Fitness of Escherichia coli during Urinary Tract Infection Requires Gluconeogenesis and the TCA Cycle

Microbial pathogenesis studies traditionally encompass dissection of virulence properties such as the bacterium''s ability to elaborate toxins, adhere to and invade host cells, cause tissue damage, or otherwise disrupt normal host immune and cellular functions. In contrast, bacterial metabolism during infection has only been recently appreciated to contribute to persistence as much as their virulence properties. In this study, we used comparative proteomics to investigate the expression of uropathogenic Escherichia coli (UPEC) cytoplasmic proteins during growth in the urinary tract environment and systematic disruption of central metabolic pathways to better understand bacterial metabolism during infection. Using two-dimensional fluorescence difference in gel electrophoresis (2D-DIGE) and tandem mass spectrometry, it was found that UPEC differentially expresses 84 cytoplasmic proteins between growth in LB medium and growth in human urine (P<0.005). Proteins induced during growth in urine included those involved in the import of short peptides and enzymes required for the transport and catabolism of sialic acid, gluconate, and the pentose sugars xylose and arabinose. Proteins required for the biosynthesis of arginine and serine along with the enzyme agmatinase that is used to produce the polyamine putrescine were also up-regulated in urine. To complement these data, we constructed mutants in these genes and created mutants defective in each central metabolic pathway and tested the relative fitness of these UPEC mutants in vivo in an infection model. Import of peptides, gluconeogenesis, and the tricarboxylic acid cycle are required for E. coli fitness during urinary tract infection while glycolysis, both the non-oxidative and oxidative branches of the pentose phosphate pathway, and the Entner-Doudoroff pathway were dispensable in vivo. These findings suggest that peptides and amino acids are the primary carbon source for E. coli during infection of the urinary tract. Because anaplerosis, or using central pathways to replenish metabolic intermediates, is required for UPEC fitness in vivo, we propose that central metabolic pathways of bacteria could be considered critical components of virulence for pathogenic microbes.     

Type 1 Fimbriae Contribute to Catheter-Associated Urinary Tract Infections Caused by Escherichia coli

Biofilm formation on catheters is thought to contribute to persistence of catheter-associated urinary tract infections (CAUTI), which represent the most frequent nosocomial infections. Knowledge of genetic factors for catheter colonization is limited, since their role has not been assessed using physicochemical conditions prevailing in a catheterized human bladder. The current study aimed to combine data from a dynamic catheterized bladder model in vitro with in vivo expression analysis for understanding molecular factors relevant for CAUTI caused by Escherichia coli. By application of the in vitro model that mirrors the physicochemical environment during human infection, we found that an E. coli K-12 mutant defective in type 1 fimbriae, but not isogenic mutants lacking flagella or antigen 43, was outcompeted by the wild-type strain during prolonged catheter colonization. The importance of type 1 fimbriae for catheter colonization was verified using a fimA mutant of uropathogenic E. coli strain CFT073 with human and artificial urine. Orientation of the invertible element (IE) controlling type 1 fimbrial expression in bacterial populations harvested from the colonized catheterized bladder in vitro suggested that the vast majority of catheter-colonizing cells (up to 88%) express type 1 fimbriae. Analysis of IE orientation in E. coli populations harvested from patient catheters revealed that a median level of ∼73% of cells from nine samples have switched on type 1 fimbrial expression. This study supports the utility of the dynamic catheterized bladder model for analyzing catheter colonization factors and highlights a role for type 1 fimbriae during CAUTI.     

Increased Antibiotic Resistance of Escherichia coli in Mature Biofilms

Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 μg/ml), kanamycin (25 μg/ml), and ofloxacin (10 μg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses.Reduced susceptibility of biofilm bacteria to antimicrobial agents is a crucial problem for treatment of chronic infections (11, 29, 48). It has been estimated that 65% of microbial infections are associated with biofilms (11, 29, 37), and biofilm cells are 100 to 1,000 times more resistant to antimicrobial agents than planktonic bacterial cells (11, 29, 32).The molecular nature of this apparent resistance has not been elucidated well, and a number of mechanisms have been proposed to explain the reduced susceptibility, such as restricted antibiotic penetration (47), decreased growth rates and metabolism (7, 52), quorum sensing and induction of a biofilm-specific phenotype (8, 29, 35, 39, 49), stress response activation (7, 52), and an increase in expression of efflux pumps (14). Biofilm resistance has generally been assumed to be due to the fact that the cells in the deeper layers of thick biofilms, which grow more slowly, have less access to antibiotics and nutrients. However, this is not the only reason in many cases. Familiar mechanisms of antibiotic resistance, such as modifying enzymes and target mutations, do not seem to be responsible for the biofilm resistance. Even sensitive bacteria that do not have a known genetic basis for resistance can exhibit profoundly reduced susceptibility when they form biofilms (48).It was reported previously that changes in gene expression induced a biofilm-specific phenotype (5, 13, 22, 35, 41, 42). Several genes have been proposed to be particularly important for biofilm formation, and the importance of the rpoS gene in Escherichia coli biofilm formation was suggested recently (1, 10, 22, 42). It has been suggested that induction of an rpoS-mediated stress response results in physiological changes that could contribute to antibiotic resistance (29). Although several mechanisms and genes have been proposed to explain biofilm resistance to antibiotics, this resistance is not still fully understood because these mechanisms seem to work together within a biofilm community. In addition, the physiology of biofilm cells is remarkably heterogeneous and varies according to the location of individual cells within biofilms (33, 34, 46).In this study, susceptibility of E. coli cells in biofilms to antibiotics was investigated. The E. coli cells in the deeper layers of mature biofilms were directly treated with three antibiotics with different molecular targets, the β-lactam ampicillin, the aminoglycoside kanamycin, and the fluoroquinolone ofloxacin. The biofilm biomass was removed before antibiotic treatment, and only the cells located in the deeper layers of the mature biofilms were directly exposed to antibiotics; thus, the effects of restricted antibiotic and nutrient penetration, as well as heterogeneous physiological states in biofilms, were reduced. Although ofloxacin and kanamycin effectively killed the biofilm cells, ampicillin could not kill the cells, which led to regrowth of biofilms. However, the cells in young colony biofilms were completely killed by ampicillin. Therefore, to determine which genes are induced in the mature biofilm cells, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. Based on the experimental data obtained, possible mechanisms of the increased biofilm resistance to ampicillin are discussed below.     

Determinants of Antimicrobial Resistance in Escherichia coli Strains Isolated from Faeces and Urine of Women with Recurrent Urinary Tract Infections

For women with recurrent urinary tract infections (rUTI), the contribution of antibiotic use versus patient-related factors in determining the presence of antimicrobial resistance in faecal and urinary Escherichia coli, obtained from the same patient population, has not been assessed yet. Within the context of the ‘Non-antibiotic prophylaxis for recurrent urinary tract infections’ (NAPRUTI) study, the present study assessed determinants of antimicrobial resistance in E. coli isolated from urinary and faecal samples of women with rUTIs collected at baseline. Potential determinants of resistance were retrieved from self-administered questionnaires. From 434 asymptomatic women, 433 urinary and 424 faecal samples were obtained. E. coli was isolated from 146 (34%) urinary samples and from 336 (79%) faecal samples, and subsequently tested for antimicrobial susceptibility. Multivariable analysis showed trimethoprim/sulfamethoxazole (SXT) use three months prior to inclusion to be associated with urine E. coli resistance to amoxicillin (OR 3.6, 95% confidence interval: 1.3–9.9), amoxicillin-clavulanic acid (OR 4.4, 1.5–13.3), trimethoprim (OR 3.9, 1.4–10.5) and SXT (OR 3.2, 1.2–8.5), and with faecal E. coli resistance to trimethoprim (OR 2.0, 1.0–3.7). The number of UTIs in the preceding year was correlated with urine E. coli resistance to amoxicillin-clavulanic acid (OR 1.11, 1.01–1.22), trimethoprim (OR 1.13, 1.03–1.23) and SXT (OR 1.10, 1.01–1.19). Age was predictive for faecal E. coli resistance to amoxicillin (OR 1.02, 1.00–1.03), norfloxacin and ciprofloxacin (both OR 1.03, 1.01–1.06). In conclusion, in women with rUTI different determinants were found for urinary and faecal E. coli resistance. Previous antibiotic use and UTI history were associated with urine E. coli resistance and age was a predictor of faecal E. coli resistance. These associations could best be explained by cumulative antibiotic use.     

Impact of rpoS Deletion on Escherichia coli Biofilms

Slow growth has been hypothesized to be an essential aspect of bacterial physiology within biofilms. In order to test this hypothesis, we employed two strains of Escherichia coli, ZK126 (ΔlacZ rpoS⁺) and its isogenic ΔrpoS derivative, ZK1000. These strains were grown at two rates (0.033 and 0.0083 h⁻¹) in a glucose-limited chemostat which was coupled either to a modified Robbins device containing plugs of silicone rubber urinary catheter material or to a glass flow cell. The presence or absence of rpoS did not significantly affect planktonic growth of E. coli. In contrast, biofilm cell density in the rpoS mutant strain (ZK1000), as measured by determining the number of CFU per square centimeter, was reduced by 50% (P < 0.05). Deletion of rpoS caused differences in biofilm cell arrangement, as seen by scanning confocal laser microscopy. In reporter gene experiments, similar levels of rpoS expression were seen in chemostat-grown planktonic and biofilm populations at a growth rate of 0.033 h⁻¹. Overall, these studies suggest that rpoS is important for biofilm physiology.     

Effect of Escherichia coli Morphogene bolA on Biofilms

Biofilm physiology is established under a low growth rate. The morphogene bolA is mostly expressed under stress conditions or in stationary phase, suggesting that bolA could be implicated in biofilm development. In order to verify this hypothesis, we tested the effect of bolA on biofilm formation. Overexpression of bolA induces biofilm development, while bolA deletion decreases biofilms.     

Assessment of the Significance of Virulence Factors of Uropathogenic Escherichia coli in Experimental Urinary Tract Infection in Mice

Four Escherichia coli strains, isolated from cystitis patients, belonging to serotype O2:H^? and possessing different combinations of urovirulence factors were examined in an experimental pyelonephritis mouse model to assess the relative importance of virulence factors in causation of urinary tract infections (UTI). The results suggest not only that the each virulence factor has a role in causation of UTI but also that the presence of P fimbriae and production of hemolysin significantly reduced the LD₅₀ and ID₅₀ of the strains in the mouse model. The results also demonstrate that the presence of additional virulence factors acts in an additive or synergetic fashion enhancing the cumulative impact of the strain.     

Presence of Bacteriophage-Like Inhibitory Particles in Escherichia coli

Bacteriophage-like particles were found in the supernatant fluids of Escherichia coli O111a and O111:B(4). Caution is urged in the study of deoxyribonucleic acid synthesis and replication in these strains.     

Cellulose as an Architectural Element in Spatially Structured Escherichia coli Biofilms

Morphological form in multicellular aggregates emerges from the interplay of genetic constitution and environmental signals. Bacterial macrocolony biofilms, which form intricate three-dimensional structures, such as large and often radially oriented ridges, concentric rings, and elaborate wrinkles, provide a unique opportunity to understand this interplay of “nature and nurture” in morphogenesis at the molecular level. Macrocolony morphology depends on self-produced extracellular matrix components. In Escherichia coli, these are stationary phase-induced amyloid curli fibers and cellulose. While the widely used “domesticated” E. coli K-12 laboratory strains are unable to generate cellulose, we could restore cellulose production and macrocolony morphology of E. coli K-12 strain W3110 by “repairing” a single chromosomal SNP in the bcs operon. Using scanning electron and fluorescence microscopy, cellulose filaments, sheets and nanocomposites with curli fibers were localized in situ at cellular resolution within the physiologically two-layered macrocolony biofilms of this “de-domesticated” strain. As an architectural element, cellulose confers cohesion and elasticity, i.e., tissue-like properties that—together with the cell-encasing curli fiber network and geometrical constraints in a growing colony—explain the formation of long and high ridges and elaborate wrinkles of wild-type macrocolonies. In contrast, a biofilm matrix consisting of the curli fiber network only is brittle and breaks into a pattern of concentric dome-shaped rings separated by deep crevices. These studies now set the stage for clarifying how regulatory networks and in particular c-di-GMP signaling operate in the three-dimensional space of highly structured and “tissue-like” bacterial biofilms.     

Effect of Phosphorus on Survival of Escherichia coli in Drinking Water Biofilms

The effect of phosphorus addition on survival of Escherichia coli in an experimental drinking water distribution system was investigated. Higher phosphorus concentrations prolonged the survival of culturable E. coli in water and biofilms. Although phosphorus addition did not affect viable but not culturable (VBNC) E. coli in biofilms, these structures could act as a reservoir of VBNC forms of E. coli in drinking water distribution systems.     

Presence and growth of naturalized Escherichia coli in temperate soils from Lake Superior watersheds

The presence of Escherichia coli in water is used as an indicator of fecal contamination, but recent reports indicate that soil populations can also be detected in tropical, subtropical, and some temperate environments. In this study, we report that viable E. coli populations were repeatedly isolated from northern temperate soils in three Lake Superior watersheds from October 2003 to October 2004. Seasonal variation in the population density of soilborne E. coli was observed; the greatest cell densities, up to 3 x 10(3) CFU/g soil, were found in the summer to fall (June to October), and the lowest numbers, < or =1 CFU/g soil, occurred during the winter to spring months (February to May). Horizontal, fluorophore-enhanced repetitive extragenic palindromic PCR (HFERP) DNA fingerprint analyses indicated that identical soilborne E. coli genotypes, those with > or =92% similarity values, overwintered in frozen soil and were present over time. Soilborne E. coli strains had HFERP DNA fingerprints that were unique to specific soils and locations, suggesting that these E. coli strains became naturalized, autochthonous members of the soil microbial community. In laboratory studies, naturalized E. coli strains had the ability to grow and replicate to high cell densities, up to 4.2 x 10(5) CFU/g soil, in nonsterile soils when incubated at 30 or 37 degrees C and survived longer than 1 month when soil temperatures were < or =25 degrees C. To our knowledge, this is the first report of the growth of naturalized E. coli in nonsterile, nonamended soils. The presence of significant populations of naturalized populations of E. coli in temperate soils may confound the use of this bacterium as an indicator of fecal contamination.     

Presence and Growth of Naturalized Escherichia coli in Temperate Soils from Lake Superior Watersheds

The presence of Escherichia coli in water is used as an indicator of fecal contamination, but recent reports indicate that soil populations can also be detected in tropical, subtropical, and some temperate environments. In this study, we report that viable E. coli populations were repeatedly isolated from northern temperate soils in three Lake Superior watersheds from October 2003 to October 2004. Seasonal variation in the population density of soilborne E. coli was observed; the greatest cell densities, up to 3 × 10³ CFU/g soil, were found in the summer to fall (June to October), and the lowest numbers, ≤1 CFU/g soil, occurred during the winter to spring months (February to May). Horizontal, fluorophore-enhanced repetitive extragenic palindromic PCR (HFERP) DNA fingerprint analyses indicated that identical soilborne E. coli genotypes, those with ≥92% similarity values, overwintered in frozen soil and were present over time. Soilborne E. coli strains had HFERP DNA fingerprints that were unique to specific soils and locations, suggesting that these E. coli strains became naturalized, autochthonous members of the soil microbial community. In laboratory studies, naturalized E. coli strains had the ability to grow and replicate to high cell densities, up to 4.2 × 10⁵ CFU/g soil, in nonsterile soils when incubated at 30 or 37°C and survived longer than 1 month when soil temperatures were ≤25°C. To our knowledge, this is the first report of the growth of naturalized E. coli in nonsterile, nonamended soils. The presence of significant populations of naturalized populations of E. coli in temperate soils may confound the use of this bacterium as an indicator of fecal contamination.     

A New Approach to Detection of the Protective Effect of Escherichia coli on Gram-Positive Bacteria in Binary Biofilms in the Presence of Antibiotics

Microbiology - Binary biofilms containing gram-positive bacteria Micrococcus thailandicus HB or Kocuria rhizophila strain 4A-2G and gram-negative bacteria Escherichia coli strain K-12 or strain...     

dsdA Does Not Affect Colonization of the Murine Urinary Tract by Escherichia coli CFT073

The urinary tract environment provides many conditions that deter colonization by microorganisms. D-serine is thought to be one of these stressors and is present at high concentrations in urine. D-serine interferes with L-serine and pantothenate metabolism and is bacteriostatic to many species. Uropathogenic Escherichia coli commonly possess the dsdCXA genetic locus, which allows them to use D-serine as a sole carbon, nitrogen, and energy source. It was previously reported that in the model UPEC strain CFT073, a dsdA mutant outcompetes wild type in the murine model of urinary tract infection. This “hypercolonization” was used to propose a model whereby UPEC strains sense D-serine in the urinary tract and subsequently up-regulate genes necessary for pathogenesis. Here, we show that inactivation of dsdA does not lead to hypercolonization. We suggest that this previously observed effect is due to an unrecognized secondary mutation in rpoS and that some D-serine specific effects described in other studies may be affected by the rpoS status of the strains used. Inactivation of dsdA in the original clinical isolate of CFT073 gives CFT073 ΔdsdA a growth defect in human urine and renders it unable to grow on minimal medium containing D-serine as the sole carbon source. However, CFT073 ΔdsdA is able to colonize the urinary tracts of CBA/J mice indistinguishably from wild type. These findings indicate that D-serine catabolism, though it may play role(s) during urinary tract infection, does not affect the ability of uropathogenic E. coli to colonize the murine urinary tract.     

Bacteriophage T4-Mediated Release of Envelope Components from Escherichia coli

When Escherichia coli B, labeled by prior growth in ¹⁴C-glucose, are infected with T4 phage there is a rapid release of ¹⁴C-nondialyzable material into the medium. About half of this material is derived from the cell envelope as evidenced by its content of phospholipid and lipopolysaccharide and its buoyant density upon isopycnic ultracentrifugation of 1.19 g/cm³. It is similar in its gross chemical and physical properties to envelope material released at a lower rate from growing uninfected cells or from cells whose protein synthesis is inhibited by chloramphenicol (22). The rate of release of this envelope material at a multiplicity of infection (MOI) of 10 is greatest in the first minute after infection, and release is completed by 4 min. The rate of its release, as a function of MOI at 2 min after infection, is greatest at low MOI (e.g., MOI 2 and 4); in addition, the release does not continue above MOI 30. The main conclusion derived from the data is that phage, as part of the process of adsorption and injection of DNA, cause an increased release of envelope substance from the cells. With the assumption that all of the envelope material released is derived from the outer envelope, it is estimated that uninfected cells release 20 to 30% of their outer envelope per hour, whereas infected cells release 30% in 2 min at MOI 30. Further, because release does not continue at high MOI, this phenomenon is not considered to be a direct cause of lysis from without. Data are also presented on the amounts of other non-dialyzable ¹⁴C-components released and on the differences in the kinetics of release from chloramphenicol-treated cells compared to phage-infected cells. To avoid the possibility that the release is due to phage lysozyme which is an adventitious “contaminant” of wild-type phage, a phage mutant (T4BeG59s) devoid of this enzyme was used in these experiments.