Escherichia coli isolated from pyometra and cystitis in the same animal exhibit a wide phenotypic similarity.

AIMS: Pyometra and cystitis caused by Escherichia coli are common diseases identified in canine or feline females. The origin of pyometra infection remains uncertain, and effective prevention strategies for this disease are still unknown. This study aimed to provide a phenotypic characterization, including antimicrobial resistance and virulence profiles, of endometrial pathogenic (EnPEC) and uropathogenic (UPEC) E. coli strains isolated simultaneously from the same animal. METHODS AND RESULTS: Sixteen E. coli strains, from eight different animals, were analyzed in this study. The antimicrobial susceptibility profile of EnPEC and UPEC strains was determined using the disc diffusion method, which showed a similar susceptibility profile among strains (EnPEC and UPEC) from the same animal. The virulence profile of the strains was assessed through biofilm formation, as well as serum resistance abilities. EnPEC and UPEC strains from the same animal exhibited slight variations in their virulence and antimicrobial resistance capabilities. Overall, most of the strain pairs showed a high similarity in their ability to establish biofilms and survive in serum complement activity. CONCLUSIONS: Overall, strains of E. coli isolated from both pyometra and cystitis in the same animal, despite presenting distinct clinical diseases, exhibit a wide phenotypic similarity, suggesting a common origin for the strains.

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.

Uropathogenic E. coli induces DNA damage in the bladder.

Urinary tract infections (UTIs) are among the most common outpatient infections, with a lifetime incidence of around 60% in women. We analysed urine samples from 223 patients with community-acquired UTIs and report the presence of the cleavage product released during the synthesis of colibactin, a bacterial genotoxin, in 55 of the samples examined. Uropathogenic Escherichia coli strains isolated from these patients, as well as the archetypal E. coli strain UTI89, were found to produce colibactin. In a murine model of UTI, the machinery producing colibactin was expressed during the early hours of the infection, when intracellular bacterial communities form. We observed extensive DNA damage both in umbrella and bladder progenitor cells. To the best of our knowledge this is the first report of colibactin production in UTIs in humans and its genotoxicity in bladder cells.

Non-lactose-fermenting uropathogenic Escherichia coli from dogs: virulence profile characterization.

Non-lactose-fermenting Escherichia coli (NLFEC) has a few descriptive studies restricted to human infections. In the present study, isolates of NLFEC obtained from urine samples of dogs with hyperadrenocorticism were characterized regarding their virulence ability, biofilm formation capacity and antimicrobial susceptibility profile. Escherichia coli lactose-fermenting strains from urinary infection in dogs with the same conditions were analysed to provide comparisons. The non-lactose-fermenting E. coli strains were classified as belonging to clade I E. coli, whereas the lactose-fermenting strains were classified in phylogroup B2. All strains presented virulence markers to adhesion, iron acquisition, toxins, colicin and cytotoxin production, and biofilm regulation. Components of the extracellular matrix in addition to the in vitro biofilm formation ability were observed in the strains. Multidrug resistance (MDR) profiles were observed by in vitro susceptibility tests to all NLFEC strains. In summary, non-lactose-fermenting uropathogenic E. coli from dogs behaves similar to lactose-fermenting E. coli, exhibiting MDR profile, and pathogenic potential of promote animal infections.

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

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

Distribution of virulence genes and phylogenetics of uropathogenic Escherichia coli among urinary tract infection patients in Addis Ababa, Ethiopia.

BACKGROUND: Urinary tract infection (UTI) is a common cause of morbidity worldwide. Uropathogenic Escherichia coli (UPEC) bacteria are the major cause of urinary tract infections. UPEC strains derive from different phylogenetic groups and possess an arsenal of virulence factors that contribute to their ability to overcome different defense mechanisms and cause disease. The objective of this study was to identify phylogroup and virulence genes of UPEC among urinary tract infection patients. METHODS: A cross sectional study was conducted from January 1, 2017 to October 9, 2017. E. coli bacteria were isolated from UTI patients using culture and conventional biochemical tests. Identification of phylogroup and genes that encodes for virulence factors was done using multiplex polymerase chain reaction (PCR). Data was processed and analyzed with SPSS version16.0 and Epi-info version 3.4.1 software. RESULTS: The most common urologic clinical manifestation combinations in this study were dysuria, urine urgency and urgency incontinence. The frequent UPEC virulence gene identified was fimH 164 (82%), followed by aer 109 (54.5%), hly 103 (51.5%), pap 59 (29.5%), cnf 58 (29%), sfa 50 (25%) and afa 24 (12%).There was significant association between pap gene and urine urgency (p-0.016); sfa and dysuria and urine urgency (p-0.019 and p-0.043 respectively); hly and suprapubic pain (p-0.002); aer and suprapubic pain, flank pain and fever (p-0.017, p-0.040, p-0.029 respectively). Majority of E. coli isolates were phylogroup B2 60(30%) followed by D 55(27.5%), B1 48(24%) and A 37(18.5%). There was significant association between E. coli phylogroup B2 and three virulence genes namely afa, pap, and sfa (p-0.014, p-0.002, p-0.004 respectively). CONCLUSION: In this study the most frequent E. coli virulence gene was fimH, followed by aer, hly, pap, cnf, sfa and afa respectively. There was significant association between E. coli virulence genes and clinical symptoms of UTI. The phylogenetic analysis indicates majority of uropathogenic E. coli isolates were phylogroup B2 followed by phylogroup D. Phylogroup B2 carries more virulence genes. Hence, targeting major UPEC phylogroup and virulence genes for potential vaccine candidates is essential for better management of UTI and further research has to be conducted in this area.

Molecular Epidemiology of Antimicrobial Resistance of Uropathogenic Escherichia coli Isolates from Patients with Urinary Tract Infections in a Tertiary Teaching Hospital in Iran.

To characterize the resistance patterns of uropathogenic Escherichia coli (UPEC) in a Tertiary Teaching Hospital in Iran, we conducted a descriptive epidemiology study using molecular techniques. The subjects consisted of patients having acute urinary tract infection, who were enrolled in the study from 2014 to 2017. The antimicrobial susceptibility profile of 101 UPEC isolates was determined by Kirby-Bauer disc diffusion method. Extended spectrum ß-lactamase (ESBL) was detected by the double-disk synergy test. Biofilm formation was done using microtiter plates. The presence of virulence genes (pai, pap, hly, traT, pai, cnf-1, sfa, and afa) was evaluated by a PCR. Molecular typing of UPEC E. coli isolates was performed with fimH and multilocus sequence typing (MLST). 70.3% of isolates were multidrug-resistant. 37.6% of isolates were Extended spectrum ß-lactamases (ESBLs) producer. Strong biofilm formation was seen in 27.7%. Forty-seven different fimH allelic variants were identified. Among identified fimH allelic variants, the most common types were f1 (18.8%) and f14 (18.8%). ST131 (54.5%) was the most prevalent clonal group significantly correlated with the pai gene. Seven sequence types (STs) were detected only once (ST405, ST410, ST450, ST636, ST648, ST1193, and ST6451). Clonal groups showed no significant differences in terms of antibiotic resistance patterns. There was no significant difference between virulence genes and antibiotic resistance patterns in the studied clonal groups. To our knowledge, the present study is the first study in Iran that investigated the genotypic diversity of UPEC isolates by MLST and fimH typing methods. The two methods might serve as a useful molecular test for surveillance and epidemiological studies of isolates.

Clonal Relatedness, Phylotyping and Antimicrobial Susceptibility of Extended- spectrum-beta-lactamase Producing Uropathogenic Escherichia coli Isolates from Outpatients and Inpatients.

OBJECTIVES: Antibiotic resistance, phylogenetic groups and Pulsed-Field Gel Electrophoresis (PFGE) patterns were evaluated in urinary tract infection (UTI) Escherichia coli (E. coli) isolates from outpatients and inpatients. METHODS: In this study, antibiotic resistance to E. coli isolated from non-hospitalized and hospitalized patients (153 outpatients and 147 inpatients ) was evaluated in Shiraz County, Iran. Phylogenetic groups and Pulse Field Gel Electrophoresis (PFGE) patterns of 143 ESBLs-producing E. coli were also assessed. RESULTS: The prevalence of ESBL-producing E. coli was shown to be 46.4% and 49% in the outpatient and inpatient UTI E. coli isolates, respectively. Most ESBL-producers were detected on patients hospitalized in clinical surgery units (66.7%) and intensive care units (62.5%). Phylogenetic group D was the dominant group in both the outpatient and inpatient isolates (67.6% and 61.1%, respectively) and also in internal, clinical surgery and ICU units. PFGE results showed more relatedness (>80% similarity) among inpatient isolates. PFGE analysis of 49 ESBL-producing inpatient E.coli in hospital units revealed 17 different pulsotypes, consisting of 11 clones and 6 single patterns. There were no clonal patterns in outpatient isolates, and similarity among the outpatient isolates and also between inpatient and outpatient isolates was less than 80% (75% and 66%, respectively). CONCLUSION: The results showed extreme genomic diversity among the ESBL-producing E. coli isolates in terms of the community and multiclonal dissemination of ESBL-producing E. coli isolated from hospital units.

Antibiotic Resistance Among Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) belong to the most common community-acquired and nosocomial infections. A main etiological factor of UTIs is uropathogenic Escherichia coli (UPEC). This review describes the current state of knowledge on the resistance of UPEC to antibiotics recommended for the treatment of UTIs based on the available literature data. Nitrofurantoin and fosfomycin are recommended as first-line therapy in the treatment of uncomplicated cystitis, and the resistance to these antimicrobial agents remains low between UPEC. Recently, in many countries, the increasing resistance is observed to trimethoprim-sulfamethoxazole, which is widely used as the first-line antimicrobial in the treatment of uncomplicated UTIs. In European countries, the resistance of UPEC to this antimicrobial agent ranges from 14.6% to 60%. The widespread use of fluoroquinolones (FQs), especially ciprofloxacin, in the outpatients is the cause of a continuous increase in resistance to these drugs. The resistance of UPEC to FQs is significantly higher in developing countries (55.5-85.5%) than in developed countries (5.1-32.0%). Amoxicillin-clavulanic acid is recommended as first line-therapy for pyelonephritis or complicated UTI. Resistance rates of UPEC to amoxicillin-clavulanic acid are regionally variable. In European countries the level of resistance to this antimicrobial ranges from 5.3% (Germany) to 37.6% (France). Increasing rates of UPEC resistance to antimicrobials indicate that careful monitoring of their use for UTI treatment is necessary.Urinary tract infections (UTIs) belong to the most common community-acquired and nosocomial infections. A main etiological factor of UTIs is uropathogenic Escherichia coli (UPEC). This review describes the current state of knowledge on the resistance of UPEC to antibiotics recommended for the treatment of UTIs based on the available literature data. Nitrofurantoin and fosfomycin are recommended as first-line therapy in the treatment of uncomplicated cystitis, and the resistance to these antimicrobial agents remains low between UPEC. Recently, in many countries, the increasing resistance is observed to trimethoprim-sulfamethoxazole, which is widely used as the first-line antimicrobial in the treatment of uncomplicated UTIs. In European countries, the resistance of UPEC to this antimicrobial agent ranges from 14.6% to 60%. The widespread use of fluoroquinolones (FQs), especially ciprofloxacin, in the outpatients is the cause of a continuous increase in resistance to these drugs. The resistance of UPEC to FQs is significantly higher in developing countries (55.5­85.5%) than in developed countries (5.1­32.0%). Amoxicillin-clavulanic acid is recommended as first line-therapy for pyelonephritis or complicated UTI. Resistance rates of UPEC to amoxicillin-clavulanic acid are regionally variable. In European countries the level of resistance to this antimicrobial ranges from 5.3% (Germany) to 37.6% (France). Increasing rates of UPEC resistance to antimicrobials indicate that careful monitoring of their use for UTI treatment is necessary.

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

Snapshot of Phylogenetic Groups, Virulence, and Resistance Markers in Escherichia coli Uropathogenic Strains Isolated from Outpatients with Urinary Tract Infections in Bucharest, Romania.

BACKGROUND: Urinary tract infections (UTIs) caused by Uropathogenic Escherichia coli (UPEC) are among the most common infections worldwide, including Romania. To the best of our knowledge, this is the first study performed on a significant number of community-acquired (CA) UPEC strains isolated from Romanian outpatients, aiming to evaluate and establish potential correlations among the phylogenetic groups (PG), resistance profiles, and the virulence factors (VF) genes of the CA-UPEC isolates. MATERIALS/METHODS: The present study was performed on a total of 787 UPEC nonrepetitive isolates consecutively isolated during one month from outpatients with CA-UTIs, visiting one of the biggest laboratories in Bucharest, Romania, receiving patients from all over the country. The strains identification was performed by MALDI TOF and the susceptibility patterns were tested using Microscan according to CLSI guidelines. PCR assays were performed to detect the presence of different VFs (fimH gene encoding for type 1 fimbriae, afaBC for A fimbriae, sfaDE for S fimbriae, KpsMTII for capsule, hlyA for haemolysin A, hlyD for haemolysin D, and cnf-1 for tumor necrosis factor), the phylogenetic groups (PG) A, B1, B2, and D, and the extended spectrum beta-lactamases (ESBLs) genes. RESULTS: The 787 CA-UPEC strains were isolated predominantly from female patients (90.95%) of >30 years (~74%). The resistance rates were 47.52% for ampicillin, 41.16% for tetracycline, 24.39% for cotrimoxazole, 19.18% for amoxicillin-clavulanic acid, 15.50% for cefazolin, 14.99% for ciprofloxacin, and 14.86% for levofloxacin; 35.19% of the investigated strains were MDR and 9.03% ESBL producers (from which 42.25% were positive for blaCTX-M, 38.02% for blaTEM, and 19.71% for blaSHV). FimH was the most frequent virulence gene (93.90%) followed by hlyD (44.34%); afaBC (38.24%); KpsMTII (32.65%); sfaDE (23.88%); hlyA (12.45%); and cnf-1 (7.75%). The distribution of the analyzed UPEC strains in phylogenetic groups was different for non-MDR and MDR strains. Overall, 35% of the strains belonged to the phylogenetic group B2 (harboring the yjaA gene); 27% to group B1 (confirmed by the presence of the TspE4C2 fragment); 16% to group D; and 22% to group A. The CA-UPEC strains included in PG B1 and PG B2 proved to be the most virulent ones, the number of strains carrying multiple VFs (>3) being significantly larger as compared to strains belonging to PG A and PG D) (p<0,0001). The presence of one or two ESBL genes was significantly associated (p =0.0024) with PGs A and D. CONCLUSIONS: Our findings showed that the community UPEC strains circulating in Bucharest, Romania, belong predominantly to group B2 and >90% harbored the fimH gene. High MDR resistance rates were observed, as well as extended VF profiles, highlighting the importance of this type of studies for improving the epidemiological surveillance and the therapeutic or prophylactic management of the respective infections, in the context of antibiotic resistance emergence.

Fosfomycin tromethamine activity on biofilm and intracellular bacterial communities produced by uropathogenic Escherichia coli isolated from patients with urinary tract infection.

Fosfomycin tromethamine (FT), an old antibiotic revived as a new strategy to overcome antibiotic resistance, is an excellent option for the treatment of lower urinary tract infection (UTI). During UTI, Escherichia coli produces biofilms and could invade the bladder epithelial cells, developing intracellular bacterial communities (IBC). The present work aimed to evaluate the activity of FT on biofilms and IBC from clinical isolates of E. coli. A total of 38 E. coli clinical UTI isolates previously characterized as biofilm and IBC producers were studied. FT susceptibility was evaluated and its activity on 48 h biofilm was determined by microtiter plate-based biofilm assay comparing three different antibiotic concentrations. Two UPEC strains were selected to evaluate FT activity on IBC in vitro using T24 bladder cells. The survival percentage of intracellular bacteria after 24 h exposure to FT was calculated and compared to the percentage of intracellular bacteria without antibiotic. All the strains were susceptible to FT. FT produced a significant reduction of biofilms at the three concentrations tested, compared to the control. However, no statistically effect on IBC was observed after 24 h of fosfomycin exposure in cell culture. FT is a good option for bacterial biofilm reduction within UTI. However, it does not affect IBC.

Identification of novel bacteriophage vB_EcoP-EG1 with lytic activity against planktonic and biofilm forms of uropathogenic Escherichia coli.

Urinary tract infections are one of the most common infectious diseases worldwide. Uropathogenic Escherichia coli (UPEC) is a major cause of unary tract infection. Due to increasing prevalence of multidrug resistance, alternative methods to eradicate the UPECs are urgently needed. In this respect, phage therapy has been demonstrated to be a good candidate. Here, we described a novel bacteriophage named vB_EcoP-EG1, which can infect several strains of UPEC. Phage morphology and genome sequencing analysis show that vB_EcoP-EG1 belongs to the T7-like Podoviridae. vB_EcoP-EG1 possesses a genome (39,919 bp) containing 51 predicted genes and 149 bp terminal repeats. vB_EcoP-EG1 genome does not encode toxic proteins or proteins related to lysogeny. And no known virulent proteins were found in purified phage particles by mass spectrometry. vB_EcoP-EG1 appeared to be relatively specific and sensitive to clinical UPEC strains, which could infect 10 out of 21 clinical multidrug-resistant UPEC strains. In addition, vB_EcoP-EG1 suspension can eliminate biofilm formed by E. coli MG1655 and multidrug-resistant UPEC strain 390G7. Therefore, we concluded that vB_EcoP-EG1 has desirable characteristics for potential therapy, which may serve as an alternative to antibiotic therapy against urinary tract infections caused by multidrug-resistant UPEC.

Characterization of uropathogenic ESBL-producing Escherichia coli isolated from hospitalized patients in western Algeria.

INTRODUCTION: The aim of this study is to assess the prevalence and molecular characterization of uropathogenic Extended spectrum ß-lactamases (ESBLs) producing Escherichia coli. METHODOLOGY: During 3 years, all hospitalized patients at the University-affiliated hospital of Tlemcen and presenting urinary tract infections caused by E. coli were considered as potential study participants. These E. coli isolates were examined phenotypically for ESBL production. ESBL strains were subjected to antimicrobial susceptibility testing and were investigated for the presence of plasmid mediated quinolone resistance genes, 16SrRNA methylase genes and virulence genes by using conventional PCR and DNA sequencing. The molecular characterization of ESBL strains was established by phylogenetic grouping method and ERIC-PCR. RESULTS: The overall prevalence of ESBL was 32.5%. The blaCTX-M-15 was the most frequently detected in ESBL isolates, followed by blaCTX-M-14, blaCTX-M-28, blaCTX-M-1 and blaSHV-12 respectively. The plasmid-mediated quinolone resistance genes were detected in the 15 ESBL strains with the aac(6')-Ib-cr gene was the most detected followed by qnrB1 and qnrA1 gene respectively. Among the 22 ESBL isolates resistant to gentamicin and amikacin, the 16SrRNA methylase genes were detected in 4 isolates. The sfa and pap virulent genes were founds in 26% and 22% of isolates receptively. The genotyping analysis of all strains revealed that almost were belonged to phylogenetic groups A1 and A0 and fourteen distinct clones. CONCLUSION: The emergence of uropathogenic ESBL isolates and the high rate of blaCTX-M are alarming in Algeria. Strict measure must be required to control the further spread of these strains in Algerian hospitals.

Multi-faceted immunomodulatory and tissue-tropic clinical bacterial isolate potentiates prostate cancer immunotherapy.

Immune checkpoint inhibitors have not been effective for immunologically "cold" tumors, such as prostate cancer, which contain scarce tumor infiltrating lymphocytes. We hypothesized that select tissue-specific and immunostimulatory bacteria can potentiate these immunotherapies. Here we show that a patient-derived prostate-specific microbe, CP1, in combination with anti-PD-1 immunotherapy, increases survival and decreases tumor burden in orthotopic MYC- and PTEN-mutant prostate cancer models. CP1 administered intra-urethrally specifically homes to and colonizes tumors without causing any systemic toxicities. CP1 increases immunogenic cell death of cancer cells, T cell cytotoxicity, and tumor infiltration by activated CD8 T cells, Th17 T cells, mature dendritic cells, M1 macrophages, and NK cells. CP1 also decreases intra-tumoral regulatory T cells and VEGF. Mechanistically, blocking CP1-recruited T cells from infiltrating the tumor inhibits its therapeutic efficacy. CP1 is an immunotherapeutic tool demonstrating how a tissue-specific microbe can increase tumor immunogenicity and sensitize an otherwise resistant cancer type to immunotherapy.

Cost-effective flow-through nanohole array-based biosensing platform for the label-free detection of uropathogenic E. coli in real time.

Rapid, inexpensive and sensitive detection of uropathogenic Escherichia coli (UPEC), a common cause of ascending urinary tract infections (UTIs) including cystitis and pyelonephritis, is critical given the increasing number of cases and its recurrence worldwide. In this paper, we present a label-free nanoplasmonic sensing platform, built with off-the-shelf optical and electronic components, which can detect intact UPEC at concentrations lower than the physiological limit for UTI diagnosis, in real time. The sensing platform consists of a red LED light source, lens assembly, CMOS detector, Raspberry Pi interface in conjugation with a metallic flow-through nanohole array-based sensor. Detection is achieved exploiting nanoplasmonic phenomena from the nanohole arrays through surface plasmon resonance imaging (SPRi) technique. The platform has a bulk sensitivity of 212 pixel intensity unit (PIU)/refractive index unit (RIU), and a resolution in the order of 10-6 RIU. We demonstrate capture and detection of UPEC with a detection limit of ~100 CFU/ml - a concentration well below the threshold limit for UTI diagnosis in clinical samples. We also demonstrate detection of UPEC in spiked human urine samples for two different concentrations of bacteria. This work is particularly relevant for point-of-care applications, especially for regions around the world where accessibility to medical facilities is heavily dependent upon economy, and availability.

Gaining Access to Bacteria through (Reversible) Control of Lipophilicity.

The development of antimicrobial photodynamic therapy (aPDT) is highly dependent on the development of suitable photosensitizers (PSs); ideally, affinity of a PS towards bacterial cells should be much higher than that towards mammalian cells. A cationic charge on a PS may lead to its selective binding to bacteria mediated through electrostatic interaction; however, the photodynamic outcome is highly dependent on the lipophilicity of the PS. Herein, we report the aPDT effect of silicon(IV) phthalocyanine derivatives bearing four positive charges and methyl, phenyl, or naphthyl substituents at the periphery of the macrocycle. We show that through modulation of lipophilicity, it is possible to find a therapeutic window in which bacteria, but not mammalian cells, are effectively killed. The photobiological activity of these PSs was significantly lower when they were deployed as host-guest complexes with cucurbit[7]uril (CB[7]). CB[7] blocks the hydrophobic part of the PS and reduces its lipophilicity, indicating that a hydrophobic interaction with the outer membrane of bacterial cells is essential for aPDT activity. The efficacies of the obtained PSs have been evaluated by using different uropathogenic E. coli isolates and human kidney epithelial carcinoma cells.

Urinary Tract Infection in a Small Animal Model: Transurethral Catheterization of Male and Female Mice.

Urinary tract infections (UTI) are extremely common worldwide, incurring significant morbidity and healthcare-associated expenses. Small animal models, which accurately reflect disease establishment and progression, permit dissection of host-pathogen interactions and generation of immunity to infection. In mice, intravesical instillation of uropathogenic E. coli, the causative agent in more than 85% of community acquired UTI, recapitulates many of the stages of infection observed in humans. Until recently, however, UTI could only be modeled in female animals. This limitation has hindered the study of sex-related differences in UTI, as well as other bladder pathologies, such as cancer. Here, we describe a method to instill male mice that allows direct comparison between female and male animals and provide a detailed protocol to assess bladder tissue by flow cytometry as a means to better understand host responses to infection. Together, these approaches will aid in the identification of host factors that contribute to sex biases observed in UTI and other bladder-associated diseases.

Molecular characterisation of uropathogenic Escherichia coli isolates at a tertiary care hospital in South India.

Uropathogenic Escherichia coli (UPEC) express a multitude of virulence factors (VFs) to break the inertia of the mucosal barrier of the urinary tract. The aim of the present study was undertaken to characterised the UPEC strains and to correlate carriage of specific virulence markers with different phylogroups and also to correlate these findings with clinical outcome of patients. A total of 156 non-repeated, clinically significant UPEC isolates were studied. Virulent genes were determined by two set of multiplex polymerase chain reaction (PCR). Phylogenetic analysis was performed by triplex PCR methods. Antibiograms and patient's clinical outcomes were collected in a structured pro forma. Of the 156 patients infected by UPEC strains with significant bacterial counts the most common predisposing factors were diabetes (45.5%) followed by carcinoma (7%). On analysis of the VF genes of the isolates, a majority of strains (140; 90%) were possessing the fimH gene followed by iutA (98; 63%), papC (76; 49%), cnf1 (46; 29.5%), hlyA (45; 29%) and neuC (8; 5%), respectively. On phylogenetic analysis, 27 (17%) isolates were belong to phylogroup A, 16 (10%) strains to Group B1, 59 (38%) were from Group B2 and 54 (35%) were from Group D. High prevalence of antibiotic resistance was observed among the isolates. The incidence of papC, cnf1 and hlyA was significantly higher (P < 0.05) among the isolates from relapse patients. Our findings indicate that virulent as well as commensal strains are capable of causing urinary tract infection. Virulence genes as well as patients-related factors are equally responsible for the development of infections and also that virulence genes may help such isolates to persist even with appropriate chemotherapy and be responsible for recurrent infections.

Selective depletion of uropathogenic E. coli from the gut by a FimH antagonist.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) affect 150 million people annually. Despite effective antibiotic therapy, 30-50% of patients experience recurrent UTIs. In addition, the growing prevalence of UPEC that are resistant to last-line antibiotic treatments, and more recently to carbapenems and colistin, make UTI a prime example of the antibiotic-resistance crisis and emphasize the need for new approaches to treat and prevent bacterial infections. UPEC strains establish reservoirs in the gut from which they are shed in the faeces, and can colonize the periurethral area or vagina and subsequently ascend through the urethra to the urinary tract, where they cause UTIs. UPEC isolates encode up to 16 distinct chaperone-usher pathway pili, and each pilus type may enable colonization of a habitat in the host or environment. For example, the type 1 pilus adhesin FimH binds mannose on the bladder surface, and mediates colonization of the bladder. However, little is known about the mechanisms underlying UPEC persistence in the gut. Here, using a mouse model, we show that F17-like and type 1 pili promote intestinal colonization and show distinct binding to epithelial cells distributed along colonic crypts. Phylogenomic and structural analyses reveal that F17-like pili are closely related to pilus types carried by intestinal pathogens, but are restricted to extra-intestinal pathogenic E. coli. Moreover, we show that targeting FimH with M4284, a high-affinity inhibitory mannoside, reduces intestinal colonization of genetically diverse UPEC isolates, while simultaneously treating UTI, without notably disrupting the structural configuration of the gut microbiota. By selectively depleting intestinal UPEC reservoirs, mannosides could markedly reduce the rate of UTIs and recurrent UTIs.