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.

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.

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.