RESUMO
Introduction: Bacterial urinary tract infections (UTI) are among the most common infectious diseases worldwide. The rise of multidrug-resistant (MDR) uropathogenic Escherichia coli (UPEC) UTI cases is a significant threat to healthcare systems. Several probiotic bacteria have been proposed as an alternative to combat MDR UTI. Lactic acid bacteria in the genus Limosilactobacillus are some of the most studied and used probiotics. However, strain-specific effects play a critical role in probiotic properties. L. reuteri KUB-AC5 (AC5), isolated from the chicken gut, confers antimicrobial and immunobiotic effects against some human pathogens. However, the antibacterial and immune modulatory effects of AC5 on UPEC have never been explored. Methods: Here, we investigated both the direct and indirect effects of AC5 against UPEC isolates (UTI89, CFT073, and clinical MDR UPEC AT31) in vitro. Using a spot-on lawn, agar-well diffusion, and competitive growth assays, we found that viable AC5 cells and cell-free components of this probiotic significantly reduced the UPEC growth of all strains tested. The human bladder epithelial cell line UM-UC-3 was used to assess the adhesion and pathogen-attachment inhibition properties of AC5 on UPEC. Results and discussion: Our data showed that AC5 can attach to UM-UC-3 and decrease UPEC attachment in a dose-dependent manner. Pretreatment of UPEC-infected murine macrophage RAW264.7 cells with viable AC5 (multiplicity of infection, MOI = 1) for 24 hours enhanced macrophage-killing activity and increased proinflammatory (Nos2, Il6, and Tnfa) and anti-inflammatory (Il10) gene expression. These findings indicate the gut-derived AC5 probiotic could be a potential urogenital probiotic against MDR UTI.
Assuntos
Limosilactobacillus reuteri , Macrófagos , Probióticos , Escherichia coli Uropatogênica , Probióticos/farmacologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/imunologia , Limosilactobacillus reuteri/fisiologia , Animais , Camundongos , Macrófagos/imunologia , Macrófagos/microbiologia , Humanos , Urotélio/microbiologia , Infecções Urinárias/microbiologia , Infecções Urinárias/prevenção & controle , Linhagem Celular , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/prevenção & controle , Células RAW 264.7 , Células Epiteliais/microbiologia , Galinhas , Aderência Bacteriana/efeitos dos fármacosRESUMO
Uropathogenic Escherichia coli, the most common cause for urinary tract infections, forms biofilm enhancing its antibiotic resistance. To assess the effects of compounds on biofilm formation of uropathogenic Escherichia coli UMN026 strain, a high-throughput combination assay using resazurin followed by crystal violet staining was optimized for 384-well microplate. Optimized assay parameters included, for example, resazurin and crystal violet concentrations, and incubation time for readouts. For the assay validation, quality parameters Z' factor, coefficient of variation, signal-to-noise, and signal-to-background were calculated. Microplate uniformity, signal variability, edge well effects, and fold shift were also assessed. Finally, a screening with known antibacterial compounds was conducted to evaluate the assay performance. The best conditions found were achieved by using 12 µg/mL resazurin for 150 min and 0.023% crystal violet. This assay was able to detect compounds displaying antibiofilm activity against UMN026 strain at sub-inhibitory concentrations, in terms of metabolic activity and/or biomass.
Assuntos
Antibacterianos , Biofilmes , Violeta Genciana , Ensaios de Triagem em Larga Escala , Oxazinas , Escherichia coli Uropatogênica , Xantenos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/fisiologia , Ensaios de Triagem em Larga Escala/métodos , Xantenos/química , Antibacterianos/farmacologia , Violeta Genciana/metabolismo , Oxazinas/farmacologia , Oxazinas/metabolismo , Oxazinas/química , Testes de Sensibilidade Microbiana , Infecções Urinárias/microbiologia , HumanosRESUMO
Urinary tract infections (UTIs) by Uropathogenic Escherichia coli (UPEC) are a significant health concern, especially due to the increasing prevalence of antibiotic resistance. This study focuses on isolating and characterizing bacteriophages specific to UPEC strains isolated from UTI samples. The isolated phages were assessed for their ability to target and lyse UPEC in vitro, focusing on their efficacy in disrupting biofilms, a key virulence factor contributing to UTI recurrence and antibiotic resistance. The morphological structure observed by TEM belongs to Myoviridae, the phage exhibited icosahedral symmetry with a long non-constricting tail, the approximate measurement of the phage head was 39 nm in diameter, and the phage tail was 105.317 nm in length. One-step growth experiments showed that the latent period was approximately 20 min, followed by a rise period of 40 min, and a growth plateau was reached within 20 min and the burst size observed was 26 phages/infected bacterial cells. These phages were capable of killing cells within the biofilms, leading to a reduction in living cell counts after a single treatment. This study highlights the potential of phages to play a significant role in disrupting, inactivating, and destroying Uropathogenic Escherichia coli (UPEC) biofilms. Such findings could be instrumental in developing treatment strategies that complement antibiotics and disinfectants. The phage-antibiotic synergistic activity was compared to have the possibility to facilitate the advancement of focused and enduring alternatives to traditional antibiotic therapies for UTIs.
Assuntos
Antibacterianos , Bacteriófagos , Biofilmes , Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/virologia , Antibacterianos/farmacologia , Humanos , Infecções por Escherichia coli/microbiologia , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Terapia por Fagos , Myoviridae/isolamento & purificação , Myoviridae/fisiologia , Sinergismo Farmacológico , Testes de Sensibilidade MicrobianaRESUMO
Probiotics offer a promising prophylactic approach against various pathogens and represent an alternative strategy to combat biofilm-related infections. In this study, we isolated vaginal commensal microbiota from 54 healthy Indian women to investigate their probiotic traits. We primarily explored the ability of cell-free supernatant (CFS) from Lactobacilli to prevent Uropathogenic Escherichia coli (UPEC) colonization and biofilm formation. Our findings revealed that CFS effectively reduced UPEC's swimming and swarming motility, decreased cell surface hydrophobicity, and hindered matrix production by downregulating specific genes (fimA, fimH, papG, and csgA). Subsequent GC-MS analysis identified Tryptamine, a monoamine compound, as the potent bioactive substance from Lactobacilli CFS, inhibiting UPEC biofilms with an MBIC of 4 µg/ml and an MBEC of 8 µg/ml. Tryptamine induced significant changes in E. coli colony biofilm morphology, transitioning from the Red, Dry, and Rough (RDAR) to the Smooth and White phenotype, indicating reduced extracellular matrix production. Biofilm time-kill assays demonstrated a four-log reduction in UPEC viability when treated with Tryptamine, highlighting its potent antibacterial properties, comparable to CFS treatment. Biofilm ROS assays indicated a significant elevation in ROS generation within UPEC biofilms, suggesting a potential antibacterial mechanism. Gene expression studies with Tryptamine-treated samples showed a reduction in expression of curli gene (csgA), consistent with CFS treatment. This study underscores the potential of Tryptamine from probiotic Lactobacilli CFS as a promising antibiofilm agent against UPEC biofilms.
Assuntos
Biofilmes , Lactobacillus , Probióticos , Triptaminas , Escherichia coli Uropatogênica , Vagina , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Humanos , Triptaminas/farmacologia , Feminino , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/fisiologia , Probióticos/farmacologia , Vagina/microbiologia , Lactobacillus/efeitos dos fármacos , Lactobacillus/metabolismo , Lactobacillus/fisiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/prevenção & controle , Adulto , Antibacterianos/farmacologiaRESUMO
Little is known about the characteristics of uropathogenic Escherichia coli (UPEC) associated with recurrent urinary tract infections (RUTIs). The present study aimed to analyze the phenotypic antimicrobial resistance of recurrent UPEC isolates attributable to either relapse or reinfection. A total of 140 E. coli strains were isolated from 70 outpatients with RUTIs. All isolates were analyzed by random amplified polymorphic DNA-polymerase chain reaction to evaluate genetic similarity between the first and second isolates. We found that 64.2% (45/70) of outpatients had a relapse with the primary infecting E. coli strain and 35.7% (25/70) had reinfection with a new E. coli strain. Compared with reinfecting strains, relapse UPEC isolates exhibited much higher antimicrobial resistance; 89% of these isolates were multidrug-resistant and 46.6% were extended-spectrum ß-lactamase producers. Our study provides evidence that RUTIs are mainly driven by the persistence of the original strain in the host (relapses) despite appropriate antibiotic treatments, and only RUTIs attributed to relapses seem to favor multidrug resistance in UPEC isolates.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli , Testes de Sensibilidade Microbiana , Recidiva , Infecções Urinárias , Escherichia coli Uropatogênica , Humanos , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/isolamento & purificação , Antibacterianos/farmacologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Farmacorresistência Bacteriana Múltipla/genética , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , beta-Lactamases/genética , Idoso , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
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.
Assuntos
Cistite , Infecções por Escherichia coli , Testes de Sensibilidade Microbiana , Tipagem de Sequências Multilocus , Pós-Menopausa , Escherichia coli Uropatogênica , Vagina , Humanos , Feminino , Cistite/microbiologia , Cistite/urina , Pós-Menopausa/urina , Vagina/microbiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/urina , Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/isolamento & purificação , Idoso , Pessoa de Meia-Idade , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Eletroforese em Gel de Campo Pulsado , Recidiva , beta-Lactamases/genética , Genótipo , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Idoso de 80 Anos ou maisRESUMO
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.
Assuntos
Antibacterianos , Cobre , Testes de Sensibilidade Microbiana , Infecções Urinárias , Escherichia coli Uropatogênica , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/genética , Cobre/farmacologia , Antibacterianos/farmacologia , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológicoRESUMO
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.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Infecções Urinárias , Escherichia coli Uropatogênica , Animais , Feminino , Camundongos , Modelos Animais de Doenças , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Flagelos/genética , Flagelos/metabolismo , Flagelina , Ferro/metabolismo , Locomoção , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/patogenicidade , VirulênciaRESUMO
Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infections, and strains that are resistant to antibiotics are a major problem in treating these infections. Phage therapy is a promising alternative approach that can be used to treat infections caused by polyresistant bacterial strains. In the present study, 16 bacteriophages isolated from sewage and surface water were investigated. Phage host specificity was tested on a collection of 77 UPEC strains. The phages infected 2-44 strains, and 80% of the strains were infected by at least one phage. The susceptible E. coli strains belonged predominantly to the B2 phylogenetic group, including strains of two clones, CC131 and CC73, that have a worldwide distribution. All of the phages belonged to class Caudoviricetes and were identified as members of the families Straboviridae, Autographiviridae, and Drexlerviridae and the genera Kagunavirus, Justusliebigvirus, and Murrayvirus. A phage cocktail composed of six phages - four members of the family Straboviridae and two members of the family Autographiviridae - was prepared, and its antibacterial activity was tested in liquid medium. Complete suppression of bacterial growth was observed after 5-22 hours of cultivation, followed by partial regrowth. At 24 hours postinfection, the cocktail suppressed bacterial growth to 43-92% of control values. Similar results were obtained when testing the activity of the phage cocktail in LB and in artificial urine medium. The results indicate that our phage cocktail has potential to inhibit bacterial growth during infection, and they will therefore be preserved in the national phage bank, serving as valuable resources for therapeutic applications.
Assuntos
Farmacorresistência Bacteriana Múltipla , Especificidade de Hospedeiro , Filogenia , Escherichia coli Uropatogênica , Escherichia coli Uropatogênica/virologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Bacteriófagos/classificação , Bacteriófagos/fisiologia , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Esgotos/virologia , Terapia por Fagos/métodos , Humanos , Antibacterianos/farmacologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/terapiaRESUMO
The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms. Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, is frequently associated with multidrug resistance and recurrent infections. To elucidate the mechanism of resistance of UPEC to beta-lactam antibiotics, we generated ampicillin-resistant UPEC strains through continuous exposure to low and high levels of ampicillin in the laboratory, referred to as Low AmpR and High AmpR, respectively. Whole-genome sequencing revealed that both Low and High AmpR strains contained mutations in the marR, acrR, and envZ genes. The High AmpR strain exhibited a single additional mutation in the nlpD gene. Using protein modeling and qRT-PCR analyses, we validated the contributions of each mutation in the identified genes to antibiotic resistance in the AmpR strains, including a decrease in membrane permeability, increased expression of multidrug efflux pump, and inhibition of cell lysis. Furthermore, the AmpR strain does not decrease the bacterial burden in the mouse bladder even after continuous antibiotic treatment in vivo, implicating the increasing difficulty in treating host infections caused by the AmpR strain. Interestingly, ampicillin-induced mutations also result in multidrug resistance in UPEC, suggesting a common mechanism by which bacteria acquire cross-resistance to other classes of antibiotics.
Assuntos
Ampicilina , Antibacterianos , Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli , Mutação , Infecções Urinárias , Escherichia coli Uropatogênica , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/efeitos dos fármacos , Animais , Farmacorresistência Bacteriana Múltipla/genética , Infecções Urinárias/microbiologia , Infecções por Escherichia coli/microbiologia , Camundongos , Antibacterianos/farmacologia , Ampicilina/farmacologia , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Humanos , Testes de Sensibilidade Microbiana , Sequenciamento Completo do GenomaRESUMO
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.
Assuntos
Biofilmes , Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Humanos , Irã (Geográfico)/epidemiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/efeitos dos fármacos , Infecções Urinárias/microbiologia , Infecções Urinárias/epidemiologia , Feminino , Fatores de Risco , Masculino , Biofilmes/crescimento & desenvolvimento , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/epidemiologia , Adulto , Pessoa de Meia-Idade , Idoso , Adulto Jovem , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Fatores de Virulência/genética , Adesinas de Escherichia coli/genética , Adolescente , Criança , Adesinas Bacterianas/genética , Idoso de 80 Anos ou mais , Farmacorresistência Bacteriana Múltipla/genética , Reação em Cadeia da Polimerase , Pré-EscolarRESUMO
Traditional bacteriocin screening methods often face limitations due to diffusion-related challenges in agar matrices, which can prevent the peptides from reaching their target organism. Turbidimetric techniques offer a solution to these issues, eliminating diffusion-related problems and providing an initial quantification of bacteriocin efficacy in producer organisms. This study involved screening the cell-free supernatant (CFS) from eight uncharacterized asymptomatic bacteriuria (ABU) isolates and Escherichia coli 83972 for antimicrobial activity against clinical uropathogenic E. coli (UPEC) strains using turbidimetric growth methods. ABU isolates exhibiting activity against five or more UPEC strains were further characterized (PUTS 37, PUTS 58, PUTS 59, S-07-4, and SK-106-1). The inhibition of the CFS by proteinase K suggested that the antimicrobial activity was proteinaceous in nature, potentially bacteriocins. The activity of E. coli PUTS 58 and SK-106-1 was enhanced in an artificial urine medium, with both inhibiting all eight UPECs. A putative microcin H47 operon was identified in E. coli SK-106-1, along with a previously identified microcin V and colicin E7 in E. coli PUTS 37 and PUTS 58, respectively. These findings indicate that ABU bacteriocin-producers could serve as viable prophylactics and therapeutics in the face of increasing antibiotic resistance among uropathogens.
Assuntos
Bacteriúria , Infecções por Escherichia coli , Escherichia coli Uropatogênica , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/genética , Bacteriúria/microbiologia , Humanos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Bacteriocinas/farmacologia , Bacteriocinas/genética , Nefelometria e Turbidimetria , Bioensaio/métodos , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Infecções Urinárias/microbiologiaRESUMO
Urinary tract infections (UTIs) are the most common bacterial infections and uropathogenic Escherichia coli (UPEC) is the main etiological agent of UTIs. UPEC can persist in bladder cells protected by immunological defenses and antibiotics and intracellular behavior leads to difficulty in eradicating the infection. The aim of this paper is to design, prepare and characterize surfactant-based nanocarriers (niosomes) able to entrap antimicrobial drug and potentially to delivery and release antibiotics into UPEC-infected cells. In order to validate the proposed drug delivery system, gentamicin, was chosen as "active model drug" due to its poor cellular penetration. The niosomes physical-chemical characterization was performed combining different techniques: Dynamic Light Scattering Fluorescence Spectroscopy, Transmission Electron Microscopy. Empty and loaded niosomes were characterized in terms of size, ζ-potential, bilayer features and stability. Moreover, Gentamicin entrapped amount was evaluated, and the release study was also carried out. In addition, the effect of empty and loaded niosomes was studied on the invasion ability of UPEC strains in T24 bladder cell monolayers by Gentamicin Protection Assay and Confocal Microscopy. The observed decrease in UPEC invasion rate leads us to hypothesize a release of antibiotic from niosomes inside the cells. The optimization of the proposed drug delivery system could represent a promising strategy to significatively enhance the internalization of antimicrobial drugs.
Assuntos
Antibacterianos , Gentamicinas , Lipossomos , Escherichia coli Uropatogênica , Gentamicinas/farmacologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Humanos , Antibacterianos/farmacologia , Portadores de Fármacos/química , Infecções Urinárias/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Testes de Sensibilidade MicrobianaRESUMO
BACKGROUND: Urinary tract infection (UTI) prevention benefits of cranberry intake are clinically validated, especially for women and children. To ensure the benefits of cranberry dietary supplement products, the anti-adhesion activity (AAA) against uropathogenic bacteria is routinely used in in vitro bioassays to determine the activity in whole product formulations, isolated compounds, and ex vivo bioassays to assess urinary activity following intake. D-mannose is another dietary supplement taken for UTI prevention, based on the anti-adhesion mechanism. OBJECTIVE: Compare the relative AAA of cranberry and D-mannose dietary supplements against the most important bacterial types contributing to the pathogenesis of UTI, and consider how certain components potentially induce in vivo activity. METHODS: The current study used a crossover design to determine ex vivo AAA against both P- and Type 1-fimbriated uropathogenic Escherichia coli of either D-mannose or a cranberry fruit juice dry extract product containing 36 mg of soluble proanthocyanidins (PACs), using bioassays that measure urinary activity following consumption. AAA of extracted cranberry compound fractions and D-mannose were compared in vitro and potential induction mechanisms of urinary AAA explored. RESULTS: The cranberry dietary supplement exhibited both P-type and Type 1 in vitro and ex vivo AAA, while D-mannose only prevented Type 1 adhesion. Cranberry also demonstrated more robust and consistent ex vivo urinary AAA than D-mannose over each 1-week study period at different urine collection time points. The means by which the compounds with in vitro activity in each supplement product could potentially induce the AAA in urines was discussed relative to the data. CONCLUSIONS: Results of the current study provide consumers and healthcare professionals with additional details on the compounds and mechanisms involved in the positive, broad-spectrum AAA of cranberry against both E. coli bacterial types most important in UTIs and uncovers limitations on AAA and effectiveness of D-mannose compared to cranberry.
Assuntos
Aderência Bacteriana , Estudos Cross-Over , Suplementos Nutricionais , Sucos de Frutas e Vegetais , Manose , Extratos Vegetais , Proantocianidinas , Infecções Urinárias , Escherichia coli Uropatogênica , Vaccinium macrocarpon , Escherichia coli Uropatogênica/efeitos dos fármacos , Manose/farmacologia , Manose/urina , Infecções Urinárias/prevenção & controle , Infecções Urinárias/microbiologia , Humanos , Extratos Vegetais/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Sucos de Frutas e Vegetais/análise , Proantocianidinas/farmacologia , Proantocianidinas/análise , Feminino , Frutas/química , Infecções por Escherichia coli/prevenção & controle , Infecções por Escherichia coli/urinaRESUMO
Reducing growth and limiting metabolism are strategies that allow bacteria to survive exposure to environmental stress and antibiotics. During infection, uropathogenic Escherichia coli (UPEC) may enter a quiescent state that enables them to reemerge after the completion of successful antibiotic treatment. Many clinical isolates, including the well-characterized UPEC strain CFT073, also enter a metabolite-dependent, quiescent state in vitro that is reversible with cues, including peptidoglycan-derived peptides and amino acids. Here, we show that quiescent UPEC is antibiotic tolerant and demonstrate that metabolic flux in the tricarboxylic acid (TCA) cycle regulates the UPEC quiescent state via succinyl-CoA. We also demonstrate that the transcriptional regulator complex integration host factor and the FtsZ-interacting protein ZapE, which is important for E. coli division during stress, are essential for UPEC to enter the quiescent state. Notably, in addition to engaging FtsZ and late-stage cell division proteins, ZapE also interacts directly with TCA cycle enzymes in bacterial two-hybrid assays. We report direct interactions between the succinate dehydrogenase complex subunit SdhC, the late-stage cell division protein FtsN, and ZapE. These interactions may enable communication between oxidative metabolism and the cell division machinery in UPEC. Moreover, these interactions are conserved in an E. coli K-12 strain. This work suggests that there is coordination among the two fundamental and essential pathways that regulate overall growth, quiescence, and antibiotic susceptibility. IMPORTANCE: Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs). Upon invasion into bladder epithelial cells, UPEC establish quiescent intracellular reservoirs that may lead to antibiotic tolerance and recurrent UTIs. Here, we demonstrate using an in vitro system that quiescent UPEC cells are tolerant to ampicillin and have decreased metabolism characterized by succinyl-CoA limitation. We identify the global regulator integration host factor complex and the cell division protein ZapE as critical modifiers of quiescence and antibiotic tolerance. Finally, we show that ZapE interacts with components of both the cell division machinery and the tricarboxylic acid cycle, and this interaction is conserved in non-pathogenic E. coli, establishing a novel link between cell division and metabolism.
Assuntos
Antibacterianos , Ciclo do Ácido Cítrico , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Escherichia coli Uropatogênica , Escherichia coli Uropatogênica/metabolismo , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Antibacterianos/farmacologia , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Ciclo do Ácido Cítrico/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana , Infecções por Escherichia coli/microbiologiaRESUMO
Urinary tract infection (UTI) is one of the most common bacterial infections worldwide. The main causative agent of UTI is uropathogenic Escherichia coli (UPEC). There is an immediate need for novel prophylactic and treatment strategies against UTI because of the increasing incidence of antimicrobial resistance among uropathogens. ABU 83972, an asymptomatic bacteriuria-causing E. coli strain, prevents UTI by suppressing the colonization of UPEC. However, the nature of competition and growth repression of UPEC by ABU 83972 is unclear and is the subject of our investigation. Here, we characterized the growth kinetics of ABU 83972 and uropathogens in human urine and laboratory media. Next, we performed a series of competitive co-culture experiments where ABU 83972 and uropathogens were inoculated at a 1:1 ratio in human urine and in various media, and their relative abundance was determined. In human urine, ABU 83972 outcompeted UPEC and additional uropathogens, reaching up to 90% of the total population after 24 hours of incubation. In contrast, UPEC outcompeted ABU 83972 in LB and M9 minimal media and exhibited superior colonization than ABU 83972 in the mouse urinary bladder. Since engineered living materials (ELMs) can be used to retain an organism of interest in a particular location, we developed ABU 83972-containing ELMs that effectively outcompeted UPEC in human urine. In summary, our work establishes that ABU 83972 outcompetes UPEC in a milieu- and cell-density-dependent manner, highlighting the importance of the metabolites and nutrients found in the human urine as determinants of the competitive fitness of ABU 83972.
Assuntos
Bacteriúria , Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Humanos , Bacteriúria/microbiologia , Animais , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/efeitos dos fármacos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/urina , Infecções Urinárias/microbiologia , Camundongos , Feminino , Urina/microbiologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacosRESUMO
Despite extensive characterisation of uropathogenic Escherichia coli (UPEC) causing urinary tract infections (UTIs), the genetic background of non-urinary extraintestinal pathogenic E. coli (ExPEC) in companion animals remains inadequately understood. In this study, we characterised virulence traits of 104 E. coli isolated from canine pyometra (n = 61) and prostatic abscesses (PAs) (n = 38), and bloodstream infections (BSIs) in dogs (n = 2), and cats (n = 3). A stronger association with UPEC of pyometra strains in comparison to PA strains was revealed. Notably, 44 isolates exhibited resistance to third-generation cephalosporins and/or fluoroquinolones, 15 were extended-spectrum ß-lactamase-producers. Twelve multidrug-resistant (MDR) strains, isolated from pyometra (n = 4), PAs (n = 5), and BSIs (n = 3), along with 7 previously characterised UPEC strains from dogs and cats, were sequenced. Genomic characteristics revealed that MDR E. coli associated with UTIs, pyometra, and BSIs belonged to international high-risk E. coli clones, including sequence type (ST) 38, ST131, ST617, ST648, and ST1193. However, PA strains belonged to distinct lineages, including ST12, ST44, ST457, ST744, and ST13037. The coreSNPs, cgMLST, and pan-genome illustrated intra-clonal variations within the same ST from different sources. The high-risk ST131 and ST1193 (phylogroup B2) contained high numbers of ExPEC virulence genes on pathogenicity islands, predominating in pyometra and UTI. Hybrid MDR/virulence IncF multi-replicon plasmids, containing aerobactin genes, were commonly found in non-B2 phylogroups from all sources. These findings offer genomic insights into non-urinary ExPEC, highlighting its potential for invasive infections in pets beyond UTIs, particularly with regards to high-risk global clones.
Assuntos
Abscesso , Doenças do Cão , Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli , Piometra , Infecções Urinárias , Cães , Animais , Infecções Urinárias/microbiologia , Infecções Urinárias/veterinária , Farmacorresistência Bacteriana Múltipla/genética , Masculino , Doenças do Cão/microbiologia , Gatos , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Piometra/microbiologia , Piometra/veterinária , Piometra/genética , Abscesso/microbiologia , Abscesso/veterinária , Feminino , Doenças do Gato/microbiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/efeitos dos fármacos , Escherichia coli Uropatogênica/patogenicidade , Escherichia coli/genética , Escherichia coli/patogenicidade , Escherichia coli/efeitos dos fármacos , Antibacterianos/farmacologia , Doenças Prostáticas/microbiologia , Doenças Prostáticas/veterinária , Doenças Prostáticas/genética , Virulência/genética , Fatores de Virulência/genéticaRESUMO
Urinary tract infections (UTI) caused by uropathogenic Escherichia coli (UPEC) pose a global health concern. Resistance mechanisms, including genetic mutations in antimicrobial target genes, efflux pumps, and drug deactivating enzymes, hinder clinical treatment. These resistance factors often spread through mobile genetic elements. Molecular techniques like whole genome sequencing (WGS), multilocus sequence typing (MLST), and phylotyping help decode bacterial genomes and categorise resistance genes. In this study, we analysed 57 UPEC isolates from different UTI patients following EUCAST guidelines. A selection of 17 representative strains underwent WGS, phylotyping, MLST, and comparative analysis to connect laboratory susceptibility data with predictive genomics based on key resistance genes and chromosomal mutations in antimicrobial targets. Trimethoprim resistance consistently correlated with dfr genes, with six different alleles detected among the isolates. These dfr genes often coexisted with class 1 integrons, with the most common gene cassette combining dfr and aadA. Furthermore, 52.9% of isolates harboured the blaTem-1 gene, rendering resistance to ampicillin and amoxicillin. Ciprofloxacin-resistant strains exhibited mutations in GyrA, GyrB and ParC, plasmid-mediated quinolone resistance genes (qnrb10), and aac(6')-Ib-cr5. Nitrofurantoin resistance in one isolate stemmed from a four amino acid deletion in NfsB. These findings illustrate the varied strategies employed by UPEC to resist antibiotics and the correlation between clinical susceptibility testing and molecular determinants. As molecular testing gains prominence in clinical applications, understanding key resistance determinants becomes crucial for accurate susceptibility testing and guiding effective antimicrobial therapy.
Assuntos
Antibacterianos , Infecções por Escherichia coli , Testes de Sensibilidade Microbiana , Infecções Urinárias , Escherichia coli Uropatogênica , Sequenciamento Completo do Genoma , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/efeitos dos fármacos , Humanos , Sequenciamento Completo do Genoma/métodos , Infecções por Escherichia coli/microbiologia , Antibacterianos/farmacologia , Infecções Urinárias/microbiologia , Irlanda , Genoma Bacteriano , Farmacorresistência Bacteriana/genética , Filogenia , Fenótipo , Tipagem de Sequências Multilocus , Feminino , MasculinoRESUMO
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.
Assuntos
Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade , Escherichia coli Uropatogênica/enzimologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Animais , Camundongos , Infecções por Escherichia coli/microbiologia , Virulência , Endopeptidases/genética , Endopeptidases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Peptidoglicano/metabolismo , Macrófagos/microbiologia , Macrófagos/imunologia , Humanos , Modelos Animais de DoençasRESUMO
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) (â¼3-4-fold change with respect to control for claudin-2 and â¼2-3-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 (â¼3.5- and â¼2-fold change with respect to control for DOPAC and â¼1.5- 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.