Raising the alarm: fosfomycin resistance associated with non-susceptible inner colonies imparts no fitness cost to the primary bacterial uropathogen.

IMPORTANCE: While fosfomycin resistance is rare, the observation of non-susceptible subpopulations among clinical Escherichia coli isolates is a common phenomenon during antimicrobial susceptibility testing (AST) in American and European clinical labs. Previous evidence suggests that mutations eliciting this phenotype are of high biological cost to the pathogen during infection, leading to current recommendations of neglecting non-susceptible colonies during AST. Here, we report that the most common route to fosfomycin resistance, as well as novel routes described in this work, does not impair virulence in uropathogenic E. coli, the major cause of urinary tract infections, suggesting a re-evaluation of current susceptibility guidelines is warranted.

Enzymatic Specificity of Conserved Rho GTPase Deamidases Promotes Invasion of Vibrio parahaemolyticus at the Expense of Infection.

Vibrio parahaemolyticus is among the leading causes of bacterial seafood-borne acute gastroenteritis. Like many intracellular pathogens, V. parahaemolyticus invades host cells during infection by deamidating host small Rho GTPases. The Rho GTPase deamidating activity of VopC, a type 3 secretion system (T3SS) translocated effector, drives V. parahaemolyticus invasion. The intracellular pathogen uropathogenic Escherichia coli (UPEC) invades host cells by secreting a VopC homolog, the secreted toxin cytotoxic necrotizing factor 1 (CNF1). Because of the homology between VopC and CNF1, we hypothesized that topical application of CNF1 during V. parahaemolyticus infection could supplement VopC activity. Here, we demonstrate that CNF1 improves the efficiency of V. parahaemolyticus invasion, a bottleneck in V. parahaemolyticus infection, across a range of doses. CNF1 increases V. parahaemolyticus invasion independent of both VopC and the T3SS altogether but leaves a disproportionate fraction of intracellular bacteria unable to escape the endosome and complete their infection cycle. This phenomenon holds true in the presence or absence of VopC but is particularly pronounced in the absence of a T3SS. The native VopC, by contrast, promotes a far less efficient invasion but permits the majority of internalized bacteria to escape the endosome and complete their infection cycle. These studies highlight the significance of enzymatic specificity during infection, as virulence factors (VopC and CNF1 in this instance) with similarities in function (bacterial uptake), catalytic activity (deamidation), and substrates (Rho GTPases) are not sufficiently interchangeable for mediating a successful invasion for neighboring bacterial pathogens. IMPORTANCE Many species of intracellular bacterial pathogens target host small Rho GTPases to initiate invasion, including the human pathogens Vibrio parahaemolyticus and uropathogenic Escherichia coli (UPEC). The type three secretion system (T3SS) effector VopC of V. parahaemolyticus promotes invasion through the deamidation of Rac1 and CDC42 in the host, whereas the secreted toxin cytotoxic necrotizing factor 1 (CNF1) drives UPEC's internalization through the deamidation of Rac1, CDC42, and RhoA. Despite these similarities in the catalytic activity of CNF1 and VopC, we observed that the two enzymes were not interchangeable. Although CNF1 increased V. parahaemolyticus endosomal invasion, most intracellular V. parahaemolyticus aborted their infection cycle and remained trapped in endosomes. Our findings illuminate how the precise biochemical fine-tuning of T3SS effectors is essential for efficacious pathogenesis. Moreover, they pave the way for future investigations into the biochemical mechanisms underpinning V. parahaemolyticus endosomal escape and, more broadly, the regulation of successful pathogenesis.

A comprehensive assessment of multi-system responses to a renal inoculation of uropathogenic E. coli in swine.

BACKGROUND: The systemic responses to infection and its progression to sepsis remains poorly understood. Progress in the field has been stifled by the shortcomings of experimental models which include poor replication of the human condition. To address these challenges, we developed and piloted a novel large animal model of severe infection that is capable of generating multi-system clinically relevant data. METHODS: Male swine (n = 5) were anesthetized, mechanically ventilated, and surgically instrumented for continuous hemodynamic monitoring and serial blood sampling. Animals were inoculated with uropathogenic E. coli by direct injection into the renal parenchyma and were maintained until a priori endpoints were met. The natural history of the infection was studied. Animals were not resuscitated. Multi-system data were collected hourly to 6 hours; all animals were euthanized at predetermined physiologic endpoints. RESULTS: Core body temperature progressively increased from mean (SD) 37.9(0.8)°C at baseline to 43.0(1.2)°C at experiment termination (p = 0.006). Mean arterial pressure did not begin to decline until 6h post inoculation, dropping from 86(9) mmHg at baseline to 28(5) mmHg (p = 0.005) at termination. Blood glucose progressively declined but lactate levels did not elevate until the last hours of the experiment. There were also temporal changes in whole blood concentrations of a number of metabolites including increases in the catecholamine precursors, tyrosine (p = 0.005) and phenylalanine (p = 0.005). Lung, liver, and kidney function parameters worsened as infection progressed and at study termination there was histopathological evidence of injury in these end-organs. CONCLUSION: We demonstrate a versatile, multi-system, longitudinal, swine model of infection that could be used to further our understanding of the mechanisms that underlie infection-induced multi-organ dysfunction and failure, optimize resuscitation protocols and test therapeutic interventions. Such a model could improve translation of findings from the bench to the bedside, circumventing a significant obstacle in sepsis research.

In silico analysis and in vivo assessment of a novel epitope-based vaccine candidate against uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) are common pathogens in urinary tract infections (UTIs), which show resistance to antibiotics. Therefore, there is a need for a vaccine to reduce susceptibility to the infection. In the present study, bioinformatics approaches were employed to predict the best B and T-cell epitopes of UPEC virulence proteins to develop a multiepitope vaccine candidate against UPEC. Then, the efficacy of the candidate was studied with and without Freund adjuvant. Using bioinformatics methods, 3 epitope-rich domains of IutA and FimH antigens were selected to construct the fusion. Molecular docking and Molecular dynamics (MD) simulation were employed to investigate in silico interaction between designed vaccine and Toll-like receptor 4 (TLR4). Our results showed that the levels of IgG and IgA antibodies were improved in the serum and mucosal samples of the vaccinated mice, and the IgG responses were maintained for at least 6 months. The fusion protein was also able to enhance the level of cytokines IFN.γ (Th1), IL.4 (Th2), and IL.17. In challenge experiments, all vaccine combinations showed high potency in the protection of the urinary tract even after 6 months post first injection. The present study indicates that the designed candidate is able to evoke strong protective responses which warrant further studies.

Low antimicrobial resistance in general practice patients in Rotterdam, the city with the largest proportion of immigrants in the Netherlands.

Antimicrobial resistance (AMR) is an increasing problem. The prevalence of antimicrobial resistance in general practice patients is expected to be relatively high in Rotterdam, the Dutch city with the largest proportion non-Western immigrants. The aim of this study was to assess the prevalence of antibiotic-resistant uropathogens (Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis) in general practices in Rotterdam, and to find a possible association between the prevalence of antibiotic-resistant E. coli and age, gender, and socioeconomic status (SES). A retrospective analysis was performed of urine samples from general practice patients in 2016. The prevalence of AMR in uropathogens was compared with national resistance data, as was the prevalence of highly and multidrug resistant and extended spectrum ß-lactamase (ESBL) producing E. coli and K. pneumoniae. Univariate logistic regression was used to study associations between antibiotic-resistant E. coli and age, gender, and SES area score. No clinically relevant differences were observed in the prevalence of antibiotic-resistant uropathogens in Rotterdam compared with the national prevalence. For E. coli and K. pneumoniae, the prevalence was 3.6% for ESBL production (both pathogens together), while the prevalence ranged between 4.2%-5.0% for high resistance and between 1.2%-3.3% for multidrug resistance. Ciprofloxacin-resistant E. coli was significantly associated with higher age. Although Rotterdam has a high percentage of non-western immigrants and a low SES, AMR is low among general practice patients. This indicates that adherence to national guidelines in general practice enables maintenance of low AMR, even in high-risk populations.

Entrapment of uropathogenic E. coli cells into ultra-thin sol-gel matrices on gold thin films: A low cost alternative for impedimetric bacteria sensing.

Bacterial infections are causing worldwide morbidity and mortality. One way to limit infectious outbreaks and optimize clinical management of infections is through the development of fast and sensitive sensing of bacteria. Most sensing approaches are currently based on immunological detection principles. We report here on an impedimetric sensor to selectively and sensitive detect uropathogenic E. coli cells (E. coli UTI89) using artificial recognition sites. We show here the possibility to imprint the rod-shape structure of E. coli UTI 89 into ultra-thin inorganic silica coatings on gold electrodes in a reproducible manner. A linear range from to 1 × 100 -1 × 104 cfu mL-1 is obtained. With a detection limit for E. coli UTI89 below 1 cfu mL-1 from five blank signals (95% confidence level) and excellent selective binding capabilities, these bacterial cell imprinted electrodes brings us closer to a low cost specific bacterial recognition surfaces.

Multiple antibiotic resistances and virulence markers of uropathogenic Escherichia coli from Mexico.

Virulence and antibiotic resistance properties related to different Escherichia coli phylogenetic groups have not been studied in detail in Mexico. We aimed to identify patterns of virulence genes and multidrug resistance in phylogenetic groups of uropathogenic strains (UPEC). Strains of E. coli were isolated from outpatients with urinary tract infections (UTIs), who went to unit of the public health sector in the State of Mexico. E. coli virulence markers and phylogenetic groups were identified by PCR. Susceptibility to 12 antimicrobials was determined by Kirby-Bauer. E. coli was identified in 60.4% (n = 194) of the patients with UTIs. Phylogroups B2 51% (n = 99), A 13.4% (n = 26) and B1 10.3% (n = 20) were the most frequent. Resistance to three or up to eleven antibiotics was detected in most phylogroups (n = 188). The genes fimH (n = 146), feoB (n = 179), iutA (n = 178), sitA (n = 121), fyuA (n = 99), and traT (n = 142) were mainly detected in strains of phylogroups B2, A, B1, C, and D. Seventy-two patterns of virulence markers were distributed across eight E. coli phylogenetic groups. A high frequency of virulence markers and the multiple antibiotic resistance phenotypes was observed in the phylogroups. The genes of extended-spectrum ß-lactamases (ESBLs) found with higher frequency among UPEC strains were blaTEM, blaSHV y blaCTX-M group 1, CIT (plasmid-mediated AmpC ß-lactamase), and blaOXA-like. In conclusion, our findings show the importance of surveillance, permanent monitoring, and particularly controlled prescription of antibiotics by physicians in the social security health system to reduce the spread of highly virulent UPEC strains that are resistant to multiple antimicrobial agents.

Population pharmacokinetics and pharmacodynamics of fosfomycin in non-critically ill patients with bacteremic urinary infection caused by multidrug-resistant Escherichia coli.

OBJECTIVES: To describe the population pharmacokinetics of fosfomycin for patients with bacteraemic urinary tract infection (BUTI). The analysis identified optimal regimens on the basis of pharmacodynamic targets and assessed the adequacy of Clinical and Laboratory Standards Institute (CLSI) and European Committee on Antimicrobial Susceptibility Testing (EUCAST) susceptibility breakpoints for Escherichia coli. METHODS: Data of 16 patients with BUTI caused by multidrug-resistant E. coli (FOREST clinical trial) received intravenous fosfomycin (4 g every 6 hours) were analysed. A population pharmacokinetic analysis was performed, and Monte Carlo simulations were undertaken using 4 g every 6 hours and 8 g every 8 hours. The probability of pharmacodynamic target attainment was assessed using pharmacodynamic targets for E. coli for static effect, 1-log drop in bacterial burden and resistance suppression. RESULTS: Sixty-four plasma samples were collected over a single dosing interval (day 2 or 3 after starting fosfomycin treatment). Fosfomycin concentrations were highly variable. Pharmacodynamic target attainment analysis showed mild improvement by increasing fosfomycin dosing (4 g every 6 hours vs. every 8 hours). These dosages showed success for decreasing 1-log bacterial burden in 89% to 96% (EUCAST breakpoints) and 33% to 54% (CLSI breakpoints) of patients, but they were unable to reach bacterial resistance suppression targets. CONCLUSIONS: Fosfomycin concentrations are highly variable-a fact partially explained by renal impairment. The present work supports the use of 4 g every 6 hours as an effective regimen for the treatment of non-critically ill patients with BUTI caused by multidrug-resistant E. coli, as higher dosages might increase toxicity but may not significantly increase efficacy. The current information may suggest that fosfomycin susceptibility breakpoints need to be reappraised.

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.

Assessment of the comparability of CLSI, EUCAST and Stokes antimicrobial susceptibility profiles for Escherichia coli uropathogenic isolates.

BACKGROUND: As many clinical laboratories convert between Stokes, Clinical and Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) methods, the problem of comparing differently derived sets of antimicrobial susceptibility testing (AST) data with each other arises, owing to a scarcity of knowledge of inter-method comparability. The purpose of the current study was to determine the comparability of CLSI, EUCAST and Stokes AST methods for determining susceptibility of uropathogenic Escherichia coli to ampicillin, amoxicillin-clavulanate, trimethoprim, cephradine/cephalexin, ciprofloxacin and nitrofurantoin. METHODS: A total of 100 E. coli isolates were obtained from boric acid urine samples from patients attending GP surgeries. For EUCAST and CLSI, the Kirby-Bauer disc diffusion method was used and results interpreted using the respective breakpoint guidelines. For the Stokes method, direct susceptibility testing was performed on the urine samples. RESULTS: The lowest levels of agreement were for amoxicillin-clavulanate (60%) and ciprofloxacin (89%) between the three AST methods, when using 2017 interpretive guidelines for CLSI and EUCAST. A comparison of EUCAST and CLSI without Stokes showed 82% agreement for amoxicillin-clavulanate and 94% agreement for ciprofloxacin. Discrepancies were compounded by varying breakpoint susceptibility guidelines issued during the period 2011-2017, and through the inclusion of a definition of intermediate susceptibility in some cases. CONCLUSIONS: Our data indicate that the discrepancies generated through using different AST methods and different interpretive guidelines may result in confusion and inaccuracy when prescribing treatment for urinary tract infection.

Urocultivos en infección clínica de vías urinarias / RESULTS OF URINE CULTURE IN PATIENTS WITH CLINICAL URINARY TRACT INFECTION

Introducción: Las infecciones de vías urinarias (IVU) constituyen un problema de salud mundial. El aumento de la resistencia bacteriana a los antimicrobianos limita la administración de antibióticos económicos y de espectro limitado, lo que afecta el costo y el acceso a la atención. El objetivo de este trabajo es determinar la sensibilidad, resistencia y germen causal en urocultivos realizados en pacientes con infección clínica de vías urinarias. Métodos: Estudio transversal. Se analizaron urocultivos de pacientes con infección clínica de vías urinarias, cada urocultivo correspondió a un paciente. Las variables fueron edad, género, microorganismo causal, resistencia y sensibilidad a los antimicrobianos. Se realizó en la Unidad de Medicina Familiar No. 222 del Instituto Mexicano del Seguro Social en Toluca Estado de México. Se evaluaron urocultivos con más de 100000 Unidades formadoras de colonias. Se realizó mediciones descriptivas, frecuencias y porcentajes en el programa SPSS v. 17 para Windows. Resultados: se incluyeron urocultivos de pacientes con infección clínica de vías urinarias. La edad promedio de los pacientes fue de 50.09 ± 19.43 años, con predominio del género femenino (211 pacientes). Los agentes causales más frecuentes fueron: Escherichia Coli (51.91%), Proteus mirabilis (14.70%) y Staphylococcus (11.11 %). Los antibióticos con mayor sensibilidad fueron: imipenem, cefotetan y meropenem (34%). Los antimicrobianos con mayor resistencia fueron: ampicilina (24%), ciprofloxacino (22%) y ampicilina con sulbactam (20%). Conclusiones: los microorganismos más frecuentemente fueron: Escherichia coli y Proteus; y los antimicrobianos a los que mostraron más resistencia bacteriana fueron: ampicilina y quinolonas.

Introduction: Urinary tract infections (UTIs) are a global health problem. Increased bacterial resistance to antimicrobials limits the administration of low-spectrum antibiotics, which affect cost and access to care. The objective of this work is to determine the sensitivity, resistance and causal germ in urine cultures in patients with clinical urinary tract infection Methods: Transversal study. Urine cultures of patients with clinical urinary tract infection were analyzed, each urine culture corresponded to one patient. The variables were age, gender, causal microorganism, resistance and sensitivity to antimicrobials. It was performed at the Family Medicine Unit No. 222 of the Mexican Institute of Social Security in Toluca State of Mexico. Urocultures were evaluated with more than 100,000 colony forming units. Measurements were made frequencies and percentages in the SPSS program version 17 for Windows. Results: there were included 558 urine cultures; the average age was 50.09 ± 19.43 years, female predominance (211 patients). The most common causative microorganisms were Escherichia coli (51.91%), Proteus mirabilis (14.70%) and Staphylococcus (11.11%). Most sensitive antibiotics were: imipenem, meropenem and cefotetan (34%). Most resistance antimicrobial were: ampicillin (24%), ciprofloxacin (22%) and ampicillin with sulbactam (20%). Conclusions: Escherichia coli and Proteus were the most commonly isolated microorganisms; Ampicillin and quinolones showed more bacterial resistence.

Development of a multiplex real-time PCR assay for phylogenetic analysis of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is among major pathogens causing 80-90% of all episodes of urinary tract infections (UTIs). Recently, E. coli strains are divided into eight main phylogenetic groups including A, B1, B2, C, D, E, F, and clade I. This study was aimed to develop a rapid, sensitive, and specific multiplex real time PCR method capable of detecting phylogenetic groups of E. coli strains. This study was carried out on E. coli strains (isolated from the patient with UTI) in which the presence of all seven target genes had been confirmed in our previous phylogenetic study. An EvaGreen-based singleplex and multiplex real-time PCR with melting curve analysis was designed for simultaneous detection and differentiation of these genes. The primers were selected mainly based on the production of amplicons with melting temperatures (Tm) ranging from 82°C to 93°C and temperature difference of more than 1.5°C between each peak.The multiplex real-time PCR assays that have been developed in the present study were successful in detecting the eight main phylogenetic groups. Seven distinct melting peaks were discriminated, with Tm value of 93±0.8 for arpA, 89.2±0.1for chuA, 86.5±0.1 for yjaA, 82.3±0.2 for TspE4C2, 87.8±0.1for trpAgpC, 85.4±0.6 for arpAgpE genes, and 91±0.5 for the internal control. To our knowledge, this study is the first melting curve-based real-time PCR assay developed for simultaneous and discrete detection of these seven target genes. Our findings showed that this assay has the potential to be a rapid, reliable and cost-effective alternative for routine phylotyping of E. coli strains.

Low biological cost of carbapenemase-encoding plasmids following transfer from Klebsiella pneumoniae to Escherichia coli.

OBJECTIVES: The objective of this study was to determine the biological cost, stability and sequence of two carbapenemase-encoding plasmids containing blaKPC-2 (pG12-KPC-2) and blaVIM-1 (pG06-VIM-1) isolated from Klebsiella pneumoniae when newly acquired by uropathogenic Escherichia coli clinical isolates of different genetic backgrounds. METHODS: The two plasmids were transferred into plasmid-free E. coli clinical isolates by transformation. The fitness effect of newly acquired plasmids on the host cell was assessed in head-to-head competitions with the corresponding isogenic strain. Plasmid stability was estimated by propagating monocultures for ∼312 generations. Plasmid nucleotide sequences were determined using next-generation sequencing technology. Assembly, gap closure, annotation and comparative analyses were performed. RESULTS: Both plasmids were stably maintained in three of four E. coli backgrounds and resulted in low to moderate reductions in host fitness ranging from 1.1% to 3.6%. A difference in fitness cost was observed for pG12-KPC-2 between two different genetic backgrounds, while no difference was detected for pG06-VIM-1 between three different genetic backgrounds. In addition, a difference was observed between pG12-KPC-2 and pG06-VIM-1 in the same genetic background. In general, the magnitude of biological cost of plasmid carriage was both host and plasmid dependent. The sequences of the two plasmids showed high backbone similarity to previously circulating plasmids in K. pneumoniae. CONCLUSIONS: The low to modest fitness cost of newly acquired and stably maintained carbapenemase-encoding plasmids in E. coli indicates a potential for establishment and further dissemination into other Enterobacteriaceae species. We also show that the fitness cost is both plasmid and host specific.

Metabolism and Fitness of Urinary Tract Pathogens.

Among common infections, urinary tract infections (UTI) are the most frequently diagnosed urologic disease. The majority of UTIs are caused by uropathogenic Escherichia coli. The primary niche occupied by E. coli is the lower intestinal tract of mammals, where it resides as a beneficial component of the commensal microbiota. Although it is well-known that E. coli resides in the human intestine as a harmless commensal, specific strains or pathotypes have the potential to cause a wide spectrum of intestinal and diarrheal diseases. In contrast, extraintestinal E. coli pathotypes reside harmlessly in the human intestinal microenvironment but, upon access to sites outside of the intestine, become a major cause of human morbidity and mortality as a consequence of invasive UTI (pyelonephritis, bacteremia, or septicemia). Thus, extraintestinal pathotypes like uropathogenic E. coli (UPEC) possess an enhanced ability to cause infection outside of the intestinal tract and colonize the urinary tract, the bloodstream, or cerebrospinal fluid of human hosts. Due to the requirement for these E. coli to replicate in and colonize both the intestine and extraintestinal environments, we posit that physiology and metabolism of UPEC strains is paramount. Here we discuss that the ability to survive in the urinary tract depends as much on bacterial physiology and metabolism as it does on the well-considered virulence determinants.

Metabolomic analysis of siderophore cheater mutants reveals metabolic costs of expression in uropathogenic Escherichia coli.

Bacterial siderophores are a group of chemically diverse, virulence-associated secondary metabolites whose expression exerts metabolic costs. A combined bacterial genetic and metabolomic approach revealed differential metabolomic impacts associated with biosynthesis of different siderophore structural families. Despite myriad genetic differences, the metabolome of a cheater mutant lacking a single set of siderophore biosynthetic genes more closely approximate that of a non-pathogenic K12 strain than its isogenic, uropathogen parent strain. Siderophore types associated with greater metabolomic perturbations are less common among human isolates, suggesting that metabolic costs influence success in a human population. Although different siderophores share a common iron acquisition function, our analysis shows how a metabolomic approach can distinguish their relative metabolic impacts in E. coli.

Quantitative assessment of human neutrophil migration across a cultured bladder epithelium.

The recruitment of immune cells from the periphery to the site of inflammation is an essential step in the innate immune response at any mucosal surface. During infection of the urinary bladder, polymorphonuclear leukocytes (PMN; neutrophils) migrate from the bloodstream and traverse the bladder epithelium. Failure to resolve infection in the absence of a neutrophilic response demonstrates the importance of PMN in bladder defense. To facilitate colonization of the bladder epithelium, uropathogenic Escherichia coli (UPEC), the causative agent of the majority of urinary tract infections (UTIs), dampen the acute inflammatory response using a variety of partially defined mechanisms. To further investigate the interplay between host and bacterial pathogen, we developed an in vitro model of this aspect of the innate immune response to UPEC. In the transuroepithelial neutrophil migration assay, a variation on the Boyden chamber, cultured bladder epithelial cells are grown to confluence on the underside of a permeable support. PMN are isolated from human venous blood and are applied to the basolateral side of the bladder epithelial cell layers. PMN migration representing the physiologically relevant basolateral-to-apical direction in response to bacterial infection or chemoattractant molecules is enumerated using a hemocytometer. This model can be used to investigate interactions between UPEC and eukaryotic cells as well as to interrogate the molecular requirements for the traversal of bladder epithelia by PMN. The transuroepithelial neutrophil migration model will further our understanding of the initial inflammatory response to UPEC in the bladder.

Assessment of immune responses of the flagellin (FliC) fused to FimH adhesin of Uropathogenic Escherichia coli.

Urinary tract infection (UTI) caused by Uropathogenic Escherichia coli (UPEC) is one of the most common infectious diseases in the world. Despite extensive efforts, a vaccine that protects humans against UTI is currently missing. In this study, the immunogenicity of flagellin (FliC) of UPEC strain in different vaccine combinations with FimH antigen of UPEC and conventional adjuvant Montanide ISA 206 was assessed. Finally, efficacy of the immune responses was evaluated for protection of the bladder and kidney of challenged immunized mice. Mice immunized with the fusion FimH·FliC induced significantly higher anti-FliC humoral (IgG1) and cellular (Th1 and Th2) immune responses than with FliC alone or FliC admixed with FimH. The Montanide enhanced the immune responses of FliC antigen and directed the anti-FliC responses preferentially toward Th1. The FliC vaccine combinations reduced bladder infection as compared to control mice. The fusion FimH·FliC and FliC admixed with FimH and Montanide combinations gave the best results in protection of kidney infection, compared to the control mice. The results of this study propose new promising vaccine combinations based on the FliC antigen and Montanide against UTI caused by UPEC.

Virulence characteristics and genetic affinities of multiple drug resistant uropathogenic Escherichia coli from a semi urban locality in India.

Extraintestinal pathogenic Escherichia coli (ExPEC) are of significant health concern. The emergence of drug resistant E. coli with high virulence potential is alarming. Lack of sufficient data on transmission dynamics, virulence spectrum and antimicrobial resistance of certain pathogens such as the uropathogenic E. coli (UPEC) from countries with high infection burden, such as India, hinders the infection control and management efforts. In this study, we extensively genotyped and phenotyped a collection of 150 UPEC obtained from patients belonging to a semi-urban, industrialized setting near Pune, India. The isolates representing different clinical categories were analyzed in comparison with 50 commensal E. coli isolates from India as well as 50 ExPEC strains from Germany. Virulent strains were identified based on hemolysis, haemagglutination, cell surface hydrophobicity, serum bactericidal activity as well as with the help of O serotyping. We generated antimicrobial resistance profiles for all the clinical isolates and carried out phylogenetic analysis based on repetitive extragenic palindromic (rep)-PCR. E. coli from urinary tract infection cases expressed higher percentages of type I (45%) and P fimbriae (40%) when compared to fecal isolates (25% and 8% respectively). Hemolytic group comprised of 60% of UPEC and only 2% of E. coli from feces. Additionally, we found that serum resistance and cell surface hydrophobicity were not significantly (p = 0.16/p = 0.51) associated with UPEC from clinical cases. Moreover, clinical isolates exhibited highest resistance against amoxicillin (67.3%) and least against nitrofurantoin (57.3%). We also observed that 31.3% of UPEC were extended-spectrum beta-lactamase (ESBL) producers belonging to serotype O25, of which four were also positive for O25b subgroup that is linked to B2-O25b-ST131-CTX-M-15 virulent/multiresistant type. Furthermore, isolates from India and Germany (as well as global sources) were found to be genetically distinct with no evidence to espouse expansion of E. coli from India to the west or vice-versa.

A multilevel analysis of trimethoprim and ciprofloxacin prescribing and resistance of uropathogenic Escherichia coli in general practice.

OBJECTIVES: A retrospective analysis of databases was performed to describe trimethoprim and ciprofloxacin prescribing and resistance in Escherichia coli within general practices in the West of Ireland from 2004 to 2008. METHODS: Antimicrobial susceptibility testing was performed by disc diffusion methods according to the CLSI methods and criteria on significant E. coli isolates (colony count >10(5) cfu/mL) from urine samples submitted from general practice. Data were collected over a 4.5 year period and aggregated at practice level. Data on antimicrobial prescribing of practices were obtained from the national Irish prescribing database, which accounts for approximately 70% of all medicines prescribed in primary care. A multilevel model (MLwiN) was fitted with trimethoprim/ciprofloxacin resistance rates as outcome and practice prescribing as predictor. Practice and individual routinely collected variables were controlled for in the model. RESULTS: Seventy-two general practices sent between 13 and 720 (median 155) samples that turned out to be E. coli positive. Prescribing at practice level was significantly correlated with the probability of antimicrobial-resistant E. coli with an odds ratio of 1.02 [95% confidence interval (CI) 1.01-1.04] for every additional prescription of trimethoprim per 1000 patients per month in the practice and 1.08 (1.04-1.11) for ciprofloxacin. Age was a significant risk factor in both models. Higher variation between practices was found for ciprofloxacin as well as a yearly increase in resistance. Comparing a 'mean' practice with 1 prescription per month with one with 10 prescriptions per month showed an increase in predicted probability of a resistant E. coli for the 'mean' patient from 23.9% to 27.5% for trimethoprim and from 3.0% to 5.5% for ciprofloxacin. CONCLUSIONS: A higher level of antimicrobial prescribing in a practice is associated with a higher probability of a resistant E. coli for the patient. The variation in antimicrobial resistance levels between practices was relatively higher for ciprofloxacin than for trimethoprim.