p = 0.0193) were higher in male patients. All the strains tested were able to form biofilms. Significant differences were found among higher optical densities (ODs) and antibiotic resistance to cefazolin (p = 0.0395), ceftazidime (p = 0.0302), and cefepime (p = 0.0420). Overall, the presence of fimH and papC coincided with strong biofilm formation by UPEC. Type 1 fimbriae (p = 0.0349), curli (p = 0.0477), and cellulose (p = 0.0253) production was significantly higher among strong biofilm formation. Our results indicated that high antibiotic resistance may be related to male infections as well as strong and moderate biofilm production. The ability of E. coli strains to produce biofilm is important for controlling urinary tract infections. [ABSTRACT FROM AUTHOR]

ABSTRACTUrinary tract infection (UTI) is a ubiquitous infectious condition, and uropathogenic Escherichia coli (UPEC) is the predominant causative agent of UTI. Copper (Cu) is implicated in innate immunity, including against UPEC. Cu is a trace element utilized as a co-factor, but excess Cu is toxic due to mismetalation of non-cognate proteins. E. coli precisely regulates Cu homeostasis via efflux systems. However, Cu import mechanisms into the bacterial cell are not clear. We hypothesized that Cu import defective mutants would exhibit increased resistance to Cu. This hypothesis was tested in a forward genetic screen with transposon (Tn5) insertion mutants in UPEC strain CFT073, and we identified 32 unique Cu-resistant mutants. Transposon and defined mutants lacking yhiM, which encodes a hypothetical inner membrane protein, were more resistant to Cu than parental strain. Loss of YhiM led to decreased cellular Cu content and increased expression of copA, encoding a Cu efflux pump. The CpxAR envelope stress response system was activated in the ΔyhiM mutant as indicated by increased expression of cpxP. Transcription of yhiM was regulated by CueR and CpxR, and the CpxAR system was essential for increased Cu resistance in the ΔyhiM mutant. Importantly, activation of CpxAR system in the ΔyhiM mutant was independent of NlpE, a known activator of this system. YhiM was required for optimal fitness of UPEC in a mouse model of UTI. Our findings demonstrate that YhiM is a critical mediator of Cu homeostasis and links bacterial adaptation to Cu stress with the CpxAR-dependent envelope stress response in UPEC.IMPORTANCEUPEC is a common bacterial infection. Bacterial pathogens are exposed to host-derived Cu during infection, including UTI. Here, we describe detection of genes involved in Cu homeostasis in UPEC. A UPEC mutant lacking YhiM, a membrane protein, exhibited dramatic increase in resistance to Cu. Our study demonstrates YhiM as a nexus between Cu stress and the CpxAR-dependent envelope stress response system. Importantly, our findings establish NlpE-independent activation of CpxAR system during Cu stress in UPEC. Collectively, YhiM emerges as a critical mediator of Cu homeostasis in UPEC and highlights the interlinked nature of bacterial adaptation to survival during Cu and envelope stress.

The dissemination of antibiotic resistance in Escherichia coli poses a significant threat to public health worldwide. This review provides a comprehensive update on the diverse mechanisms employed by E. coli in developing resistance to antibiotics. We primarily focus on pathotypes of E. coli (e.g., uropathogenic E. coli) and investigate the genetic determinants and molecular pathways that confer resistance, shedding light on both well-characterized and recently discovered mechanisms. The most prevalent mechanism continues to be the acquisition of resistance genes through horizontal gene transfer, facilitated by mobile genetic elements such as plasmids and transposons. We discuss the role of extended-spectrum β-lactamases (ESBLs) and carbapenemases in conferring resistance to β-lactam antibiotics, which remain vital in clinical practice. The review covers the key resistant mechanisms, including: 1) Efflux pumps and porin mutations that mediate resistance to a broad spectrum of antibiotics, including fluoroquinolones and aminoglycosides; 2) adaptive strategies employed by E. coli, including biofilm formation, persister cell formation, and the activation of stress response systems, to withstand antibiotic pressure; and 3) the role of regulatory systems in coordinating resistance mechanisms, providing insights into potential targets for therapeutic interventions. Understanding the intricate network of antibiotic resistance mechanisms in E. coli is crucial for the development of effective strategies to combat this growing public health crisis. By clarifying these mechanisms, we aim to pave the way for the design of innovative therapeutic approaches and the implementation of prudent antibiotic stewardship practices to preserve the efficacy of current antibiotics and ensure a sustainable future for healthcare.

ABSTRACTSensory systems allow pathogens to differentiate between different niches and respond to stimuli within them. A major mechanism through which bacteria sense and respond to stimuli in their surroundings is two-component systems (TCSs). TCSs allow for the detection of multiple stimuli to lead to a highly controlled and rapid change in gene expression. Here, we provide a comprehensive list of TCSs important for the pathogenesis of uropathogenic Escherichia coli (UPEC). UPEC accounts for >75% of urinary tract infections (UTIs) worldwide. UTIs are most prevalent among people assigned female at birth, with the vagina becoming colonized by UPEC in addition to the gut and the bladder. In the bladder, adherence to the urothelium triggers E. coli invasion of bladder cells and an intracellular pathogenic cascade. Intracellular E. coli are safely hidden from host neutrophils, competition from the microbiota, and antibiotics that kill extracellular E. coli. To survive in these intimately connected, yet physiologically diverse niches E. coli must rapidly coordinate metabolic and virulence systems in response to the distinct stimuli encountered in each environment. We hypothesized that specific TCSs allow UPEC to sense these diverse environments encountered during infection with built-in redundant safeguards. Here, we created a library of isogenic TCS deletion mutants that we leveraged to map distinct TCS contributions to infection. We identify—for the first time—a comprehensive panel of UPEC TCSs that are critical for infection of the genitourinary tract and report that the TCSs mediating colonization of the bladder, kidneys, or vagina are distinct.IMPORTANCEWhile two-component system (TCS) signaling has been investigated at depth in model strains of Escherichia coli, there have been no studies to elucidate—at a systems level—which TCSs are important during infection by pathogenic Escherichia coli. Here, we report the generation of a markerless TCS deletion library in a uropathogenic E. coli (UPEC) isolate that can be leveraged for dissecting the role of TCS signaling in different aspects of pathogenesis. We use this library to demonstrate, for the first time in UPEC, that niche-specific colonization is guided by distinct TCS groups.

ABSTRACTUropathogenic Escherichia coli (UPEC) is the primary causative agent of lower urinary tract infection (UTI). UTI presents a serious health risk and has considerable secondary implications including economic burden, recurring episodes, and overuse of antibiotics. A safe and effective vaccine would address this widespread health problem and emerging antibiotic resistance. Killed, whole-cell vaccines have shown limited efficacy to prevent recurrent UTI in human trials. We explored photochemical inactivation with psoralen drugs and UVA light (PUVA), which crosslinks nucleic acid, as an alternative to protein-damaging methods of inactivation to improve whole-cell UTI vaccines. Exposure of UPEC to the psoralen drug AMT and UVA light resulted in a killed but metabolically active (KBMA) state, as reported previously for other PUVA-inactivated bacteria. The immunogenicity of PUVA-UPEC as compared to formalin-inactivated UPEC was compared in mice. Both generated high UPEC-specific serum IgG titers after intramuscular delivery. However, using functional adherence as a measure of surface protein integrity, we found differences in the properties of PUVA- and formalin-inactivated UPEC. Adhesion mediated by Type-1 and P-fimbriae was severely compromised by formalin but was unaffected by PUVA, indicating that PUVA preserved the functional conformation of fimbrial proteins, which are targets of protective immune responses. In vitro assays indicated that although they retained metabolic activity, PUVA-UPEC lost virulence properties that could negatively impact vaccine safety. Our results imply the potential for PUVA to improve killed, whole-cell UTI vaccines by generating bacteria that more closely resemble their live, infectious counterparts relative to vaccines generated with protein-damaging methods.IMPORTANCELower urinary tract infection (UTI), caused primarily by uropathogenic Escherichia coli, represents a significant health burden, accounting for 7 million primary care and 1 million emergency room visits annually in the United States. Women and the elderly are especially susceptible and recurrent infection (rUTI) is common in those populations. Lower UTI can lead to life-threatening systemic infection. UTI burden is manifested by healthcare dollars spent (1.5 billion annually), quality of life impact, and resistant strains emerging from antibiotic overuse. A safe and effective vaccine to prevent rUTI would address a substantial healthcare issue. Vaccines comprised of inactivated uropathogenic bacteria have yielded encouraging results in clinical trials but improvements that enhance vaccine performance are needed. To that end, we focused on inactivation methodology and provided data to support photochemical inactivation, which targets nucleic acid, as a promising alternative to conventional protein-damaging inactivation methods to improve whole-cell UTI vaccines.

During 2015–2022, a genetic cluster of OXA-48–producing uropathogenic Escherichia coli sequence type 127 spread throughout the Netherlands. The 20 isolates we investigated originated mainly from urine, belonged to Clermont phylotype B2, and carried 18 genes encoding putative uropathogenicity factors. The isolates were susceptible to first-choice antimicrobial drugs for urinary tract infections.

Recurrent urinary tract infections (UTIs) are associated primarily with the ability of Escherichia coli to form biofilms. The interaction of neutrophils, factors of innate immunity, with microorganisms in biofilms is difficult compared to planktonic forms due to the lack of direct contact, as well as due to the antiphagocytic action of the extracellular matrix of biofilms. The purpose of this study was evaluation of neutrophils phagocytic and oxidative activity during interaction with biofilms of uropathogenic E. coli (UPEC) DL82 and R44. Peripheral blood neutrophils from healthy men were isolated using ficoll-urographin double gradient, incubated for 1 h with bacterial cells from biofilms or their supernatants, then leukocytes functional activity was evaluated. Phagocytic activity of neutrophils was determined by the degree of bioluminescence inhibition of bioluminescent strain E. coli K12 TG1 lux+ (pXen) upon their absorption by neutrophils. Production of extracellular reactive oxygen species (ROS) was analyzed by the intensity of luminol-dependent chemiluminescence in spontaneous and stimulated by E. coli K12 variants. Significance of differences was determined using Student’s t-test at p < 0.05. It was found that neutrophils interaction with UPEC biofilm cells or supernatants did not affect the phagocytic activity. E. coli DL82 supernatants reduce neutrophils spontaneous ROS production compared to control and biofilm cells. E. coli R44 supernatants with a low virulence potential did not affect ROS production, while biofilm cells stimulated it. When assessing stimulated ROS production, exposure to R44 strain supernatants did not cause a decrease in neutrophils activation in response to an external stimulus (E. coli K12 cells). Preliminary contact of neutrophils with E. coli R44 bacteria resulted in a high and prolonged level of ROS production compared to the control. Neutrophils interaction with DL82 cells resulted in a higher level of ROS compared to supernatants, however a subsequent rapid depletion of neutrophils oxidative potential was observed. Thus, cells and supernatants of UPEC biofilms can determine the activation of neutrophils.

IntroductionRecurrent urinary tract infections (RUTIs) caused by uropathogenic Escherichia coli are costly public health problems impacting patients’ quality of life.AimIn this work, a comparative genomics analysis of three clinical RUTI strains isolated from bladder biopsy specimens was performed.Materials and methodsOne hundred seventy-two whole genomes of urinary tract E. coli strains were selected from the NCBI database. The search for virulence factors, fitness genes, regions of interest, and genetic elements associated with resistance was manually carried out. The phenotypic characterization of antibiotic resistance, haemolysis, motility, and biofilm formation was performed. Moreover, adherence and invasion assays with human bladder HTB-5 cells, and transmission electron microscopy (TEM) were performed.ResultsThe UTI-1_774U and UTI-3_455U/ST1193 strains were associated with the extraintestinal pathotypes, and the UTI-2_245U/ST295 strain was associated with the intestinal pathotype, according to a phylogenetic analysis of 172 E. coli urinary strains. The three RUTI strains were of clinical, epidemiological, and zoonotic relevance. Several resistance genes were found within the plasmids of these strains, and a multidrug resistance phenotype was revealed. Other virulence genes associated with CFT073 were not identified in the three RUTI strains (genes for type 1 and P fimbriae, haemolysin hlyA, and sat toxin). Quantitative adherence analysis showed that UTI-1_774U was significantly (p

Urinary tract infections (UTIs) are the second most common bacterial infection with high recurrence rates and can involve biofilm formation on patient catheters. Biofilms are inherently tolerant to antimicrobials, making them difficult to eradicate. Many antibiofilm agents alone do not have bactericidal activity; therefore, linking them to antibiotics is a promising antibiofilm strategy. However, many of these hybrid agents have not been tested in relevant preclinical settings, limiting their potential for clinical translation. Here, we evaluate a ciprofloxacin di-nitroxide hybrid (CDN11), previously reported to have antibiofilm activity against uropathogenic Escherichia coli (UPEC) strain UTI89 in vitro, as a potential UTI therapeutic using multiple preclinical models that reflect various aspects of UTI pathogenesis. We report improved in vitro activity over the parent drug ciprofloxacin against mature UTI89 biofilms formed inside polyethylene catheters. In bladder cell monolayers infected with UTI89, treatment with CDN11 afforded significant reduction in bacterial titers, including intracellular UPEC. Infected mouse bladders containing biofilm-like intracellular reservoirs of UPEC UTI89 showed decreased bacterial loads after ex vivo bladder treatment with CDN11. Activity for CDN11 was reported across different models of UTI, showcasing nitroxide–antibiotic hybridization as a promising antibiofilm approach. The pipeline we described here could be readily used in testing other new therapeutic compounds, fast-tracking the development of novel antibiofilm therapeutics. [ABSTRACT FROM AUTHOR]

Background: Necrotizing enterocolitis (NEC) is still one of the leading causes of neonatal death. The present study reports the data from a French case–control prospective multicenter study. Methods: A total of 146 preterm neonates (PNs) with or without NEC were included. Bacterial 16S rRNA gene sequencing was performed on stool samples (n = 103). Specific culture media were used to isolate Escherichia coli, Clostridium butyricum, and Clostridium neonatale, and strains were phenotypically characterized. Results: The gut microbiota of PNs was dominated by Firmicutes and Proteobacteria, and five enterotypes were identified. The microbiota composition was similar between NEC cases and PN controls. However, differences were observed in the relative abundance of Lactobacillus genus, which was significantly lower in the NEC group, whereas that of the Clostridium cluster III was significantly higher (p < 0.05). Within enterotypes, several phylotypes were significantly more abundant in NEC cases (p < 0.05). Regarding perinatal factors, a statistical association was found between the gut microbiota and cesarean delivery and antifungal therapy. In NEC cases and PN controls, the carriage rates and virulence genes of uropathogenic E. coli were equivalent based on culture. No correlation was found between E. coli, C. butyricum, and C. neonatale carriages, beta-lactam resistance, and antibiotic treatment. Conclusions: At disease onset, our data support a microbiota dysbiosis between NEC and control infants at the genus level. In addition, it provides valuable information on bacterial antimicrobial susceptibility. [ABSTRACT FROM AUTHOR]

Uropathogenic Escherichia coli (UPEC) is a nosocomial pathogen associated with urinary tract infections and biofilm formation, which contributes to antibiotic resistance. Discovering potent antibacterial agents is crucial. This study aimed to assess the antibacterial and antibiofilm effects of gold and silver nanoparticles on UPEC using Scanning Electron Microscopy (SEM). UPEC biofilms were cultivated on nitrocellulose membranes for 48 hours at 37°C, then treated with gold nanoparticles (50 ppm and 100 ppm) and silver nanoparticles (50 ppm and 100 ppm) for another 48 hours. Antibacterial and antibiofilm activities were evaluated through cell density and SEM analysis. SEM revealed lower cell density, reduced biofilm formation, and altered cell morphology with rough, wrinkled surfaces after nanoparticle treatment. In conclusion, gold and silver nanoparticles exhibit antibacterial and antibiofilm properties, as observed in SEM analysis. SEM is a valuable tool for studying the antimicrobial effects of nano gold and silver on bacterial cell morphology and biofilm populations.

The modification of lipopolysaccharide (LPS) in Escherichia coli and Salmonella spp. is primarily controlled by the two-component system PmrAB. LPS modification allows bacteria to avoid killing by positively charged antibiotics like polymyxin B (PMB). We previously demonstrated that in uropathogenic E. coli (UPEC), the sensor histidine kinase PmrB also activates a non-cognate transcription factor, QseB, and this activation somehow augments PMB tolerance in UPEC. Here, we demonstrate—for the first time—that in the absence of the canonical LPS transcriptional regulator, PmrA, QseB can direct some modifications on the LPS. In agreement with this observation, transcriptional profiling analyses demonstrate regulatory overlaps between PmrA and QseB in terms of regulating LPS modification genes. However, both PmrA and QseB must be present for UPEC to mount robust tolerance to PMB. Transcriptional and metabolomic analyses also reveal that QseB transcriptionally regulates the metabolism of glutamate and 2-oxoglutarate, which are consumed and produced during the modification of lipid A. We show that deletion of qseB alters glutamate levels in the bacterial cells. The qseB deletion mutant, which is susceptible to positively charged antibiotics, is rescued by exogenous addition of 2-oxoglutarate. These findings uncover a previously unknown mechanism of metabolic control of antibiotic tolerance that may be contributing to antibiotic treatment failure in the clinic. IMPORTANCE Although antibiotic prescriptions are guided by well-established susceptibility testing methods, antibiotic treatments oftentimes fail. The presented work is significant because it uncovers a mechanism by which bacteria transiently avoid killing by antibiotics. This mechanism involves two closely related transcription factors, PmrA and QseB, which are conserved across Enterobacterales. We demonstrate that PmrA and QseB share regulatory targets in lipid A modification pathway and prove that QseB can orchestrate modifications of lipid A in Escherichia coli in the absence of PmrA. Finally, we show that QseB controls glutamate metabolism during the antibiotic response. These results suggest that rewiring of QseB-mediated metabolic genes could lead to stable antibiotic resistance in subpopulations within the host, thereby contributing to antibiotic treatment failure.

In this study, the data of the antagonistic action of the enzyme L-lysine-α-oxidase in relation to clinical isolates of multiresistant uropathogenic Escherichia coli isolated from patients aged 2 to 17 years of various genders with an established diagnosis of infectious urethritis and/or cystitis are presented. According to the results obtained, the top priority drugs for the treatment of infectious urethritis and/or cystitis are FO (Fosfomycin, 200 µg/disc), IMP (Imipenem, 10 mg/disc), and CIP (Ciprofloxacin, 30 µg/disc). It was found that out of 70 clinical isolates, only 36 of them formed biofilms using the plate method, which is equal to 51.4% of the total number of isolates studied. Despite polyresistance, clinical isolates of E. coli have moderate sensitivity to both the homogeneous enzyme and the culture fluid of the producer. The introduction of an enzyme or culture liquid at an early stage of strain cultivation significantly inhibits the formation of biofilms (91–100%). When introduced at later stages of the experiment—24 h and 48 h—inhibition is less pronounced—20–36% and 5–22%, respectively. [ABSTRACT FROM AUTHOR]

Uropathogenic Escherichia coli (UPEC) is a nosocomial pathogen associated with urinary tract infections and biofilm formation, which contributes to antibiotic resistance. Discovering potent antibacterial agents is crucial. This study aimed to assess the antibacterial and antibiofilm effects of gold and silver nanoparticles on UPEC using Scanning Electron Microscopy (SEM). UPEC biofilms were cultivated on nitrocellulose membranes for 48 hours at 37°C, then treated with gold nanoparticles (50 ppm and 100 ppm) and silver nanoparticles (50 ppm and 100 ppm) for another 48 hours. Antibacterial and antibiofilm activities were evaluated through cell density and SEM analysis. SEM revealed lower cell density, reduced biofilm formation, and altered cell morphology with rough, wrinkled surfaces after nanoparticle treatment. In conclusion, gold and silver nanoparticles exhibit antibacterial and antibiofilm properties, as observed in SEM analysis. SEM is a valuable tool for studying the antimicrobial effects of nano gold and silver on bacterial cell morphology and biofilm populations. [ABSTRACT FROM AUTHOR]

Background: The ability of Escherichia coli to build biofilms leads to the development of numerous diseases and makes their removal challenging. In addition to being the most common cause of urinary tract infections (UTIs), E. coli has been linked to disease in almost every area of the human body. Objective: The objective of the study was to evaluate the uropathogenic E. coli biofilm development using molecular and biochemical methods. Materials and Methods: Out of the total 117 urine samples obtained from UTI patients and diagnosed by selective media EMB (eosin methylene blue agar) and Vitek2 system, antibiotic sensitivity test, biofilm formation assay, and molecular detection of genes encoding for adhesion factors using polymerase chain reaction (PCR) technique were done. Results: Fifty E. coli isolates from both sexes of different ages were isolated; the UTI rate in females was 82% and in males was 18%. The result of the antibiotic sensitivity test, in terms of the percentage of resistance, was as follows: ampicillin, 50%; amoxicillin-clavulanate, 98%; ceftazidime, 72%; cefotaxime, 68%; aztreonam, 26%; gentamicin, 48%; levofloxacin, 36%; and trimethoprim, 52%, so that a high percentage of multidurg resistance resulted in the current study was 88%. The results of the quantification of biofilm formation revealed that all isolates produced biofilm with the following percentages: five (10%) as strong adherents, 36 (72%) as moderate biofilm producers, and nine (18%) were weak producers. The prevalence of genes fimH, csgA, and ag43 was 92%, 98%, and 92%, respectively, the result of detection of genes encoding for adhesion factors using PCR technique. Conclusions: The biofilm phenotype was indicated in all E. coli isolates and can confer virulence behavior and considered as a great challenging health problem and there is a significant association between adherent factor’s genes (fimH, csgA, ag43) and the ability to produce biofilm within E. coli isolates.

Urinary tract infection (UTI) poses a serious health issue for all age groups from neonates to geriatric age groups. Majority of the women experience urinary tract infections once in their lifetime. Escherichia coli (E.coli) is the most common uropathogen causing UTI which is followed by Klebsiella and Enterococci. Amoxicillin is the most resistant antibiotic against both E.coli and Klebsiella pneumonia whereas Gentamycin and Nitrofurantoin have been sensitive to both E.coli and Klebsiella pneumonia. Gram-negative bacteria such as E. coli, Klebsiella species, Proteus species, Acinetobacter species, Enterobacter species, Pseudomonas aeruginosa, and Citrobacter species are the most common causes of UTI. Enterococcus species, Staphylococcus saprophyticus, and Coagulase-negative bacteria are among Gram-positive bacteria. Staphylococcus aureus is a coagulase-positive gram-positive bacteria that cause urinary tract infections (UTIs). Hence, we come to the serious conclusion that Uropathogens are becoming increasingly resistant to a different group of antibiotics. The misuse and indiscriminate use of antimicrobials have led to the emergence of antibiotic resistance in bacteria all over the world, posing a threat to the public. Estimating the local etiology and sensitivity pattern could help in successful treatment. Inquiring into UTI epidemiology, such as risk factors, bacterial strains, and antimicrobial susceptibility, could help healthcare planners determine the best course of action.

With the global increase of infections caused by antibiotic‐resistant bacterial strains, there is an urgent need for new methods of tackling the issue. Genomic analysis of bacterial strains can help to understand their virulence and antibiotic resistance profile. Bioinformatic skills are in great demand across the biological sciences. We designed a workshop that allows university students to learn the process of genome assembly using command‐line tools within a virtual machine on a Linux operating system. We use Illumina and Nanopore short and long‐read raw sequences to reveal the advantages and disadvantages of short, long, and hybrid assembly methods. The workshop teaches how to assess read and assembly quality, perform genome annotation, and analyze pathogenicity, antibiotic and phage resistance. The workshop is intended for a five‐week teaching period and is concluded by a student poster presentation assessment. [ABSTRACT FROM AUTHOR]

Uropathogenic Escherichia coli is a major cause of urinary tract infections. Analysis of the innate immune response in immortalized urothelial cells suggests that the bacterial flagellar subunit, flagellin, is key in inducing host defenses. A panel of 48 clinical uro-associated E. coli isolates recovered from either cystitis, pyelonephritis asymptomatic bacteriuria (ABU) or UTI-associated bacteraemia infections were characterized for motility and their ability to induce an innate response in urothelial cells stably transfected with a NF-κB luciferase reporter. Thirty-two isolates (67%) were identified as motile with strains recovered from cystitis patients exhibiting an uneven motility distribution pattern; seven of the cystitis isolates were associated with a  > 5-fold increase in NF-κB signaling. To explore whether the NF-κB signaling response reflected antigenic variation, flagellin was purified from 14 different isolates. Purified flagellin filaments generated comparable NF-κ signaling responses, irrespective of either the source of the isolate or H-serotype. These data argued against any variability between isolates being related to flagellin itself. Investigations also argued that neither TLR4 dependent recognition of bacterial lipopolysaccharide nor growth fitness of the isolates played key roles in leading to the variable host response. To determine the roles, if any, of flagellar abundance in inducing these variable responses, flagellar hook numbers of a range of cystitis and ABU isolates were quantified. Images suggested that up to 60% of the isolate population exhibited flagella with the numbers averaging between 1 and 2 flagella per bacterial cell. These data suggest that selective pressures exist in the urinary tract that allow uro-associated E. coli strains to maintain motility, but exploit population heterogeneity, which together function to prevent host TLR5 recognition and bacterial killing. [ABSTRACT FROM AUTHOR]

Urinary tract infections (UTIs) are among the most common infections in humans and are often caused by uropathogenic E. coli (UPEC). Trimethylamine N-oxide (TMAO) is a proinflammatory metabolite that has been linked to vascular inflammation, atherosclerosis, and chronic kidney disease. As of today, no studies have investigated the effects of TMAO on infectious diseases like UTIs. The aim of this study was to investigate whether TMAO can aggravate bacterial colonization and the release of inflammatory mediators from bladder epithelial cells during a UPEC infection. We found that TMAO aggravated the release of several key cytokines (IL-1β and IL-6) and chemokines (IL-8, CXCL1 and CXCL6) from bladder epithelial cells during a CFT073 infection. We also found that CFT073 and TMAO mediate increased release of IL-8 from bladder epithelial cells via ERK 1/2 signaling and not bacterial growth. Furthermore, we showed that TMAO enhances UPEC colonization of bladder epithelial cells. The data suggest that TMAO may also play a role in infectious diseases. Our results can be the basis of further research to investigate the link between diet, gut microbiota, and urinary tract infection. [ABSTRACT FROM AUTHOR]

ABSTRACT Macrophages are innate immune cells that patrol tissues and are the first responders to detect infection. They orchestrate the host immune response in eliminating invading pathogens and the subsequent transition from inflammation to tissue repair. Macrophage dysfunction contributes to age-related pathologies, including low-grade inflammation in advanced age that is termed “inflammaging.” Our laboratory has previously identified that macrophage expression of a fatty acid desaturase, stearoyl-CoA desaturase 2 (SCD2), declines with age. Herein, we delineate the precise cellular effects of SCD2 deficiency in murine macrophages. We found that deletion of Scd2 from macrophages dysregulated basal and bacterial lipopolysaccharide (LPS)-stimulated transcription of numerous inflammation-associated genes. Specifically, deletion of Scd2 from macrophages decreased basal and LPS-induced expression of Il1b transcript that corresponded to decreased production of precursor IL1B protein and release of mature IL1B. Furthermore, we identified disruptions in autophagy and depletion of unsaturated cardiolipins in SCD2-deficient macrophages. To assess the functional relevance of SCD2 in the macrophage response to infection, we challenged SCD2-deficient macrophages with uropathogenic Escherichia coli and found that there was impaired clearance of intracellular bacteria. This increased burden of intracellular bacteria was accompanied by increased release of pro-inflammatory cytokines IL6 and TNF but decreased IL1B. Taken together, these results indicate that macrophage expression of Scd2 is necessary for maintaining the macrophage response to inflammatory stimuli. This link between fatty acid metabolism and fundamental macrophage effector functions may potentially be relevant to diverse age-related pathologies. IMPORTANCE Macrophages are immune cells that respond to infection, but their dysfunction is implicated in many age-related diseases. Recent evidence showed that macrophage expression of a fatty acid enzyme, stearoyl-CoA desaturase 2, declines in aged organisms. In this work, we characterize the effects when stearoyl-CoA desaturase 2 is deficient in macrophages. We identify aspects of the macrophage inflammatory response to infection that may be affected when expression of a key fatty acid enzyme is decreased, and these findings may provide cellular insight into how macrophages contribute to age-related diseases.