Lactobacillus crispatus Limits Bladder Uropathogenic E. coli Infection by Triggering a Host Type I Interferon Response.

Many urinary tract infections (UTIs) are recurrent because uropathogens persist within the bladder epithelial cells (BECs) for extended periods between bouts of infection. Because persistent uropathogens are intracellular, they are often refractive to antibiotic treatment. The recent discovery of endogenous Lactobacillus spp. in the bladders of healthy humans raised the question of whether these endogenous bacteria directly or indirectly impact intracellular bacterial burden in the bladder. Here, we report that in contrast to healthy women, female patients experiencing recurrent UTIs have a bladder population of Lactobacilli that is markedly reduced. Exposing infected human BECs to L. crispatus in vitro markedly reduced the intracellular uropathogenic Escherichia coli (UPEC) load. The adherence of Lactobacilli to BECs was found to result in increased type I interferon (IFN) production, which in turn enhanced the expression of cathepsin D within lysosomes harboring UPECs. This lysosomal cathepsin D-mediated UPEC killing was diminished in germ-free mice and type I IFN receptor-deficient mice. Secreted metabolites of L. crispatus seemed to be responsible for the increased expression of type I IFN in human BECs. Intravesicular administration of Lactobacilli into UPEC-infected murine bladders markedly reduced their intracellular bacterial load suggesting that components of the endogenous microflora can have therapeutic effects against UTIs.

Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

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.

The RyfA small RNA regulates oxidative and osmotic stress responses and virulence in uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are a common bacterial infectious disease in humans, and strains of uropathogenic Escherichia coli (UPEC) are the most frequent cause of UTIs. During infection, UPEC must cope with a variety of stressful conditions in the urinary tract. Here, we demonstrate that the small RNA (sRNA) RyfA of UPEC strains is required for resistance to oxidative and osmotic stresses. Transcriptomic analysis of the ryfA mutant showed changes in expression of genes associated with general stress responses, metabolism, biofilm formation and genes coding for cell surface proteins. Inactivation of ryfA in UPEC strain CFT073 decreased urinary tract colonization in mice and the ryfA mutant also had reduced production of type 1 and P fimbriae (pili), adhesins which are known to be important for UTI. Furthermore, loss of ryfA also reduced UPEC survival in human macrophages. Thus, ryfA plays a key regulatory role in UPEC adaptation to stress, which contributes to UTI and survival in macrophages.

DNA methylation by three Type I restriction modification systems of Escherichia coli does not influence gene regulation of the host bacterium.

DNA methylation is a common epigenetic mark that influences transcriptional regulation, and therefore cellular phenotype, across all domains of life. In particular, both orphan methyltransferases and those from phasevariable restriction modification systems (RMSs) have been co-opted to regulate virulence epigenetically in many bacteria. We now show that three distinct non-phasevariable Type I RMSs in Escherichia coli have no measurable impact on gene expression, in vivo virulence, or any of 1190 in vitro growth phenotypes. We demonstrated this using both Type I RMS knockout mutants as well as heterologous installation of Type I RMSs into two E. coli strains. These data provide three clear and currently rare examples of restriction modification systems that have no impact on their host organism's gene regulation. This leads to the possibility that other such nonregulatory methylation systems may exist, broadening our view of the potential role that RMSs may play in bacterial evolution.

Modeling the Temperature Effect on the Growth of Uropathogenic Escherichia coli in Sous-Vide Chicken Breast.

Uropathogenic Escherichia coli (UPEC) is known to cause 65-75% of human urinary tract infection (UTI) cases. Poultry meat is a reservoir of UPEC, which is suspected to cause foodborne UTIs. In the present study, we aimed to determine the growth potential of UPEC in ready-to-eat chicken breasts prepared by sous-vide processing. Four reference strains isolated from the urine of UTI patients (Bioresource Collection and Research Center [BCRC] 10,675, 15,480, 15,483, and 17,383) were tested by polymerase chain reaction assay for related genes to identify their phylogenetic type and UPEC specificity. A cocktail of these UPEC strains was inoculated into sous-vide cooked chicken breast at 103-4 colony-forming unit (CFU)/g and stored at 4°C, 10°C, 15°C, 20°C, 30°C, and 40°C. Changes in the populations of UPEC during storage were analyzed by a one-step kinetic analysis method using the U.S. Department of Agriculture [USDA] Integrated Pathogen Modeling Program-Global Fit [IPMP-Global Fit]. The results showed that the combination of the no lag phase primary model and the Huang square-root secondary model fitted well with the growth curves to obtain the appropriate kinetic parameters. This combination for predicting UPEC growth kinetics was further validated using it to study additional growth curves at 25°C and 37°C, which showed that the root mean square error, bias factor, and accuracy factor were 0.49-0.59 (log CFU/g), 0.941-0.984, and 1.056-1.063, respectively. In conclusion, the models developed in this study are acceptable and can be used to predict the growth of UPEC in sous-vide chicken breast.

The inhibitory receptor CD300a is essential for neutrophil-mediated clearance of urinary tract infection in mice.

Neutrophils play a crucial role in immune defense against and clearance of uropathogenic Escherichia coli (UPEC)-mediated urinary tract infection, the most common bacterial infection in healthy humans. CD300a is an inhibitory receptor that binds phosphatidylserine and phosphatidylethanolamine, presented on the membranes of apoptotic cells. CD300a binding to phosphatidylserine and phosphatidylethanolamine, also known as the "eat me" signal, mediates immune tolerance to dying cells. Here, we demonstrate for the first time that CD300a plays an important role in the neutrophil-mediated immune response to UPEC-induced urinary tract infection. We show that CD300a-deficient neutrophils have impaired phagocytic abilities and despite their increased accumulation at the site of infection, they are unable to reduce bacterial burden in the bladder, which results in significant exacerbation of infection and worse host outcome. Finally, we demonstrate that UPEC's pore forming toxin α-hemolysin induces upregulation of the CD300a ligand on infected bladder epithelial cells, signaling to neutrophils to be cleared.

YdiV regulates Escherichia coli ferric uptake by manipulating the DNA-binding ability of Fur in a SlyD-dependent manner.

Iron is essential for all bacteria. In most bacteria, intracellular iron homeostasis is tightly regulated by the ferric uptake regulator Fur. However, how Fur activates the iron-uptake system during iron deficiency is not fully elucidated. In this study, we found that YdiV, the flagella gene inhibitor, is involved in iron homeostasis in Escherichia coli. Iron deficiency triggers overexpression of YdiV. High levels of YdiV then transforms Fur into a novel form which does not bind DNA in a peptidyl-prolyl cis-trans isomerase SlyD dependent manner. Thus, the cooperation of YdiV, SlyD and Fur activates the gene expression of iron-uptake systems under conditions of iron deficiency. Bacterial invasion assays also demonstrated that both ydiV and slyD are necessary for the survival and growth of uropathogenic E. coli in bladder epithelial cells. This reveals a mechanism where YdiV not only represses flagella expression to make E. coli invisible to the host immune system, but it also promotes iron acquisition to help E. coli overcome host nutritional immunity.

The bacteria and the host: a story of purinergic signaling in urinary tract infections.

The local environment forces a selection of bacteria that might invade the urinary tract, allowing only the most virulent to access the kidney. Quite similar to the diet in setting the stage for the gut microbiome, renal function determines the conditions for bacteria-host interaction in the urinary tract. In the kidney, the term local environment or microenvironment is completely justified because the environment literally changes within a few micrometers. The precise composition of the urine is a function of the epithelium lining the microdomain, and the microenvironment in the kidney shows more variation in the content of nutrients, ion composition, osmolality, and pH than any other site of bacteria-host interaction. This review will cover some of the aspects of bacterial-host interaction in this unique setting and how uropathogenic bacteria can alter the condition for bacteria-host interaction. There will be a particular focus on the recent findings regarding how bacteria specifically trigger host paracrine signaling, via release of extracellular ATP and activation of P2 purinergic receptors. These finding will be discussed from the perspective of severe urinary tract infections, including pyelonephritis and urosepsis.

Osteopontin Deficiency Ameliorates Prostatic Fibrosis and Inflammation.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

Rapid Growth and Metabolism of Uropathogenic Escherichia coli in Relation to Urine Composition.

Uropathogenic Escherichia coli (UPEC) strains cause a majority of urinary tract infections (UTIs). Since UPEC strains can become antibiotic resistant, adjunct or alternate therapies are urgently needed. UPEC strains grow extremely rapidly in patients with UTIs. Thus, this review focuses on the relation between urine composition and UPEC growth and metabolism. Compilation of urinary components from two major data sources suggests the presence of sufficient amino acids and carbohydrates as energy sources and abundant phosphorus, sulfur, and nitrogen sources. In a mouse UTI model, mutants lacking enzymes of the tricarboxylic acid cycle, gluconeogenesis, and the nonoxidative branch of the pentose cycle are less competitive than the corresponding parental strains, which is consistent with amino acids as major energy sources. Other evidence suggests that carbohydrates are required energy sources. UPEC strains in urine ex vivo and in vivo express transporters for peptides, amino acids, carbohydrates, and iron and genes associated with nitrogen limitation, amino acid synthesis, nucleotide synthesis, and nucleotide salvage. Mouse models confirm the requirement for many, but not all, of these genes. Laboratory evolution studies suggest that rapid nutrient uptake without metabolic rewiring is sufficient to account for rapid growth. Proteins and pathways required for rapid growth should be considered potential targets for alternate or adjunct therapies.

Peptidoglycan Sensing Prevents Quiescence and Promotes Quorum-Independent Colony Growth of Uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) is the leading cause of human urinary tract infections (UTIs), and many patients experience recurrent infection after successful antibiotic treatment. The source of recurrent infections may be persistent bacterial reservoirs in vivo that are in a quiescent state and thus are not susceptible to antibiotics. Here, we show that multiple UPEC strains require a quorum to proliferate in vitro with glucose as the carbon source. At low cell density, the bacteria remain viable but enter a quiescent, nonproliferative state. Of the clinical UPEC isolates tested to date, 35% (51/145) enter this quiescent state, including isolates from the recently emerged, multidrug-resistant pandemic lineage ST131 (i.e., strain JJ1886) and isolates from the classic endemic lineage ST73 (i.e., strain CFT073). Moreover, quorum-dependent UPEC quiescence is prevented and reversed by small-molecule proliferants that stimulate colony formation. These proliferation cues include d-amino acid-containing peptidoglycan (PG) tetra- and pentapeptides, as well as high local concentrations of l-lysine and l-methionine. Peptidoglycan fragments originate from the peptidoglycan layer that supports the bacterial cell wall but are released as bacteria grow. These fragments are detected by a variety of organisms, including human cells, other diverse bacteria, and, as we show here for the first time, UPEC. Together, these results show that for UPEC, (i) sensing of PG stem peptide and uptake of l-lysine modulate the quorum-regulated decision to proliferate and (ii) quiescence can be prevented by both intra- and interspecies PG peptide signaling.IMPORTANCE Uropathogenic Escherichia coli (UPEC) is the leading cause of urinary tract infections (UTIs). During pathogenesis, UPEC cells adhere to and infiltrate bladder epithelial cells, where they may form intracellular bacterial communities (IBCs) or enter a nongrowing or slowly growing quiescent state. Here, we show in vitro that UPEC strains at low population density enter a reversible, quiescent state by halting division. Quiescent cells resume proliferation in response to sensing a quorum and detecting external signals, or cues, including peptidoglycan tetra- and pentapeptides.

Disperse red 15 (DR15) impedes biofilm formation of uropathogenic Escherichia coli.

Catheter associated urinary tract infection (CAUTI) is a highly prevalent hospital-acquired infection that is predominantly caused by uropathogenic Escherichia coli (UPEC). It adheres on catheter surface using type I pili as the initial step of pathogenesis that progresses to form biofilm. In this study, potential inhibitors against FimH adhesin of type I pili were screened computationally that yielded ten compounds. These were further validated in vitro against adhesion and biofilm formation. The compounds, 1-Amino-4-hydroxyanthraquinone (Disperse Red 15 or DR15) and 4-(4'-chloro-4-biphenylylsulfonylamino) benzoic acid (CB1) impaired adhesion and biofilm formation without inhibiting the planktonic growth. Also, both compounds inhibited cell assemblages like autoaggregation and swarming motility by unknown mechanisms. DR15 was further derivatised into N-(4-hydroxy-9,10-dioxo-9,10-dihydroanthracen-1-yl) undec-10-enamide that self-assembled with linseed oil, which was used as the coating material on urinary Foley catheters. The thin-film coating on the catheter did not leach when incubated in artificial urine and effectively restricted biofilm formation of UPEC. Altogether, the thin-film coating of urinary catheter with DR15 inhibited biofilm formation of UPEC and this application could potentially help to reduce CAUTI incidents in healthcare facilities.

Synergistic mode of action of catechin, vanillic and protocatechuic acids to inhibit the adhesion of uropathogenic Escherichia coli on silicone surfaces.

AIMS: To study the individual and combined contribution of catechin, protocatechuic and vanillic acids to inhibit the adhesion of uropathogenic Escherichia coli (UPEC) on the surface of silicone catheters. METHODS AND RESULTS: The adhesion of UPEC to silicone catheters during the exposure to nonlethal concentrations of phenolic compounds was measured, as well as changes in motility, presence of fimbriae, extra-cellular polymeric substances, surface charge, hydrophobicity and membrane fluidity. The phenolic combination reduced 26-51% of motility, 1 log CFU per cm2 of adhered bacteria and 20-40% the carbohydrate and protein content in the biofilm matrix. Curli fimbriae, surface charge and cell hydrophobicity were affected to a greater extent by the phenolic combination. In the mixture, vanillic acid was the most effective for reducing bacterial adhesion, extra-polymeric substance production, motility, curli fimbriae and biofilm structure. Notwithstanding, protocatechuic acid caused major changes in the bacterial cell surface properties, whereas catechin affected the cell membrane functionality. CONCLUSION: Catechin, protocatechuic and vanillic acids have different bacterial cell targets, explaining the synergistic effect of their combination against uropathogenic E. coli. SIGNIFICANCE AND IMPACT OF STUDY: This study shows the contribution of catechin, protocatechuic and vanillic acids in producing a synergistic mixture against the adhesion of uropathogenic E. coli on silicone catheters. The action of catechin, vanillic and protocatechuic acids included specific contributions of each compound against the E. coli membrane's integrity, motility, surface properties and production of extracellular polymeric substances. Therefore, the studied mixture of phenolic compounds could be used as an antibiotic alternative to reduce urinary tract infections associated with silicone catheters.

In Vitro and In Vivo Biological Activity of Berberine Chloride against Uropathogenic E. coli Strains Using Galleria mellonella as a Host Model.

Berberine is an alkaloid of the protoberberine type used in traditional oriental medicine. Its biological activities include documented antibacterial properties against a wide variety of microorganisms; nonetheless, its use against Escherichia coli strains isolated from urinary infections has not yet been widely investigated in vivo. The emergence of antimicrobial resistance requires new therapeutic approaches to ensure the continued effectiveness of antibiotics for the treatment and prevention of urinary infections. Moreover, uropathogenic Escherichia coli (UPEC) has developed several virulence factors and resistance to routine antibiotic therapy. To this end, several in vitro and in vivo tests were conducted to assess the activity of berberine on uropathogenic E. coli strains. Galleria mellonella as an infection model was employed to confirm the in vivo translatability of in vitro data on berberine activity and its influence on adhesion and invasion proprieties of E. coli on human bladder cells. In vitro pre-treatment with berberine was able to decrease the adhesive and invasive UPEC ability. In vivo treatment increased the larvae survival infected with UPEC strains and reduced the number of circulating pathogens in larvae hemolymph. These preliminary findings demonstrated the efficacy and reliability of G. mellonella as in vivo model for pre-clinical studies of natural substances.

Flexible Metabolism and Suppression of Latent Enzymes Are Important for Escherichia coli Adaptation to Diverse Environments within the Host.

Bacterial metabolism is necessary for adaptation to the host microenvironment. Flexible metabolic pathways allow uropathogenic Escherichia coli (UPEC) to harmlessly reside in the human intestinal tract and cause disease upon extraintestinal colonization. E. coli intestinal colonization requires carbohydrates as a carbon source, while UPEC extraintestinal colonization requires gluconeogenesis and the tricarboxylic acid cycle. UPEC containing disruptions in two irreversible glycolytic steps involving 6-carbon (6-phosphofructokinase; pfkA) and 3-carbon (pyruvate kinase; pykA) substrates have no fitness defect during urinary tract infection (UTI); however, both reactions are catalyzed by isozymes: 6-phosphofructokinases Pfk1 and Pfk2, encoded by pfkA and pfkB, and pyruvate kinases Pyk II and Pyk I, encoded by pykA and pykF UPEC strains lacking one or both phosphofructokinase-encoding genes (pfkB and pfkA pfkB) and strains lacking one or both pyruvate kinase genes (pykF and pykA pykF) were investigated to determine their regulatory roles in carbon flow during glycolysis by examining their fitness during UTI and in vitro growth requirements. Loss of a single phosphofructokinase-encoding gene has no effect on fitness, while the pfkA pfkB double mutant outcompeted the parental strain in the bladder. A defect in bladder and kidney colonization was observed with loss of pykF, while loss of pykA resulted in a fitness advantage. The pykA pykF mutant was indistinguishable from wild-type in vivo, suggesting that the presence of Pyk II rather than the loss of Pyk I itself is responsible for the fitness defect in the pykF mutant. These findings suggest that E. coli suppresses latent enzymes to survive in the host urinary tract.IMPORTANCE Urinary tract infections are the most frequently diagnosed urologic disease, with uropathogenic Escherichia coli (UPEC) infections placing a significant financial burden on the health care system by generating more than two billion dollars in annual costs. This, in combination with steadily increasing antibiotic resistances to present day treatments, necessitates the discovery of new antimicrobial agents to combat these infections. By broadening our scope beyond the study of virulence properties and investigating bacterial physiology and metabolism, we gain a better understanding of how pathogens use nutrients and compete within host microenvironments, enabling us to cultivate new therapeutics to exploit and target pathogen growth requirements in a specific host environment.

O-Antigen-Dependent Colicin Insensitivity of Uropathogenic Escherichia coli.

The outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted by Escherichia coli, can target other E. coli cells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of various E. coli strains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why do E. coli strains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenic E. coli sequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen into E. coli K-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizing E. coli toward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coli infections can be a major health burden, especially with the organism becoming increasingly resistant to "last-resort" antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenic E. coli strain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity among E. coli organisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenic E. coli such as uropathogenic E. coli (UPEC).

Bacterial Microcompartment-Mediated Ethanolamine Metabolism in Escherichia coli Urinary Tract Infection.

Urinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenic Escherichia coli (UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinal E. coli, both pathogenic enterohemorrhagic E. coli and commensal strains. During a UTI, urease-negative E. coli bacteria thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTIs is understudied. We evaluated the role of the metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analyzed infected urine samples by culture, high-performance liquid chromatography, reverse transcription-quantitative PCR, and genomic sequencing. The ethanolamine concentration in urine was comparable to the concentration of the most abundant reported urinary amino acid, d-serine. Transcription of the eut operon was detected in the majority of urine samples containing E. coli screened. All sequenced UPEC strains had conserved eut operons, while metabolic genotypes previously associated with UTI (dsdCXA, metE) were mainly limited to phylogroup B2. In vitro ethanolamine was found to be utilized as a sole source of nitrogen by UPEC strains. The metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly, eutE (which encodes acetaldehyde dehydrogenase) was required for UPEC strains to utilize ethanolamine to gain a growth advantage in AUM, suggesting that ethanolamine is also utilized as a carbon source. These data suggest that urinary ethanolamine is a significant additional carbon and nitrogen source for infecting E. coli strains.

Hydrogen peroxide stimulates uropathogenic Escherichia coli strains to cellulose production.

Reactive oxygen intermediates, such as hydrogen peroxide, are toxic molecules produced by immune cells in response to bacterial invasion into the host. Bacteria try to protect themselves against the immune system through specific properties such as biofilm formation. This phenomenon occurs also during urinary tract infections. Cellulose is an important factor of Escherichia coli biofilm and contributes to building a protective shield around bacterial cells upon the host immune response. In this study, we aimed to analyze the effect of hydrogen peroxide on the production of this biofilm component. To achieve this goal, 25 clinical E. coli strains isolated from patients with urinary tract infections were used. These bacterial strains were characterized based on their growth characteristics, their ability to form biofilm and their capacity to produce cellulose upon exposure to sub-lethal concentrations of hydrogen peroxide growth, and the biofilm formation of these strains was analyzed. Our results revealed that the analyzed uropathogenic E. coli strains slightly, but significantly, reduced growth and biofilm production upon hydrogen peroxide treatment. However, when separating these strains regarding their ability to produce cellulose, we found that general biofilm production was reduced but cellulose expression was induced upon peroxide treatment. This finding contributes to a better understanding of how bacterial biofilm formation is triggered and provides interesting insights into how uropathogenic E. coli protect themselves in an inhospitable environment.

The roles of hormones in the modulation of growth and virulence genes' expressions in UPEC strains.

BACKGROUND: Host factors such as hormones are known to modulate growth, virulence and antibiotic susceptibility of bacteria. In the present study, the effect of norepinephrine (NE) and estradiol (Est) on growth and expression levels of virulence genes (usp, sfa/foc, cnf1, aer) of uropathogenic E. coli (UPEC) strains C7 and C149 were investigated. METHODS: E. coli C7 and C149 were grown in serum based SAPI broth with and without three different concentrations of norepinephrine and estradiol. Growths were determined via optical density measurement in a spectrophotometer. Real-time polymerase chain reaction was used to determine gene expression levels. Statistical analyses were performed by one way Anova Tukey's post hoc-test. RESULTS: According to our results it has been shown that, growths of bacteria could be affected in the presence of hormones which are variable according to incubation period and hormones' concentrations. Up regulation of usp, sfa/foc, cnf1 were shown to be statistically significant (p < 0.05) in the presence of low, medium levels NE and all concentrations of Est. The expression of aer was down regulated significantly in the presence of low (p < 0.001) and medium level of Est; but all levels of NE was shown to be increased the expression of aer significantly (p < 0.05). CONCLUSIONS: The results of the present study has shown once more that host factors (norepinephrine and estradiol) could influence the growth of a bacterium as well as gene expressions.

Direct Evaluation of Live Uropathogenic Escherichia coli Adhesion and Efficiency of Antiadhesive Compounds Using a Simple Microarray Approach.

Many pathogens use host glycans as docking points for adhesion. Therefore, the use of compounds blocking carbohydrate-binding adhesins is a promising strategy for fighting infections. In this work, we describe a simple and rapid microarray approach for assessing the bacterial adhesion and efficiency of antiadhesive compounds targeting uropathogenic Escherichia coli UTI89, which displays mannose-specific adhesin FimH at the tip of fimbriae. The approach consisted in direct detection of live fluorescently labeled bacteria bound to mannan printed onto microarray slides. The utility of the arrays for binding/inhibition assays was first validated by comparing array-derived results for the model mannose-binding lectin concanavalin A with data obtained by isothermal titration calorimetry. Growth phase-dependent binding of UTI89 to the arrays was observed, proving the usefulness of the setup for detecting differences in FimH expression. Importantly, bacteria labeling and binding assays entailed minimal manipulation, helping to preserve the integrity of fimbriae. The efficiency of three different dodecamannosylated fullerenes as FimH-targeted antiadhesives was next evaluated in competition assays. The results revealed a superior activity of the mannofullerenes (5- to 18-fold per mannose residue) over methyl α-d-mannopyranoside. Moreover, differences in activity were detected for mannofullerenes differing in the structure/length of the spacer used for grafting mannose onto the fullerene core, further demonstrating the sensitivity of the assay. Overall, the approach combines straightforward and time-saving protocols for microarray preparation, bacteria labeling, and binding assays, and it can be easily tailored to other bacteria bearing carbohydrate-binding adhesins.