Bacteria metabolic adaptation to oxidative stress: the case of silica.

Although the presence of silica in many living organisms offers advanced properties including cell protection, the different in vitro attempts to build living materials in pure silica never favoured the cells viability. Thus, little attention has been paid to host-guest interactions to modify the expected biologic response. Here we report the physiological changes undergone by Escherichia coli K-12 in silica from colloidal solution to gel confinement. We show that the physiological alterations in growing cultures are not triggered by the initial oxidative Reactive Oxygen Species (ROS) response. Silica promotes the induction of alternative metabolic pathways along with an increase of growth suggesting the existence of rpoS polymorphisms. Since the functionality of hybrid materials depends on the specific biologic responses of their guests, such cell physiological adaptation opens perspectives in the design of bioactive devices attracting for a large field of sciences.

Genotypic and phenotypic characteristics of Escherichia coli involved in transfusion-transmitted bacterial infections: implications for preventive strategies.

BACKGROUND: Transfusion-transmitted bacterial infections (TTBIs) are the main residual infectious complications of transfusions. Escherichia coli and platelet (PLT) concentrates may be epidemiologically associated, leading to severe, if not lethal, TTBIs. We investigated the genotypic and phenotypic reasons for this clinically deleterious combination. STUDY DESIGN AND METHODS: We investigated a French national E. coli strain collection related to six independent episodes of TTBIs. Their phenotypic characterizations included antibiotic susceptibility testing, growth testing under different culture conditions, serum survival assays, and virulence in a sepsis mouse model. Their genotypic characterizations included polymerase chain reaction phylotyping, whole genome sequencing, and a subsequent in silico analysis. RESULTS: We highlighted a selection process of highly extraintestinal virulent strains, mainly belonging to the B2 phylogroup, adapted to the hostile environment (high citrate concentration and a bactericidal serum effect) of apheresis-collected platelet concentrates (PCs). Compared to controls, the E. coli TTBI strains grew faster in the PCs due to a superior ability to capture iron. The in vitro growth performances were highly compatible with blood-derived product real-life conditions, including storage conditions and delays. The consistent serum resistance of TTBI strains promotes their survival in both the donor's and the receiver's blood and in the PCs. CONCLUSION: This study pointed out that E. coli strains responsible for TTBI exhibit very specific traits. They belong to the extraintestinal pathogenic phylogroups and have a high intrinsic virulence. They can be resistant to complement, capture iron, and grow in the apheresis-collected PCs. These findings therefore support the reinforcement of the postdonation information.

Diversity of the auxotrophic requirements in natural isolates of Escherichia coli.

Isolates of Escherichia coli, except Shigella, are generally prototrophic; they do not require any growth factors to grow in mineral medium. However, a nicotinic acid requirement is common among B2 phylogroup STc95 O18 E. coli clone strains. Nicotinic acid is a precursor of nicotinamide adenine dinucleotide (NAD), an essential molecule that plays central role in cellular metabolism. The defect in NAD synthesis of these strains is due to alterations in de novo biosynthesis pathway nadB gene. Here, by studying growth on minimal medium with glycolytic (glucose) or gluconeogenic (pyruvate or succinate) substrates as the carbon supply in a large panel of E. coli natural isolates representative of the species diversity, we identify an absolute nicotinic acid requirement in non-STc95 strains due in one case to a nadA inactivation. The growth on glucose medium of some extraintestinal pathogenic E. coli strains belonging to various non-O18 B2 phylogroup STc95 clones is restored either by aspartate or nicotinate, demonstrating that the nicotinic acid requirement can also be due to an intracellular aspartate depletion. The auxotrophic requirements depend on the carbon source available in the environment. Moreover, some strains prototrophic in glucose medium become auxotrophic in succinate medium, and conversely, some strainsauxotrophic in glucose medium become prototrophic in succinate medium. Finally, a partial depletion of intracellular aspartate can be observed in some prototrophic strains belonging to various phylogroups. The observed more or less significant depletion according to isolates may be due to differences in tricarboxylic acid cycle enzyme activities. These metabolic defects could be involved in the adaptation of E. coli to its various niches.

Links between Transcription, Environmental Adaptation and Gene Variability in Escherichia coli: Correlations between Gene Expression and Gene Variability Reflect Growth Efficiencies.

Gene expression is known to be the principle factor explaining how fast genes evolve. Highly transcribed genes evolve slowly because any negative impact caused by a particular mutation is magnified by protein abundance. However, gene expression is a phenotype that depends both on the environment and on the strains or species. We studied this phenotypic plasticity by analyzing the transcriptome profiles of four Escherichia coli strains grown in three different culture media, and explored how expression variability was linked to gene allelic diversity. Genes whose expression changed according to the media and not to the strains were less polymorphic than other genes. Genes for which transcription depended predominantly on the strain were more polymorphic than other genes and were involved in sensing and responding to environmental changes, with an overrepresentation of two-component system genes. Surprisingly, we found that the correlation between transcription and gene diversity was highly variable among growth conditions and could be used to quantify growth efficiency of a strain in a medium. Genetic variability was found to increase with gene expression in poor growth conditions. As such conditions are also characterized by down-regulation of all DNA repair systems, including transcription-coupled repair, we suggest that gene expression under stressful conditions may be mutagenic and thus leads to a variability in mutation rate among genes in the genome which contributes to the pattern of protein evolution.

Interactions between genotype and environment drive the metabolic phenotype within Escherichia coli isolates.

To gain insights into the adaptation of the Escherichia coli species to different environments, we monitored protein abundances using quantitative proteomics and measurements of enzymatic activities of central metabolism in a set of five representative strains grown in four contrasted culture media including human urine. Two hundred and thirty seven proteins representative of the genome-scale metabolic network were identified and classified into pathway categories. We found that nutrient resources shape the general orientation of metabolism through coordinated changes in the average abundances of proteins and in enzymatic activities that all belong to the same pathway category. For example, each culture medium induces a specific oxidative response whatever the strain. On the contrary, differences between strains concern isolated proteins and enzymes within pathway categories in single environments. Our study confirms the predominance of genotype by environment interactions at the proteomic and enzyme activity levels. The buffering of genetic variation when considering life-history traits suggests a multiplicity of evolutionary strategies. For instance, the uropathogenic isolate CFT073 shows a deregulation of iron demand and increased oxidative stress response.

Hepcidin as a Major Component of Renal Antibacterial Defenses against Uropathogenic Escherichia coli.

The iron-regulatory peptide hepcidin exhibits antimicrobial activity. Having previously shown hepcidin expression in the kidney, we addressed its role in urinary tract infection (UTI), which remains largely unknown. Experimental UTI was induced in wild-type (WT) and hepcidin-knockout (Hepc-/-) mice using the uropathogenic Escherichia coli CFT073 strain. Compared with infected WT mice, infected Hepc-/- mice showed a dramatic increase in renal bacterial load. Moreover, bacterial invasion was significantly dampened by the pretreatment of WT mice with hepcidin. Infected Hepc-/- mice exhibited decreased iron accumulation in the renal medulla and significant attenuation of the renal inflammatory response. Notably, we demonstrated in vitro bacteriostatic activity of hepcidin against CFT073. Furthermore, CFT073 repressed renal hepcidin, both in vivo and in cultured renal cells, and reduced phosphorylation of SMAD kinase in vivo, suggesting a bacterial strategy to escape the antimicrobial activities of hepcidin. In conclusion, we provide new mechanisms by which hepcidin contributes to renal host defense and suggest that targeting hepcidin offers a strategy to prevent bacterial invasion.

Effects of Proanthocyanidins on Adhesion, Growth, and Virulence of Highly Virulent Extraintestinal Pathogenic Escherichia coli Argue for Its Use to Treat Oropharyngeal Colonization and Prevent Ventilator-Associated Pneumonia.

OBJECTIVE: In the context of increasing microbial resistance and limited new antimicrobials, we aimed to study the antimicrobial effects of cranberry proanthocyanidin extracts on Escherichia coli growth, adhesion to epithelial cells, and lung infection. DESIGN: Experimental in vitro and in vivo investigation. SETTING: University research laboratory. SUBJECTS: Seventy-eight 6- to 8-week-old male Balb/C mice. INTERVENTIONS: In vitro, the effect of increasing concentrations of cranberry proanthocyanidin on bacterial growth of different clinical E. coli isolates was evaluated. Ex vivo, adhesion of E. coli to fresh human buccal epithelial cells was measured in the presence or absence of cranberry proanthocyanidin using microscopy. In vivo, lung bacterial count, pulmonary immune response (neutrophil murine chemokine keratinocyte-derived cytokine measurement and polymorphonuclear recruitment in bronchoalveolar lavage fluid), and lethality were evaluated in a pneumonia mouse model with E. coli precultured with or without cranberry proanthocyanidin. E. coli isolates originated from ventilated ICU patients with respiratory tract colonization or ventilator- associated pneumonia. They differed in number of virulence genes. MEASUREMENTS AND MAIN RESULTS: A significant inhibition of bacterial growth was observed with increasing concentration of cranberry proanthocyanidin, affecting both time to maximal growth and maximal growth rate (p<0.0001 for both). The minimal concentration at which this effect occurred was 250 µg/mL. Cranberry proanthocyanidin significantly reduced E. coli adhesion to fresh buccal epithelial cells by up to 80% (p<0.001). Bacterial counts in homogenized lungs and bronchoalveolar lavage fluid were decreased after cranberry proanthocyanidin exposition (p<0.05 and p<0.01, respectively). Cranberry proanthocyanidin also decreased KC concentrations and polymorphonuclear cell recruitment in bronchoalveolar lavage fluid (p<0.05 for both). At identical inoculum, mortality was reduced by more than half in mice inoculated with E. coli exposed to cranberry proanthocyanidin (p<0.01). CONCLUSION: Cranberry proanthocyanidins exhibit potent effects on growth, adhesion, and virulence of oropharyngeal and lung isolates of E. coli, suggesting that cranberry proanthocyanidin could be of clinical interest to reduce oropharyngeal colonization and prevent lung infection.

Changes in urine composition after trauma facilitate bacterial growth.

BACKGROUND: Critically ill patients including trauma patients are at high risk of urinary tract infection (UTI). The composition of urine in trauma patients may be modified due to inflammation, systemic stress, rhabdomyolysis, life support treatment and/or urinary catheter insertion. METHODS: Prospective, single-centre, observational study conducted in patients with severe trauma and without a history of UTIs or recent antibiotic treatment. The 24-hour urine samples were collected on the first and the fifth days and the growth of Escherichia coli in urine from patients and healthy volunteers was compared. Biochemical and hormonal modifications in urine that could potentially influence bacterial growth were explored. RESULTS: Growth of E. coli in urine from trauma patients was significantly higher on days 1 and 5 than in urine of healthy volunteers. Several significant modifications of urine composition could explain these findings. On days 1 and 5, trauma patients had an increase in glycosuria, in urine iron concentration, and in the concentrations of several amino acids compared to healthy volunteers. On day 1, the urinary osmotic pressure was significantly lower than for healthy volunteers. CONCLUSION: We showed that urine of trauma patients facilitated growth of E. coli when compared to urine from healthy volunteers. This effect was present in the first 24 hours and until at least the fifth day after trauma. This phenomenon may be involved in the pathophysiology of UTIs in trauma patients. Further studies are required to define the exact causes of such modifications.

Variation in endogenous oxidative stress in Escherichia coli natural isolates during growth in urine.

BACKGROUND: Uropathogenic strains of Escherichia coli cause symptomatic infections whereas asymptomatic bacteriuria (ABU) strains are well adapted for growth in the human urinary tract, where they establish long-term bacteriuria. Human urine is a very complex growth medium that could be perceived by certain bacteria as a stressful environment. To investigate a possible imbalance between endogenous oxidative response and antioxidant mechanisms, lipid oxidative damage estimated as thiobarbituric acid reactive substances (TBARS) content was evaluated in twenty-one E. coli belonging to various pathovars and phylogenetic groups. Antioxidant defense mechanisms were also analysed. RESULTS: During exponential growth in urine, TBARS level differs between strains, without correlation with the ability to grow in urine which was similarly limited for commensal, ABU and uropathogenic strains. In addition, no correlation between TBARS level and the phylogroup or pathogenic group is apparent. The growth of ABU strain 83972 was associated with a high level of TBARS and more active antioxidant defenses that reduce the imbalance. CONCLUSIONS: Our results indicate that growth capacity in urine is not a property of ABU strains. However, E. coli isolates respond very differently to this stressful environment. In strain ABU 83972, on one hand, the increased level of endogenous reactive oxygen species may be responsible for adaptive mutations. On the other hand, a more active antioxidant defense system could increase the capacity to colonize the bladder.

Core and panmetabolism in Escherichia coli.

Escherichia coli exhibits a wide range of lifestyles encompassing commensalism and various pathogenic behaviors which its highly dynamic genome contributes to develop. How environmental and host factors shape the genetic structure of E. coli strains remains, however, largely unknown. Following a previous study of E. coli genomic diversity, we investigated its diversity at the metabolic level by building and analyzing the genome-scale metabolic networks of 29 E. coli strains (8 commensal and 21 pathogenic strains, including 6 Shigella strains). Using a tailor-made reconstruction strategy, we significantly improved the completeness and accuracy of the metabolic networks over default automatic reconstruction processes. Among the 1,545 reactions forming E. coli panmetabolism, 885 reactions were common to all strains. This high proportion of core reactions (57%) was found to be in sharp contrast to the low proportion (13%) of core genes in the E. coli pangenome, suggesting less diversity of metabolic functions compared to that of all gene functions. Core reactions were significantly overrepresented among biosynthetic reactions compared to the more variable degradation processes. Differences between metabolic networks were found to follow E. coli phylogeny rather than pathogenic phenotypes, except for Shigella networks, which were significantly more distant from the others. This suggests that most metabolic changes in non-Shigella strains were not driven by their pathogenic phenotypes. Using a supervised method, we were yet able to identify small sets of reactions related to pathogenicity or commensalism. The quality of our reconstructed networks also makes them reliable bases for building metabolic models.

Molecular and evolutionary bases of within-patient genotypic and phenotypic diversity in Escherichia coli extraintestinal infections.

Although polymicrobial infections, caused by combinations of viruses, bacteria, fungi and parasites, are being recognised with increasing frequency, little is known about the occurrence of within-species diversity in bacterial infections and the molecular and evolutionary bases of this diversity. We used multiple approaches to study the genomic and phenotypic diversity among 226 Escherichia coli isolates from deep and closed visceral infections occurring in 19 patients. We observed genomic variability among isolates from the same site within 11 patients. This diversity was of two types, as patients were infected either by several distinct E. coli clones (4 patients) or by members of a single clone that exhibit micro-heterogeneity (11 patients); both types of diversity were present in 4 patients. A surprisingly wide continuum of antibiotic resistance, outer membrane permeability, growth rate, stress resistance, red dry and rough morphotype characteristics and virulence properties were present within the isolates of single clones in 8 of the 11 patients showing genomic micro-heterogeneity. Many of the observed phenotypic differences within clones affected the trade-off between self-preservation and nutritional competence (SPANC). We showed in 3 patients that this phenotypic variability was associated with distinct levels of RpoS in co-existing isolates. Genome mutational analysis and global proteomic comparisons in isolates from a patient revealed a star-like relationship of changes amongst clonally diverging isolates. A mathematical model demonstrated that multiple genotypes with distinct RpoS levels can co-exist as a result of the SPANC trade-off. In the cases involving infection by a single clone, we present several lines of evidence to suggest diversification during the infectious process rather than an infection by multiple isolates exhibiting a micro-heterogeneity. Our results suggest that bacteria are subject to trade-offs during an infectious process and that the observed diversity resembled results obtained in experimental evolution studies. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies.

Norepinephrine-dependently released Dr fimbriae of diffusely adhering Escherichia coli strain IH11128 promotes a mitogen-activated protein kinase ERK1/2-dependent production of pro-inflammatory cytokine, IL-8 in human intestinal Caco-2/TC7 cells.

The diffusely adhering Escherichia coli (Afa/Dr DAEC) are associated with recurrent urinary tract infections in adults as well as with diarrheal disease in infants. We previously demonstrated that in wild-type strain IH11128, the Dr fimbriae is released in the extracellular medium in response to multiple environmental signals such as temperature, low aeration and rich medium. A number of molecules of eukaryotic origin, such as catecholamines, have been reported to stimulate bacterial growth and virulence factor production. We show that norepinephrine affects the production and release of Dr fimbriae in Afa/Dr DAEC WT-IH11128 bacteria. The regulatory mechanism involved with norepinephrine-induced Dr fimbriae liberation was apparently due to a differential induction of genes draC, encoding the usher, and draE, encoding the major fimbrial subunit. In addition, we show that the released Dr fimbriae induces the phosphorylation of the mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2 (ERK1/2) and the production of the pro-inflammatory cytokine, IL-8 in fully differentiated cultured human intestinal Caco-2/TC7 cells.

Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths.

The Escherichia coli species represents one of the best-studied model organisms, but also encompasses a variety of commensal and pathogenic strains that diversify by high rates of genetic change. We uniformly (re-) annotated the genomes of 20 commensal and pathogenic E. coli strains and one strain of E. fergusonii (the closest E. coli related species), including seven that we sequenced to completion. Within the approximately 18,000 families of orthologous genes, we found approximately 2,000 common to all strains. Although recombination rates are much higher than mutation rates, we show, both theoretically and using phylogenetic inference, that this does not obscure the phylogenetic signal, which places the B2 phylogenetic group and one group D strain at the basal position. Based on this phylogeny, we inferred past evolutionary events of gain and loss of genes, identifying functional classes under opposite selection pressures. We found an important adaptive role for metabolism diversification within group B2 and Shigella strains, but identified few or no extraintestinal virulence-specific genes, which could render difficult the development of a vaccine against extraintestinal infections. Genome flux in E. coli is confined to a small number of conserved positions in the chromosome, which most often are not associated with integrases or tRNA genes. Core genes flanking some of these regions show higher rates of recombination, suggesting that a gene, once acquired by a strain, spreads within the species by homologous recombination at the flanking genes. Finally, the genome's long-scale structure of recombination indicates lower recombination rates, but not higher mutation rates, at the terminus of replication. The ensuing effect of background selection and biased gene conversion may thus explain why this region is A+T-rich and shows high sequence divergence but low sequence polymorphism. Overall, despite a very high gene flow, genes co-exist in an organised genome.

A carbon monoxide-releasing molecule (CORM-3) exerts bactericidal activity against Pseudomonas aeruginosa and improves survival in an animal model of bacteraemia.

The search for new molecules to fight Pseudomonas aeruginosa is of paramount importance. Carbon monoxide (CO) is known to act as an effective inhibitor of the respiratory chain in P. aeruginosa, but the practical use of this gas as an antibacterial molecule is hampered by its toxicity and difficulty to manipulate. Here, we show that a water-soluble CO releaser (CORM-3) possesses bactericidal properties against laboratory and antibiotic-resistant P. aeruginosa. CORM-3 reduced the bacterial count by 4 logs 180 min after in vitro treatment. CORM-3-treated bacteria had a lower O(2) consumption than vehicle-treated bacteria, and the decrease in O(2) consumption temporally preceded the bactericidal action of CORM-3. These results support the hypothesis that the antimicrobial effect of CORM-3 is mediated by an interaction of CO liberated by the carrier with the bacterial respiratory chain. The antibacterial effect occurred at concentrations of CORM-3 that are 50-fold lower than toxic concentrations for eukaryotic cells. CORM-3 treatment compared to vehicle treatment decreased bacterial counts in the spleen and increased survival in immunocompetent and immunosuppressed mice following P. aeruginosa bacteremia. Our results suggest that CORMs could form the basis for developing a new therapeutic strategy against P. aeruginosa-induced infection.

Environmental signals implicated in Dr fimbriae release by pathogenic Escherichia coli.

Afa/Dr diffusely adhering Escherichia coli have been shown to cause urinary tract infections and enteric infections. Virulence of Dr-positive IH11128 bacteria is associated with the presence of Dr fimbriae. In this report, we show for the first time that the Dr fimbriae are released in the extracellular medium in response to multiple environmental signals. Production and secretion of Dr fimbriae are clearly thermoregulated. A comparison of the amounts of secreted fimbriae showed that the secretion is drastically increased during anaerobic growth in minimal medium. The effect of anaerobiosis on secretion seemed to depend on both the growth phase and the culture medium. The secretion was maximal during the logarithmic-phase growth and corresponded to 27 and 57% of total Dr fimbriae produced by bacteria grown in mineral medium+glucose and LB broth, respectively. Thus, the anaerobic environment of the colon would favour the secretion of Dr fimbriae during bacterial multiplication. The controlled release of the Dr fimbriae, which is carried out in the absence of cellular lysis, appears independent of the action of proteases or a process of maturation. The mechanism employed in the liberation of Dr fimbriae thus seems different from that described for the adhesins FHA and Hap of Bordetella pertussis and Haemophilus influenzae.

On the relationship between the physiological state of bacteria and rapid enzymatic assays of fecal coliforms in the environment.

Culturable cells and non-culturable cells of fecal coliforms, obtained by irradiation at 312 nm were submitted to the combined stress conditions of salinity and starvation. After 14 days, beta-galactosidase activity of UV-irradiated cells was at least twice the value of non-irradiated cells. UV-irradiated cells thus contribute more than non-irradiated cells to the enzyme assay after incubation in saline water. This finding is essential for the interpretation of quantitative investigations into the environment using enzymatic methods.

Are UV-induced nonculturable Escherichia coli K-12 cells alive or dead?

Cells that have lost the ability to grow in culture could be defined operationally as either alive or dead depending on the method used to determine cell viability. As a consequence, the interpretation of the state of 'nonculturable' cells is often ambiguous. Escherichia coli K12 cells inactivated by UV-irradiation with a low (UV1) and a high (UV2) dose were used as a model of nonculturable cells. Cells inactivated by the UV1 dose lost 'culturability' but they were not lysed and maintained the capacity to respond to nutrient addition by protein synthesis and cell wall synthesis. The cells also retained both a high level of glucose transport and the capacity for metabolizing glucose. Moreover, during glucose incorporation, UV1-treated cells showed the capacity to respond to aeration conditions modifying their metabolic flux through the Embden-Meyerhof and pentose-phosphate pathways. However, nonculturable cells obtained by irradiation with the high UV2 dose showed several levels of metabolic imbalance and retained only residual metabolic activities. Nonculturable cells obtained by irradiation with UV1 and UV2 doses were diagnosed as active and inactive (dying) cells, respectively.

Escherichia coli response to exogenous pyrophosphate and analogs.

The addition of exogenous pyrophosphate increases the growth yield and cAMP synthesis in stationary phase when Escherichia coli is grown in minimal medium. Pyrophosphate increases the yield by altering the enterobactin uptake system. We studied the physiological effects and examined how the E. coli transcriptome was modified when two structural analogs of pyrophosphate were added to the growth medium. Methylenediphosphonic acid or a high concentration of iron had the same positive effects as pyrophosphate on growth yield, cAMP synthesis and the repression of Fur-regulated genes. In contrast, imidodiphosphate did not affect these cellular processes significantly. The transcriptome modifications generated by pyrophosphate or methylenediphosphonic acid were more similar than those generated by imidodiphosphate or excess iron. The transcriptome data also indicated that processes other than iron uptake might be involved in the cellular response to exogenous pyrophosphate or methylenediphosphonic acid.

Accumulation of poly(3-hydroxybutyrate) from octanoate in different pseudomonas belonging to the rRNA homology group I.

It is admitted that one of the characteristics of pseudomonads is their inability to accumulate poly(3-hydroxybutyrate). In this paper, we show that poly(3-hydroxyoctanoate) synthesis is restricted to Pseudomonas rRNA homology group I, which includes both fluorescent and nonfluorescent species. However, within the genus Pseudomonas, the P. aeruginosa complex can be subdivided into two groups: the "P. aeruginosa group", which includes P. aeruginosa, P. alcaligenes, P. citronellolis, P. mendocina, produce poly(3-hydroxyoctanoate) from octanoate and the "P. oleovorans group" which includes the type strain of P. oleovorans, P. pseudoalcaligenes and two Pseudomonas sp., produce poly(3-hydroxybutyrate) during cultivation on octanoate. Strain GPo1 (ATCC 29347) formely identified as P. oleovorans and known to produce various medium-side-chain PHAs such as poly(3-hydroxyoctanoate) has been reclassified in the P. putida complex.