Homology of Escherichia coli isolated from urine and vagina and their antimicrobial susceptibility in postmenopausal women with recurrent cystitis.

Uropathogenic Escherichia coli (UPEC) is a typical cystitis-causing organism that can migrate from the vagina to the bladder and cause recurrent cystitis (RC). Few reports have compared the characteristics of urinary and vaginal UPEC in patients with RC. We carried out molecular biological analyses of Escherichia coli (E. coli) strains and their antimicrobial susceptibility to assess the association between urinary and vaginally UPEC. We included E. coli isolated from urinary and vaginal samples at the onset of cystitis in postmenopausal women with RC between 2014 and 2019 in our hospital. Pulsed-field gel electrophoresis (PFGE) was performed using a restriction enzyme (Xba I). These sequences were compared with 17 antimicrobial susceptibilities determined by a micro-liquid dilution method. Multilocus sequence typing (MLST) and classification of extended-spectrum ß-lactamase (ESBL) genotypes by multiplex polymerase chain reaction (PCR) were performed on ESBL-producing E. coli. We analyzed 14 specimens (each seven urine and vaginal) from seven patients in total. On PFGE, the similarity of urinary and vaginal E. coli per patient ranged from 89.5 to 100 %, including four patients with 100 % matches. MLST demonstrated that 29 % (4/14 specimens) were strain sequence type 131. Two specimens contained ESBL-producing strains and identified the CTX-M-27 genotype for each specimen. For each patient, antimicrobial susceptibilities between urinary and vaginal E. coli were mostly identical. Thus, urinary- and vaginally-derived E. coli were identical in postmenopausal women with RC. Management targeting both urinary and vaginal UPEC is essential for RC, indicating the importance of a vagina-targeted approach.

Identification of a copper-responsive small molecule inhibitor of uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are a major global health problem and are caused predominantly by uropathogenic Escherichia coli (UPEC). UTIs are a leading cause of prescription antimicrobial use. Incessant increase in antimicrobial resistance in UPEC and other uropathogens poses a serious threat to the current treatment practices. Copper is an effector of nutritional immunity that impedes the growth of pathogens during infection. We hypothesized that copper would augment the toxicity of select small molecules against bacterial pathogens. We conducted a small molecule screening campaign with a library of 51,098 molecules to detect hits that inhibit a UPEC ΔtolC mutant in a copper-dependent manner. A molecule, denoted as E. coli inhibitor or ECIN, was identified as a copper-responsive inhibitor of wild-type UPEC strains. Our gene expression and metal content analysis results demonstrate that ECIN works in concert with copper to exacerbate Cu toxicity in UPEC. ECIN has a broad spectrum of activity against pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. Subinhibitory levels of ECIN eliminate UPEC biofilm formation. Transcriptome analysis of UPEC treated with ECIN reveals induction of multiple stress response systems. Furthermore, we demonstrate that L-cysteine rescues the growth of UPEC exposed to ECIN. In summary, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC.IMPORTANCEUrinary tract infection (UTI) is a ubiquitous infectious condition affecting millions of people annually. Uropathogenic Escherichia coli (UPEC) is the predominant etiological agent of UTI. However, UTIs are becoming increasingly difficult to resolve with antimicrobials due to increased antimicrobial resistance in UPEC and other uropathogens. Here, we report the identification and characterization of a novel copper-responsive small molecule inhibitor of UPEC. In addition to E. coli, this small molecule also inhibits pathogens of medical and veterinary significance including Acinetobacter baumannii, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus.

Iron limitation induces motility in uropathogenic E. coli CFT073 partially through action of LpdA.

More than half of women will experience a urinary tract infection (UTI) with most cases caused by uropathogenic Escherichia coli (UPEC). Bacterial swimming motility enhances UPEC pathogenicity, resulting in more severe disease outcomes including kidney infection. Surprisingly, the connection between motility and iron limitation is mostly unexplored despite the lack of free iron available in the host. We sought to investigate a potential connection between iron restriction and regulation of motility in UPEC. We cultured E. coli CFT073, a prototypical UPEC strain, under iron limitation and observed that CFT073 had elevated fliC (flagella) promoter activity, and this iron-specific response was repressed by the addition of exogenous iron. We confirmed increased flagellar expression in CFT073 by measuring fliC transcript, FliC protein, and surface-expressed flagella under iron-limited conditions. Interestingly, known motility regulator flhDC did not have altered transcription under these conditions. To define the regulatory mechanism of this response, we constructed single knockouts of eight master regulators and found the iron-regulated response was lost in crp, arcA, and fis mutants. Thus, we focused on the five genes regulated by all three regulators. Of the five genes knocked out, the iron-regulated motility response was most strongly dysregulated in the lpdA mutant, which also resulted in significantly lowered fitness in the murine model of ascending UTI, both against the WT and a non-motile fliC mutant. Collectively, we demonstrated that iron-mediated motility in CFT073 is partially regulated by lpdA, which contributes to the understanding of how uropathogens differentially regulate motility mechanisms in the iron-restricted host. IMPORTANCE: Urinary tract infections (UTIs) are ubiquitous and responsible for over five billion dollars in associated health care costs annually. Both iron acquisition and motility are highly studied virulence factors associated with uropathogenic Escherichia coli (UPEC), the main causative agent of uncomplicated UTI. This work is innovative by providing mechanistic insight into the synergistic relationship between these two critical virulence properties. Here, we demonstrate that iron limitation has pleiotropic effects with consequences that extend beyond metabolism and impact other virulence mechanisms. Indeed, targeting iron acquisition as a therapy may lead to an undesirable enhancement of UPEC pathogenesis through increased motility. It is vital to understand the full breadth of UPEC pathogenesis to adequately respond to this common infection, especially with the increase of antibiotic-resistant pathogens.

Evaluation of adhesin genes and risk factors associated with urinary tract infections by drug-resistant uropathogenic Escherichia coli in North of Iran.

BACKGROUND: Uropathogenic Escherichia coli (UPEC) isolates, have a wide variety of virulence factors to promote colonization and survival in the urinary tract. This study aimed to evaluate adhesin genes, biofilm formation ability, antibiotic resistance profiles of UPEC strains, and the related risk factors in patients with UTIs caused by drug-resistant UPEC. METHODOLOGY: A total of 105 UPEC isolates were evaluated for biofilm formation using 96-well microtiter plates, the presence of adhesin genes by PCR assay and the antimicrobial susceptibility pattern using the disk diffusion method. Demographic and clinical characteristics of patients were investigated to identify predisposing factors for drug-resistant isolates. RESULTS: Out of 105 UPEC isolates, 84.8% were positive for biofilm formation. Biofilm-producing isolates exhibited a significantly higher prevalence of fimH, kpsMTII, csgA, afa/draBC, and pap adhesin genes compared to non-biofilm-producing strains (p < 0.05). The results also revealed that 52.4% of the isolates were ESBL-producing, and 84.8% were multidrug-resistant (MDR). Further analysis of antibiotic susceptibility among ESBL-producing strains showed the highest resistance rates to ampicillin, ciprofloxacin, and trimethoprim-sulfamethoxazole. Conversely, the highest susceptibility, in addition to carbapenems, was observed for fosfomycin, amikacin, cefoxitin, and nitrofurantoin. We identified hypertension as a potential risk factor for infection with ESBL-producing UPEC strains. CONCLUSIONS: Our results revealed a significant rate of drug resistance among UPEC isolates obtained from UTIs in our region. This underscores the importance of monitoring the empirical use of antibiotics and identifying specific risk factors in our geographical area to guide the selection of appropriate empirical treatment for UTIs.

A novel chaperone-effector-immunity system identified in uropathogenic Escherichia coli UMN026.

Background: Urinary tract infections (UTIs) are very common worldwide. According to their symptomatology, these infections are classified as pyelonephritis, cystitis, or asymptomatic bacteriuria (AB). Approximately 75-95% of UTIs are caused by uropathogenic Escherichia coli (UPEC), which is an extraintestinal bacterium that possesses virulence factors for bacterial adherence and invasion in the urinary tract. In addition, UPEC possesses type 6 secretion systems (T6SS) as virulence mechanisms that can participate in bacterial competition and in bacterial pathogenicity. UPEC UMN026 carries three genes, namely, ECUMN_0231, ECUMN_0232, and ECUMN_0233, which encode three uncharacterized proteins related to the T6SS that are conserved in strains from phylogroups B2 and D and have been proposed as biomarkers of UTIs. Aim: To analyze the frequency of the ECUMN_0231, ECUMN_0232, ECUMN_0233, and vgrG genes in UTI isolates, as well as their expression in Luria Bertani (LB) medium and urine; to determine whether these genes are related to UTI symptoms or bacterial competence and to identify functional domains on the putative proteins. Methods: The frequency of the ECUMN and vgrG genes in 99 clinical isolates from UPEC was determined by endpoint PCR. The relationship between gene presence and UTI symptomatology was determined using the chi2 test, with p < 0.05 considered to indicate statistical significance. The expression of the three ECUMN genes and vgrG was analyzed by RT-PCR. The antibacterial activity of strain UMN026 was determined by bacterial competence assays. The identification of functional domains and the docking were performed using bioinformatic tools. Results: The ECUMN genes are conserved in 33.3% of clinical isolates from patients with symptomatic and asymptomatic UTIs and have no relationship with UTI symptomatology. Of the ECUMN+ isolates, only five (15.15%, 5/33) had the three ECUMN and vgrG genes. These genes were expressed in LB broth and urine in UPEC UMN026 but not in all the clinical isolates. Strain UMN026 had antibacterial activity against UPEC clinical isolate 4014 (ECUMN-) and E. faecalis but not against isolate 4012 (ECUMN+). Bioinformatics analysis suggested that the ECUMN genes encode a chaperone/effector/immunity system. Conclusions: The ECUMN genes are conserved in clinical isolates from symptomatic and asymptomatic patients and are not related to UTI symptoms. However, these genes encode a putative chaperone/effector/immunity system that seems to be involved in the antibacterial activity of strain UMN026.

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.

Cranberry Polyphenols and Prevention against Urinary Tract Infections: New Findings Related to the Integrity and Functionality of Intestinal and Urinary Barriers.

This work seeks to generate new knowledge about the mechanisms underlying the protective effects of cranberry against urinary tract infections (UTI). Using Caco-2 cells grown in Transwell inserts as an intestinal barrier model, we found that a cranberry-derived digestive fluid (containing 135 ± 5 mg of phenolic compounds/L) increased transepithelial electrical resistance with respect to control (ΔTEER = 54.5 Ω cm2) and decreased FITC-dextran paracellular transport by about 30%, which was related to the upregulation of the gene expression of tight junction (TJ) proteins (i.e., occludin, zonula occludens-1 [ZO-1], and claudin-2) (∼3-4-fold change with respect to control for claudin-2 and ∼2-3-fold for occludin and ZO-1). Similar protective effects, albeit to a lesser extent, were observed when Caco-2 cells were previously infected with uropathogenic Escherichia coli (UPEC). In a urinary barrier model comprising T24 cells grown in Transwell inserts and either noninfected or UPEC-infected, treatments with the cranberry-derived phenolic metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and phenylacetic acid (PAA) (250 µM) also promoted favorable changes in barrier integrity and permeability. In this line, incubation of noninfected T24 cells with these metabolites induced positive regulatory effects on claudin-2 and ZO-1 expression (∼3.5- and ∼2-fold change with respect to control for DOPAC and ∼1.5- and >2-fold change with respect to control for PAA, respectively). Overall, these results suggest that the protective action of cranberry polyphenols against UTI might involve molecular mechanisms related to the integrity and functionality of the urothelium and intestinal epithelium.

Biofilm formation and antimicrobial resistance pattern of uropathogenic E. coli ST131 isolated from children with malignant tumors.

The multidrug-resistant clone identified as Escherichia coli sequence type 131 (E. coli ST131) has spread world-wide. This study sought to ascertain the frequency and biofilm formation of E. coli ST131 isolated from children with various malignancies. A total of 60 uropathogenic E. coli (UPEC) isolates from children without cancer and 30 UPEC isolates from children with cancer were assessed in this study. The microdilution method was used to investigate the sensitivity of bacteria to antibiotics. The microtiter plate (MTP) approach was used to phenotypically assess biofilm formation. The lasR, pelA, and lecA biofilm-encoding genes were detected by PCR in biofilm-producing isolates of E. coli. Thirty-seven out of 90 E. coli isolates were found to be ST131 (41.1%), with 17 (56.7%) from cancer-affected children and 20 (33.3%) from children without cancer, respectively (P-value = 0.036). The frequency of antimicrobial resistance was higher in ST131 strains were compared to non-ST131 strains and when they were isolated from healthy children vs. those who had cancer. In contrast to non-ST131 isolates, ST131 isolates were more biofilm-producers. There was a significant difference between the percentage of biofilm producers between the 22 (100%) ST131-O16 isolates and the 13 (86.7%) ST131-O25b isolates (P-value = 0.04). Children with cancer are more likely than children without cancer to develop biofilm forming E. coli ST131, the latter having a higher profile of antibiotic resistance. Interestingly, E. coli ST131 isolates from non-cancer patients had higher levels of overall antibiotic resistance and while more E. coli ST131isolates from cancer patients formed biofilms.

Secretory leukocyte protease inhibitor protects against severe urinary tract infection in mice.

Millions suffer from urinary tract infections (UTIs) worldwide every year with women accounting for the majority of cases. Uropathogenic Escherichia coli (UPEC) causes most of these primary infections and leads to 25% becoming recurrent or chronic. To repel invading pathogens, the urinary tract mounts a vigorous innate immune response that includes the secretion of antimicrobial peptides (AMPs), rapid recruitment of phagocytes, and exfoliation of superficial umbrella cells. Here, we investigate secretory leukocyte protease inhibitor (SLPI), an AMP with antiprotease, antimicrobial, and immunomodulatory functions, known to play protective roles at other mucosal sites, but not well characterized in UTIs. Using a preclinical model of UPEC-caused UTI, we show that urine SLPI increases in infected mice and that SLPI is localized to bladder epithelial cells. UPEC-infected SLPI-deficient (Slpi-/-) mice suffer from higher urine bacterial burdens, prolonged bladder inflammation, and elevated urine neutrophil elastase (NE) levels compared to wild-type (Slpi+/+) controls. Combined with bulk bladder RNA sequencing, our data indicate that Slpi-/- mice have a dysregulated immune and tissue repair response following UTI. We also measure SLPI in urine samples from a small group of female subjects 18-49 years old and find that SLPI tends to be higher in the presence of a uropathogen, except in patients with a history of recent or recurrent UTI, suggesting a dysregulation of SLPI expression in these women. Taken together, our findings show SLPI promotes clearance of UPEC in mice and provides preliminary evidence that SLPI is likewise regulated in response to uropathogen exposure in women.IMPORTANCEAnnually, millions of people suffer from urinary tract infections (UTIs) and more than $3 billion are spent on work absences and treatment of these patients. While the early response to UTI is known to be important in combating urinary pathogens, knowledge of host factors that help curb infection is still limited. Here, we use a preclinical model of UTI to study secretory leukocyte protease inhibitor (SLPI), an antimicrobial protein, to determine how it protects the bladder against infection. We find that SLPI is increased during UTI, accelerates the clearance of bacteriuria, and upregulates genes and pathways needed to fight an infection while preventing prolonged bladder inflammation. In a small clinical study, we show SLPI is readily detectable in human urine and is associated with the presence of a uropathogen in patients without a previous history of UTI, suggesting SLPI may play an important role in protecting from bacterial cystitis.

Genomic epidemiology and antibiotic susceptibility profiling of uropathogenic Escherichia coli among children in the United States.

Escherichia coli is the most common cause of urinary tract infections (UTIs) in children, and yet the underlying mechanisms of virulence and antibiotic resistance and the overall population structure of the species is poorly understood within this age group. To investigate whether uropathogenic E. coli (UPEC) from children who developed pyelonephritis carried specific genetic markers, we generated whole-genome sequence data for 96 isolates from children with UTIs. This included 57 isolates from children with either radiologically confirmed pyelonephritis or cystitis and 27 isolates belonging to the well-known multidrug-resistant sequence type ST131, selected to investigate their population structure and antibiotic resistance characteristics. We observed a UPEC population structure that is similar to those reported in adults. In comparison with prior investigations, we found that the full pap operon was more common among UPEC from pediatric cases of pyelonephritis. Further, in contrast with recent reports that the P-fimbriae adhesin-encoding papGII allele is substantially more prevalent in invasive UPEC from adults, we found papGII was common to both invasive and non-invasive UPEC from children. Among the set of ST131 isolates from children with UTIs, we found antibiotic resistance was correlated with known genetic markers of resistance, as in adults. Unexpectedly, we observed that fimH30, an allele of the fimbrial gene fimH often used as a proxy to type ST131 isolates into the most drug-resistant subclade C, was carried by some of the subclade A and subclade B isolates, suggesting that the fimH30 allele could confer a selective advantage for UPEC. IMPORTANCE Urinary tract infections (UTIs), which are most often caused by Escherichia coli, are not well studied in children. Here, we examine genetic characteristics that differentiate UTI-causing bacteria in children that either remain localized to the bladder or are involved in more serious kidney infections. We also examine patterns of antibiotic resistance among strains from children that are part of E. coli sequence type 131, a group of bacteria that commonly cause UTIs and are known to have high levels of drug resistance. This work provides new insight into the virulence and antibiotic resistance characteristics of the bacteria that cause UTIs in children.

Contribution of Antibiotic Susceptibility Testing and CH Typing Compared to Next-Generation Sequencing for the Diagnosis of Recurrent Urinary Tract Infections Due to Genetically Identical Escherichia coli Isolates: a Prospective Cohort Study of Cystitis in Women.

Recurrent cystitis is a common disease in women, mainly due to uropathogenic Escherichia coli (UPEC). For decades, typing methods now considered obsolete suggested that relapse by the same clone is dominant over reinfection, most UPEC strains being otherwise fully susceptible to antibiotics. We aimed to update these data. Thanks to a prospective study over 17 months, we recruited 323 women with cystitis. Of these, 251 of them had sporadic infection and 72 had recurrence, with 2 to 9 episodes per patient for a total of 131 UPEC isolates and 145 UPEC pairs at patient level. Phylogroups B2 (52.4%) and D (14.1%) were overall dominant, as expected due to their particular urovirulence. CH typing identified 119 distinct profiles with no CH type particularly associated with recurrence. Relapse was attested by CH typing for only 30.6% (22 out of 72), with very diverse situations ranging from all episodes due to the same clone to alternating reinfections and relapses. Next-generation sequencing confirmed the clonality for all but two of the 145 UPEC pairs. Antibiotic resistance was common for recurrent cystitis isolates (only 25 [17.2%] out of 145 UPEC pairs were fully susceptible), allowing us to predict UPEC clonality. Indeed, antibiotic susceptibility profile matched CH typing for 104 (71.7%) pairs. Finally, we demonstrated a large genetic diversity among UPEC isolates responsible for cystitis in women, even in cases of recurrence for which reinfection appeared dominant over relapse. Recurrent cystitis appears to be a heterogeneous disease requiring tailored treatment and prevention. IMPORTANCE More than half of women will experience cystitis during their lifetime. Among these women, 25% will experience a second episode within the following 6 months. It is epidemiologically important to discriminate relapses from reinfections. Relapse identification relies on long and laborious methods and might influence treatment. Therefore, the designation of time- and cost-effective strategies for this goal is of particular interest. Our work suggests using CH typing and antibiotic susceptibility profiles to type Escherichia coli, the main uropathogen.

Genotypic characteristics of Uropathogenic Escherichia coli isolated from complicated urinary tract infection (cUTI) and asymptomatic bacteriuria-a relational analysis.

Background: Uropathogenic Escherichia coli (UPEC) is the predominant agent causing various categories of complicated urinary tract infections (cUTI). Although existing data reveals that UPEC harboured numerous virulence determinants to aid its survival in the urinary tract, the reason behind the occurrence of differences in the clinical severity of uninary tract infections (UTI) demonstrated by the UPEC infection is poorly understood. Therefore, the present study aims to determine the distribution of virulence determinants and antimicrobial resistance among different phylogroups of UPEC isolated from various clinical categories of cUTI and asymptomatic bacteriuria (ASB) E. coli isolates. The study will also attempt a relational analysis of the genotypic characteristics of cUTI UPEC and ASB E. coli isolates. Methods: A total of 141 UPEC isolates from cUTI and 160 ASB E. coli isolates were obtained from Universiti Malaya Medical Centre (UMMC). Phylogrouping and the occurrence of virulence genes were investigated using polymerase chain reaction (PCR). Antimicrobial susceptibility of the isolates to different classes of antibiotics was determined using the Kirby Bauer Disc Diffusion method. Results: The cUTI isolates were distributed differentially among both Extraintestinal Pathogenic E. coli (ExPEC) and non-ExPEC phylogroups. Phylogroup B2 isolates were observed to possess the highest average aggregative virulence score (7.17), a probable representation of the capability to cause severe disease. Approximately 50% of the cUTI isolates tested in this study were multidrug resistant against common antibiotics used to treat UTI. Analysis of the occurrence of virulence genes among different cUTI categories demonstrated that UPEC isolates of pyelonephritis and urosepsis were highly virulent and had the highest average aggregative virulence scores of 7.80 and 6.89 respectively, compared to other clinical categories. Relational analysis of the occurrence of phylogroups and virulence determinants of UPEC and ASB E. coli isolates showed that 46.1% of UPEC and 34.3% of ASB E. coli from both categories were distributed in phylogroup B2 and had the highest average aggregative virulence score of 7.17 and 5.37, respectively. The data suggest that UPEC isolates which carry virulence genes from all four virulence genes groups studied (adhesions, iron uptake systems, toxins and capsule synthesis) and isolates from phylogroup B2 specifically could predispose to severe UTI involving the upper urinary tract. Therefore, specific analysis of the genotypic characteristics of UPEC could be further explored by incorporating the combination of virulence genes as a prognostic marker for predicting disease severity, in an attempt to propose a more evidence driven treatment decision-making for all UTI patients. This will go a long way in enhancing favourable therapeutic outcomes and reducing the antimicrobial resistance burden among UTI patients.

Uropathogenic Escherichia coli infection-induced epithelial trained immunity impacts urinary tract disease outcome.

Previous urinary tract infections (UTIs) can predispose one to future infections; however, the underlying mechanisms affecting recurrence are poorly understood. We previously found that UTIs in mice cause differential bladder epithelial (urothelial) remodelling, depending on disease outcome, that impacts susceptibility to recurrent UTI. Here we compared urothelial stem cell (USC) lines isolated from mice with a history of either resolved or chronic uropathogenic Escherichia coli (UPEC) infection, elucidating evidence of molecular imprinting that involved epigenetic changes, including differences in chromatin accessibility, DNA methylation and histone modification. Epigenetic marks in USCs from chronically infected mice enhanced caspase-1-mediated cell death upon UPEC infection, promoting bacterial clearance. Increased Ptgs2os2 expression also occurred, potentially contributing to sustained cyclooxygenase-2 expression, bladder inflammation and mucosal wounding-responses associated with severe recurrent cystitis. Thus, UPEC infection acts as an epi-mutagen reprogramming the urothelial epigenome, leading to urothelial-intrinsic remodelling and training of the innate response to subsequent infection.

Testicular exosomes disturb the immunosuppressive phenotype of testicular macrophages mediated by miR-155-5p in uropathogenic Escherichia coli-induced orchitis.

Male reproductive infections are known to shape the immunological homeostasis of the testes, leading to male infertility. However, the specific pathogenesis of these changes remains poorly understood. Exosomes released in the inflammatory microenvironment are important in communication between the local microenvironment and recipient cells. Here, we aim to identify the immunomodulatory properties of inflammatory testes-derived exosomes (IT-exos) and explore their underlying mechanisms in orchitis. IT-exos were isolated using a uropathogenic Escherichia coli (UPEC)-induced orchitis model and confirmed that IT-exos promoted proinflammatory M1 activation with increasing expression of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in vitro. We further used small RNA sequencing to identify the differential miRNA profiles in exosomes and primary testicular macrophages (TMs) from normal and UPEC-infected testes, respectively, and identified that miR-155-5p was highly enriched in IT-exos and TMs from inflammatory testes. Further study of bone marrow derived macrophages (BMDMs) transfected with miR-155-5p mimic showed that macrophages polarized to proinflammatory phenotype. In addition, the mice that were administrated IT-exos showed remarkable activation of TM1-like macrophages; however, IT-exos with silencing miR-155-5p showed a decrease in proinflammatory responses. Overall, we demonstrate that miR-155-5p delivered by IT-exos plays an important role in the activation of TM1 in UPEC-induced orchitis. Our study provides a new perspective on the immunological mechanisms underlying inflammation-related male infertility.

Identification of Virulence Markers and Phylogenetic Groups' Association, and Antimicrobial Susceptibility of Uropathogenic Escherichia coli Isolates.

BACKGROUND: Urinary tract infections represent a world public health problem, which is caused mainly by Uropathogenic Escherichia coli. Although they are originally found in the intestinal microbiota in the majority of the cases, urinary tract infections can also be caused by intra-intestinal pathogenic E. coli. OBJECTIVE: The main objective of our research is to identify the virulence factors generally associated with different pathotypes across phylogenetic groups. METHODS: E. coli were isolated from patients with urinary tract infections. Antimicrobial susceptibility tests, virulence genes and phylogroups were prospected. The data analysis were performed using the chi-square and Fisher exact test. RESULTS: In total, 72.2% of isolates showed multidrug resistant. We have also depicted an important association between E. coli from inpatients with UTIs and pap and hlyA genes (p-0.041 and p-0.019 respectively). The predominant phylogenetic group in our isolates is B2 (45.4%) followed by D (12.4%). Our results showed that 9.3% of isolates have an unknown phylogroup which shows a significant association with astA gene (p-0.008). We have as well found a significant association between B2 and three virulence genes namely pap, hlyA and invE (p-0.002, p-0.001, p-0.025 respectively); B1 and pap, hlyA genes (p-0.049 and p-0.021 respectively); E and afa gene (p-0.024). CONCLUSION: Certain virulence factors have been shown to be potential targets for drug design and therapeutic pathways in order to deal with the antimicrobial resistance problem enhanced by antibiotic therapy.

Intracellular uropathogenic Escherichia coli are undetectable in urinary bladders after oral mecillinam treatment: An experimental study in a pig model of cystitis.

OBJECTIVES: Experiments in murine models of urinary tract infection (UTI) show that uropathogenic Escherichia coli (UPEC) form bacterial reservoirs in the bladder tissue that can survive beta lactam antibiotics and give rise to reinfection. The observed reinfection cascade suggests intracellular bacterial persistence as a possible explanation for recurrent UTI in humans. To test this hypothesis in an animal model closer to humans, we here investigated whether UPEC infecting the bladders of experimentally inoculated pigs are able to survive standard oral mecillinam treatment. Moreover, we analyzed the infected pig bladders by microscopy for the presence of intracellular UPEC colonies. METHODS: Seven pigs were experimentally inoculated with the UPEC cystitis strain, UTI89, to induce cystitis. After 5 days of infections, a 3-day oral treatment with the extracellularly active ß-lactam, mecillinam, was initiated. The infection was monitored with regular urine and blood samples. When terminated, whole bladders were removed and homogenized to quantify viable intracellular bacteria. In addition, two pigs were inoculated with UTI89pMAN01 constitutively expressing green fluorescent protein and the bladders subsequently analyzed by microscopy for bacterial location and morphology. RESULTS: Experimental inoculation resulted in cystitis in all animals. After 3-day treatment with mecillinam, no viable UPEC were detectable in urine or bladder homogenates. Microscopy analysis of pig bladders at 12 h post infection, revealed no detectable intracellular bacterial colonies and no filamentous UPEC phenotypes were identified. CONCLUSIONS: Pigs experimentally infected with UPEC completely clear their infection upon mecillinam treatment, which contrasts earlier findings from similar experiments in mice. Moreover, the hallmarks of induced UTI in mice, i.e. intracellular bacterial communities and bacterial filamentation, could not be identically reproduced in a pig model of acute UTI. This result suggests that significant differences might exist between UTI in mice and larger mammals, and therefore perhaps also between mice and humans. Additional studies are needed to reveal details on the Escherichia coli acute UTI pathogenesis cascade in larger mammals to assess to which extent observations in mice can be transferred to humans.

High mannose level in bladder cancer enhances type 1 fimbria-mediated attachment of uropathogenic E. coli.

Bladder cancer is the 4th leading cancer in men. Tumor resection followed by bladder instillation of Bacillus Calmette-Guérin (BCG) is the primary treatment for high-risk patients with Non-Muscle Invasive Bladder Cancer (NMIBC) to prevent recurrence and progression to muscle-invasive disease. This treatment, however, lacks efficiency and causes severe adverse effects. Mannose residues are expressed on bladder surfaces and their levels were indicated to be higher in bladder cancer. Intravesical instillations of a recombinant Pseudomonas aeruginosa (PA) overexpressing the mannose-sensitive hemagglutination fimbriae (PA-MSHA), and of a mannose-specific lectin-drug conjugate showed efficiency against NMIBC in murine models of bladder cancer. Urothelial mannosylation facilitates bladder colonization by Uropathogenic E. coli (UPEC) via the interaction with the FimH mannose lectin, positioned at the tip of type 1 fimbria. A recombinant BCG strain overexpressing FimH on its outer surface, exhibited higher attachment and internalization to bladder cancer cells and increased effectivity in treating bladder cancer in mice. Investigating the pattern of mannose expression in NMIBC is important for improving treatment. Here, using tissue microarrays containing multiple normal and cancerous bladder samples, and lectins, we confirm that human bladder cancer cells express high mannose levels. Using UPEC mutants lacking or overexpressing type 1 fimbria, we also demonstrate that tumor-induced hypermannosylation increases type 1 fimbria mediated UPEC attachment to human and mouse bladder cancer. Our results provide an explanation for the effectiveness of PA-MSHA and the FimH-overexpressing BCG and support the hypothesis that mannose-targeted therapy holds potential for improving bladder cancer treatment.

Genome-wide analysis of fitness factors in uropathogenic Escherichia coli in a pig urinary tract infection model.

Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs) in animals and humans. We applied Transposon-Directed Insertion Site sequencing (TraDIS) to determine the fitness genes in two well-characterized UPEC strains, UTI89 and CFT073, in order to identify fitness factors during UTI in a pig model. This novel animal model better reflects the course of UTI in humans than the commonly used mouse model, and facilitates the differentiation between sessile and planktonic UPEC populations. A total of 854 and 483 genes in UTI89 and CFT073, respectively, were predicted to contribute to growth in pig urine, and 1257 and 764, were scored as required for colonization of the bladder. The combined list of fitness genes for growth in urine and cystitis contained 741 (UTI89) and 439 (CFT073) genes. The essential genes for growth on LB agar media supplemented with kanamycin and the fitness factors during growth in human urine were also analyzed in CFT073. A total of 457 essential genes were identified and the pool of fitness genes for growth in human urine included 215 genes. The gene rfaG, which is involved in lipopolysaccharide biosynthesis, was included in all the fitness-gene-lists and was further confirmed to be relevant for all the conditions tested regardless of the host and the strain. Thus, this gene may represent a promising target for the development of new therapeutic strategies against UTI UPEC-associated. Besides this important observation, the study revealed strain-specific differences in gene-essentiality as well as in the fitness-gene-repertoire for growth in human urine and UTI of the pig model, and it identified novel factors required for UPEC-induced UTIs.

TcpC Inhibits M1 but Promotes M2 Macrophage Polarization via Regulation of the MAPK/NF-κB and Akt/STAT6 Pathways in Urinary Tract Infection.

TcpC is a multifunctional virulence factor of Uropathogenic Escherichia coli (UPEC). Macrophages can differentiate into two different subsets M1 and M2 that play distinct roles in anti-infection immunity. Here, we investigate the influence of TcpC on M1/M2 polarization and the potential mechanisms. Our data showed that M1 markers CD86 and iNOS were significantly inhibited, while the M2 markers CD163, CD206 and Arg-1 were enhanced in macrophages in kidneys from the TcpC-secreting wild-type CFT073 (CFT073wt)-infected pyelonephritis mouse model, compared with those in macrophages in kidneys from TcpC knockout CFT073 mutant (CFT073Δtcpc)-infected mice. CFT073wt or recombinant TcpC (rTcpC) treatment inhibits LPS + IFN-γ-induced CD80, CD86, TNF-α and iNOS expression, but promotes IL-4-induced CD163, CD206, Arg-1 and IL-10 expression in both human and mouse macrophage cell lines THP-1 and J774A.1. Moreover, rTcpC significantly attenuated LPS + IFN-γ-induced phosphorylation of p38, ERK, p50 and p65 but enhanced IL-4-induced phosphorylation of Akt and STAT6. These data suggest that TcpC inhibits M1 but promotes M2 macrophage polarization by down-regulation of p38, ERK/NF-κB and up-regulation of the Akt/STAT6 signaling pathway, respectively. Our findings not only illuminate the regulatory effects of TcpC on macrophage M1/M2 polarization and its related signaling pathways, but also provide a novel mechanism underlying TcpC-mediated immune evasion of macrophage-mediated innate immunity.

Distribution of papG alleles among uropathogenic Escherichia coli from reproductive age women.

BACKGROUND: Extraintestinal Escherichia coli (E. coli) causing urinary tract infections (UTIs), and often referred to as uropathogenic E. coli (UPEC), are a major contributor to the morbidity of UTIs and associated healthcare costs. UPEC possess several virulence factors (VFs) for infecting and injuring the host. We studied the papG allele distribution, and its association with other VF genes and phylogenetic groups, amongst 836 UPEC and fecal isolates from reproductive age women. RESULTS: The papGII gene was highly prevalent amongst pyelonephritis isolates (68%), whilst the majority, albeit smaller proportion, of cystitis isolates (31%) harboured the papGIII gene. Among the pyelonephritis and cystitis isolates, papG positive isolates on average had higher VF gene scores, and were more likely to belong to phylogenetic group B2, than their negative counterparts. This was mostly due to the contribution of papGII isolates, which on average contained more VF genes than their papGIII counterparts, irrespective of the uro-clinical syndrome. However, the papGII isolates from the pyelonephritis cohort had higher VF gene scores than the cystitis ones, suggesting presence of possible papGII clones with differing inferred virulence potential. Furthermore, papGII isolates were more likely to possess an intact pap gene operon than their papGIII counterparts. Also of note was the high proportion of isolates with the papGI allele which was not associated with other pap operon genes; and this finding has not been described before. CONCLUSIONS: The association of the papGII gene with several VF genes compared to the papGIII gene, appears to explain the abundance of these genes in pyelonephritis and cystitis isolates, respectively.