Escherichia coli O157:H7 and Other E. coli Strains Share Physiological Properties Associated with Intestinal Colonization

Escherichia coli isolates (72 commensal and 10 O157:H7 isolates) were compared with regard to physiological and growth parameters related to their ability to survive and persist in the gastrointestinal tract and found to be similar. We propose that nonhuman hosts in E. coli O157:H7 strains function similarly to other E. coli strains in regard to attributes relevant to gastrointestinal colonization.Escherichia coli is well known for its ecological versatility (15). A life cycle which includes both gastrointestinal and environmental stages has been stressed by both Savageau (15) and Adamowicz et al. (1). The gastrointestinal stage would be subjected to acid and detergent stress. The environmental stage is implicit in E. coli having transport systems for fungal siderophores (4) as well as pyrroloquinoline quinone-dependent periplasmic glucose utilization (1) because their presence indicates evolution in a location containing fungal siderophores and pyrroloquinoline quinone (1).Since its recognition as a food-borne pathogen, there have been numerous outbreaks of food-borne infection due to E. coli O157:H7, in both ground beef and vegetable crops (6, 13). Cattle are widely considered to be the primary reservoir of E. coli O157:H7 (14), but E. coli O157:H7 does not appear to cause disease in cattle. To what extent is E. coli O157:H7 physiologically unique compared to the other naturally occurring E. coli strains? We feel that the uniqueness of E. coli O157:H7 should be evaluated against a backdrop of other wild-type E. coli strains, and in this regard, we chose the 72-strain ECOR reference collection originally described by Ochman and Selander (10). These strains were chosen from a collection of 2,600 E. coli isolates to provide diversity with regard to host species, geographical distribution, and electromorph profiles at 11 enzyme loci (10).In our study we compared the 72 strains of the ECOR collection against 10 strains of E. coli O157:H7 and six strains of E. coli which had been in laboratory use for many years (Table ​(Table1).1). The in vitro comparisons were made with regard to factors potentially relevant to the bacteria''s ability to colonize animal guts, i.e., acid tolerance, detergent tolerance, and the presence of the Entner-Doudoroff (ED) pathway (Table ​(Table2).2). Our longstanding interest in the ED pathway (11) derives in part from work by Paul Cohen''s group (16, 17) showing that the ED pathway is important for E. coli colonization of the mouse large intestine. Growth was assessed by replica plating 88 strains of E. coli under 40 conditions (Table ​(Table2).2). These included two LB controls (aerobic and anaerobic), 14 for detergent stress (sodium dodecyl sulfate [SDS], hexadecyltrimethylammonium bromide [CTAB], and benzalkonium chloride, both aerobic and anaerobic), 16 for acid stress (pH 6.5, 6.0, 5.0, 4.6, 4.3, 4.2, 4.1, and 4.0), four for the ability to grow in a defined minimal medium (M63 glucose salts with and without thiamine), and four for the presence or absence of a functional ED pathway (M63 with gluconate or glucuronate). All tests were done with duplicate plates in two or three separate trials. The data are available in Tables S1 to S14 in the supplemental material, and they are summarized in Table ​Table22.

TABLE 1.

E. coli strains used in this study

Parameters Influencing Baseline HIV-1 Genotypic Tropism Testing Related to Clinical Outcome in Patients on Maraviroc

ObjectivesWe analysed the impact of different parameters on genotypic tropism testing related to clinical outcome prediction in 108 patients on maraviroc (MVC) treatment.Methods87 RNA and 60 DNA samples were used. The viral tropism was predicted using the geno2pheno_[coreceptor] and T-CUP tools with FPR cut-offs ranging from 1%-20%. Additionally, 27 RNA and 28 DNA samples were analysed in triplicate, 43 samples with the ESTA assay and 45 with next-generation sequencing. The influence of the genotypic susceptibility score (GSS) and 16 MVC-resistance mutations on clinical outcome was also studied.ResultsConcordance between single-amplification testing compared to ESTA and to NGS was in the order of 80%. Concordance with NGS was higher at lower FPR cut-offs. Detection of baseline R5 viruses in RNA and DNA samples by all methods significantly correlated with treatment success, even with FPR cut-offs of 3.75%-7.5%. Triple amplification did not improve the prediction value but reduced the number of patients eligible for MVC. No influence of the GSS or MVC-resistance mutations but adherence to treatment, on the clinical outcome was detected.ConclusionsProviral DNA is valid to select candidates for MVC treatment. FPR cut-offs of 5%-7.5% and single amplification from RNA or DNA would assure a safe administration of MVC without excluding many patients who could benefit from this drug. In addition, the new prediction system T-CUP produced reliable results.     

Independent external validation of nomograms for predicting risk of low-trauma fracture and hip fracture

Background

A set of nomograms based on the Dubbo Osteoporosis Epidemiology Study predicts the five- and ten-year absolute risk of fracture using age, bone mineral density and history of falls and low-trauma fracture. We assessed the discrimination and calibration of these nomograms among participants in the Canadian Multicentre Osteoporosis Study.

Methods

We included participants aged 55–95 years for whom bone mineral density measurement data and at least one year of follow-up data were available. Self-reported incident fractures were identified by yearly postal questionnaire or interview (years 3, 5 and 10). We included low-trauma fractures before year 10, except those of the skull, face, hands, ankles and feet. We used a Cox proportional hazards model.

Results

Among 4152 women, there were 583 fractures, with a mean follow-up time of 8.6 years. Among 1606 men, there were 116 fractures, with a mean follow-up time of 8.3 years. Increasing age, lower bone mineral density, prior fracture and prior falls were associated with increased risk of fracture. For low-trauma fractures, the concordance between predicted risk and fracture events (Harrell C) was 0.69 among women and 0.70 among men. For hip fractures, the concordance was 0.80 among women and 0.85 among men. The observed fracture risk was similar to the predicted risk in all quintiles of risk except the highest quintile of women, where it was lower. The net reclassification index (19.2%, 95% confidence interval [CI] 6.3% to 32.2%), favours the Dubbo nomogram over the current Canadian guidelines for men.

Interpretation

The published nomograms provide good fracture-risk discrimination in a representative sample of the Canadian population.Current recommendations for the treatment of osteoporosis are in transition. The T-score-based definition of osteoporosis and osteopenia by the expert committee of the World Health Organization on bone mineral density has been used in many guidelines to set intervention thresholds for treatment. However, studies have consistently reported that the highest number of fractures in a given population occurs in those with osteopenic or normal bone mineral density.^1,2 In fact, the National Osteoporosis Foundation has singled out people with osteopenic bone mineral density as a population in which assessment for fracture risk is merited.³Nevertheless, appropriate prevention and treatment strategies for such people are uncertain.⁴ Recent developments include the assessment of absolute fracture risk based on bone mineral density and other risk factors. Current Canadian methodology determines categorical risk based on age, sex, T-score, fracture history and glucocorticoid use.⁵ These criteria were derived from Swedish data, but have been assessed and validated in a cohort of Manitoba women.⁶ Newer nomograms based on the Australian cohort of the Dubbo Osteoporosis Epidemiology Study⁷ are now available for the calculation of low-trauma hip fracture⁸ and any fracture.⁹ These nomograms provide continuous estimates for five- and 10-year absolute fracture risk in both men and women (available at http://fractureriskcalculator.com). The use of factors in addition to bone mineral density may provide a better assessment of fracture risk for people who are near the T-score thresholds and facilitate decisions regarding therapeutic intervention.A key step in the development of any prediction model is the assessment of its validity.¹⁰ The aim of our study was to assess the performance of the Australian-derived nomogram among community-dwelling Canadians aged 55–95 years old. The first part of this assessment was a comparison of the nomogram model using the same variables, but using data from a Canadian population — participants in the Canadian Multicentre Osteoporosis Study (www.camos.org). The second part involved computing the calibration and discrimination of the nomogram in a Canadian cohort. The final part was comparison of the new assessments with the existing Canadian risk classification system.     

Temporally regulated expression of Cre recombinase in neural stem cells

The use of mouse gene targeting to study molecules important in neural development is oftentimes impaired by early embryonic lethality. In order to address later roles for such molecules, specifically in neural stem cells, we generated transgenic mice that express both the tetracycline-inducible molecule rtTA-M2 and GFP under the control of the neural precursor specific form of nestin. Developmental analysis of these mice demonstrates that GFP expression is exclusive to the neural tube. Adult expression of GFP is seen only in known areas of adult neurogenesis, namely, the subventricular zone and the dentate gyrus. When crossed with a second transgenic mouse (TetOp-Cre) that expresses the Cre recombinase under the control of the tetracycline responsive promotor, we demonstrate temporal induction of Cre in bigenic animals exposed to doxycycline. We further demonstrate the feasibility of this approach by using the ROSA-26 reporter mouse to mediate recombination in neural precursor cells.     

Nucleotide sequence of the head assembly gene cluster of bacteriophage L and decoration protein characterization

The temperate Salmonella enterica bacteriophage L is a close relative of the very well studied bacteriophage P22. In this study we show that the L procapsid assembly and DNA packaging genes, which encode terminase, portal, scaffold, and coat proteins, are extremely close relatives of the homologous P22 genes (96.3 to 99.1% identity in encoded amino acid sequence). However, we also identify an L gene, dec, which is not present in the P22 genome and which encodes a protein (Dec) that is present on the surface of L virions in about 150 to 180 molecules/virion. We also show that the Dec protein is a trimer in solution and that it binds to P22 virions in numbers similar to those for L virions. Its binding dramatically stabilizes P22 virions against disruption by a magnesium ion chelating agent. Dec protein binds to P22 coat protein shells that have expanded naturally in vivo or by sodium dodecyl sulfate treatment in vitro but does not bind to unexpanded procapsid shells. Finally, analysis of phage L restriction site locations and a number of patches of nucleotide sequence suggest that phages ST64T and L are extremely close relatives, perhaps the two closest relatives that have been independently isolated to date among the lambdoid phages.     

Structure and secretion of CofJ,a putative colonization factor of enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) colonize the human gut, causing severe cholera‐like diarrhoea. ETEC utilize a diverse array of pili and fimbriae for host colonization, including the Type IVb pilus CFA/III. The CFA/III pilus machinery is encoded on the cof operon, which is similar in gene sequence and synteny to the tcp operon that encodes another Type IVb pilus, the Vibrio cholerae toxin co‐regulated pilus (TCP). Both pilus operons possess a syntenic gene encoding a protein of unknown function. In V. cholerae, this protein, TcpF, is a critical colonization factor secreted by the TCP apparatus. Here we show that the corresponding ETEC protein, CofJ, is a soluble protein secreted via the CFA/III apparatus. We present a 2.6 Å resolution crystal structure of CofJ, revealing a large β‐sandwich protein that bears no sequence or structural homology to TcpF. CofJ has a cluster of exposed hydrophobic side‐chains at one end and structural homology to the pore‐forming proteins perfringolysin O and α‐haemolysin. CofJ binds to lipid vesicles and epithelial cells, suggesting a role in membrane attachment during ETEC colonization.     

The Diversity and Evolution of Wolbachia Ankyrin Repeat Domain Genes

Ankyrin repeat domain-encoding genes are common in the eukaryotic and viral domains of life, but they are rare in bacteria, the exception being a few obligate or facultative intracellular Proteobacteria species. Despite having a reduced genome, the arthropod strains of the alphaproteobacterium Wolbachia contain an unusually high number of ankyrin repeat domain-encoding genes ranging from 23 in wMel to 60 in wPip strain. This group of genes has attracted considerable attention for their astonishing large number as well as for the fact that ankyrin proteins are known to participate in protein-protein interactions, suggesting that they play a critical role in the molecular mechanism that determines host-Wolbachia symbiotic interactions. We present a comparative evolutionary analysis of the wMel-related ankyrin repeat domain-encoding genes present in different Drosophila-Wolbachia associations. Our results show that the ankyrin repeat domain-encoding genes change in size by expansion and contraction mediated by short directly repeated sequences. We provide examples of intra-genic recombination events and show that these genes are likely to be horizontally transferred between strains with the aid of bacteriophages. These results confirm previous findings that the Wolbachia genomes are evolutionary mosaics and illustrate the potential that these bacteria have to generate diversity in proteins potentially involved in the symbiotic interactions.     

The use of different eye regions in the mantis shrimp Hemisquilla californiensis Stephenson, 1967 (Crustacea: Stomatopoda) for detecting objects

A behavioral assay was used to assess the ability of the stomatopod Hemisquilla californiensis to perceive and respond to a moving target under different wavelengths and intensities of light illumination. Subjects responded to targets rotating horizontally across their visual field by a brief startle response of their eyes or antennules but did not track the targets. Under white light responses were elicited down to a light intensity of 0.9 μW cm^− 2. Responses were seen in blue light at intensities as low as 0.5 μW cm^− 2, and in green light down to 1.0 μW cm^− 2. The animals were less sensitive to red light, with no responses seen at intensities below 3.0 μW cm^− 2. Subjects did not respond to the targets at all under infrared light. This response pattern mirrors the computed sensitivity spectrum of ommatidia in the species' peripheral hemispheres but not that in most of the central bands. We conclude that this species uses the monochromatic vision in the peripheral hemispheres of its eyes to recognize objects and that the sharply tuned color receptors of the central band serve to add supplemental information if light conditions allow.     

Geographic variation in cardiovascular inflammation among healthy women in the Women's Health Study

Background

Geographic variation in traditional cardiovascular disease (CVD) risk factors has been observed among women in the US. It is not known whether state-level variation in cardiovascular inflammation exists or could be explained by traditional clinical risk factors and behavioral lifestyle factors.

Methods and Results

We used multilevel linear regression to estimate state-level variation in inflammatory biomarker patterns adjusted for clinical and lifestyle characteristics among 26,029 women free of CVD. Participants derived from the Women''s Health Study, a national cohort of healthy middle-aged and older women. Inflammatory biomarker patterns (plasma levels of high-sensitivity C-reactive protein (hsCRP), soluble intercellular adhesion molecule-1 (sICAM-1), and fibrinogen) were compared to state-level patterns of traditional CVD risk factors and global risk scores. We found that all three inflammatory biomarkers exhibited significant state-level variation including hsCRP (lowest vs. highest state median 1.3 mg/L vs. 2.7 mg/L, unadjusted random effect estimate 1^st to 99^th percentile range for log hsCRP 0.52, p<.001), sICAM-1 (325 ng/ml vs. 366ng/ml, unadjusted random effect estimate 1^st to 99^th percentile range 0.44, p<.001), and fibrinogen (322 mg/dL vs. 367 mg/dL, unadjusted random effect estimate 1^st to 99^th percentile range 0.41, p = .001). Neither demographic, clinical or lifestyle characteristics explained away state-level effects in biomarker patterns. Southern and Appalachian states (Arkansas, West Virginia) had the highest inflammatory biomarker values. Regional geographic patterns of traditional CVD risk factors and risk scores did not completely overlap with biomarkers of inflammation.

Conclusions

There is state-level geographic variation in inflammatory biomarkers among otherwise healthy women that cannot be completely attributed to traditional clinical risk factors or lifestyle characteristics. Future research should aim to identify additional factors that may explain geographic variation in biomarkers of inflammation among healthy women.