Exploring Social Determinants of Health in Healthy Aging Among Older Adults: A Qualitative Study.

BACKGROUND: The U.S. population is aging and diversifying. Older Black Americans comprise the largest racial minority group and experience greater disability than White Americans. OBJECTIVES: Within a long-standing, community-based research partnership, we explored the determinants of healthy aging in Flint Michigan, a low-income, predominantly Black American community recovering from a water crisis. METHODS: Focus groups were conducted among older adults residing in Flint, Michigan. A grounded theory approach and constant comparison method was utilized for data analysis. RESULTS: Five focus groups were conducted with 49 total participants. We identified four themes that impacted healthy aging: economic instability, health care access and quality, neighborhood and built environment, and social and community context. Economic instability heavily influenced the other themes. CONCLUSIONS: Economic instability is a barrier to healthy aging. As a result, we are testing an innovative cross-sector partnership combining older adult affordable housing and health care.

Psychological Support Strategies for Adults With Type 2 Diabetes in a Very Low-Carbohydrate Web-Based Program: Randomized Controlled Trial.

BACKGROUND: A very low-carbohydrate (VLC) nutritional strategy may improve glycemic control and weight loss in adults with type 2 diabetes (T2D). However, the supplementary behavioral strategies that might be able to improve outcomes using this nutritional strategy are uncertain. OBJECTIVE: This study aims to compare the impact of adding 3 different supplementary behavioral strategies to a web-based VLC diet intervention. To our knowledge, this is the first trial to randomize participants to different frequencies of dietary self-monitoring. METHODS: The study included 112 overweight adults with T2D (hemoglobin A1c ≥6.5%) taking no antiglycemic medications or only metformin. They received a remotely delivered 12-month VLC diet intervention. Participants were randomly assigned through a full factorial 2×2×2 design to supplementary strategies: either daily or monthly dietary self-monitoring, either mindful eating training or not, and either positive affect skills training or not. Our research goal was to determine whether 3 different supplemental strategies had at least a medium effect size (Cohen d=0.5). RESULTS: Overall, the VLC intervention led to statistically significant improvements in glycemic control (-0.70%, 95% CI -1.04% to -0.35%; P<.001), weight loss (-6.82%, 95% CI -8.57% to -5.08%; P<.001), and depressive symptom severity (Cohen d -0.67, 95% CI -0.92 to -0.41; P<.001). Furthermore, 30% (25/83) of the participants taking metformin at baseline reduced or discontinued their metformin. Only 1 Cohen d point estimate reached 0.5; daily (vs monthly) dietary self-monitoring had a worse impact on depressive symptoms severity (Cohen d=0.47, 95% CI -0.02 to 0.95; P=.06). None of the strategies had a statistically significant effect on outcomes. For changes in our primary outcome, hemoglobin A1c, the daily (vs monthly) dietary self-monitoring impact was 0.42% (95% CI -0.28% to 1.12%); for mindful eating, it was -0.47% (95% CI -1.15% to 0.22%); and for positive affect, it was 0.12% (95% CI -0.57% to 0.82%). Other results for daily (vs monthly) dietary self-monitoring were mixed, suggesting an increase in weight (0.98%) and depressive symptoms (Cohen d=0.47), less intervention satisfaction (Cohen d=-0.20), more sessions viewed (3.02), and greater dietary adherence (Cohen d=0.24). For mindful eating, the results suggested a benefit for dietary adherence (Cohen d=0.24) and intervention satisfaction (Cohen d=0.30). For positive affect, the results suggested a benefit for depressive symptoms (Cohen d=-0.32), the number of sessions viewed (3.68), dietary adherence (Cohen d=0.16), and intervention satisfaction (Cohen d=0.25). CONCLUSIONS: Overall, our results support the use of a VLC diet intervention in adults with T2D. The addition of monthly (not daily) dietary self-monitoring, mindful eating, and positive affect skills training did not show a definitive benefit, but it is worth further testing. TRIAL REGISTRATION: ClinicalTrials.gov NCT03037528; https://clinicaltrials.gov/ct2/show/NCT03037528.

Comparing Very Low-Carbohydrate vs DASH Diets for Overweight or Obese Adults With Hypertension and Prediabetes or Type 2 Diabetes: A Randomized Trial.

PURPOSE: Adults with a triple multimorbidity (hypertension, prediabetes or type 2 diabetes, and overweight or obesity), are at increased risk of serious health complications, but experts disagree on which dietary patterns and support strategies should be recommended. METHODS: We randomized 94 adults from southeast Michigan with this triple multimorbidity using a 2 × 2 diet-by-support factorial design, comparing a very low-carbohydrate (VLC) diet vs a Dietary Approaches to Stop Hypertension (DASH) diet, as well as comparing results with and without multicomponent extra support (mindful eating, positive emotion regulation, social support, and cooking). RESULTS: Using intention-to-treat analyses, compared with the DASH diet, the VLC diet led to greater improvement in estimated mean systolic blood pressure (-9.77 mm Hg vs -5.18 mm Hg; P = .046), greater improvement in glycated hemoglobin (-0.35% vs -0.14%; P = .034), and greater improvement in weight (-19.14 lb vs -10.34 lb; P = .0003). The addition of extra support did not have a statistically significant effect on outcomes. CONCLUSIONS: For adults with hypertension, prediabetes or type 2 diabetes, and overweight or obesity, the VLC diet resulted in greater improvements in systolic blood pressure, glycemic control, and weight over a 4-month period compared with the DASH diet. These findings suggest that larger trials with longer follow-up are warranted to determine whether the VLC diet might be more beneficial for disease management than the DASH diet for these high-risk adults.

Testing a very low-carbohydrate adaption of the Diabetes Prevention Program among adults with prediabetes: study protocol for the Lifestyle Education about prediabetes (LEAP) trial.

BACKGROUND: The Center for Disease Control and Prevention's National Diabetes Prevention Program (NDPP) aims to help individuals with prediabetes avoid progression to type 2 diabetes mellitus (T2DM) through weight loss. Specifically, the NDPP teaches individuals to follow a low-fat, calorie-restricted diet and to engage in regular physical activity to achieve ≥ 5% body weight loss. Most NDPP participants, however, do not achieve this weight loss goal, and glycemic control remains largely unchanged. One promising opportunity to augment the NDPP's weight loss and glycemic effectiveness may be to teach participants to follow a very low-carbohydrate diet (VLCD), which can directly reduce post-prandial glycemia and facilitate weight loss by reducing circulating insulin and enabling lipolysis. To date, there have been no high-quality, randomized controlled trials to test whether a VLCD can prevent progression to T2DM among individuals with prediabetes. The aim of this study is to test the effectiveness of a VLCD version the NDPP (VLC-NDPP) versus the standard NDPP. We hypothesize the VLC-NDPP will demonstrate greater improvements in weight loss and glycemic control. METHODS: We propose to conduct a 12-month, 1:1, randomized controlled trial that will assign 300 adults with overweight or obesity and prediabetes to either the NDPP or VLC-NDPP. The primary outcome will be glycemic control as measured by change in hemoglobin A1c (HbA1c) from baseline to 12 months. Secondary outcomes will include percent body weight change and changes in glycemic variability, inflammatory markers, lipids, and interim HbA1c. We will evaluate progression to T2DM and initiation of anti-hyperglycemic agents. We will conduct qualitative interviews among a purposive sample of participants to explore barriers to and facilitators of dietary adherence. The principal quantitative analysis will be intent-to-treat using hierarchical linear mixed effects models to assess differences over time. DISCUSSION: The NDPP is the dominant public health strategy for T2DM prevention. Changing the program's dietary advice to include a carbohydrate-restricted eating pattern as an alternative option may enhance the program's effectiveness. If the VLC-NDPP shows promise, this trial would be a precursor to a multi-site trial with incident T2DM as the primary outcome. TRIAL REGISTRATION: NCT05235425. Registered February 11, 2022.

Impact of an online multicomponent very-low-carbohydrate program in women with polycystic ovary syndrome: a pilot study.

OBJECTIVE: To study the impact of a very-low-carbohydrate (VLC) diet for 16 weeks in overweight or obese women with polycystic ovary syndrome (PCOS). DESIGN: Single-arm prospective pilot study. SETTING: We recruited participants using medical records from an academic medical center. PATIENTS: Twenty-nine overweight or obese women (body mass index, 25-50 kg/m2) with PCOS. INTERVENTIONS: We taught participants to follow a VLC diet and provided information about a variety of behavioral skills including mindfulness and positive affect using an online 16-week intervention. MAIN OUTCOME MEASURES: Changes in body weight, glycated hemoglobin, and PCOS-related quality of life. RESULTS: The intervention led to positive health outcomes including decreases in percent weight (mean difference = -7.67, SD = 6.10) and glycated hemoglobin level (mean difference = -0.21%, SD = 0.27), an increase in sex hormone binding globulin level (mean difference = 9.24 nmol/L, SD = 16.34), and increases in PCOS-related quality of life measures, including menstrual predictability (mean difference = 2.10, SD = 2.76) and body hair (mean difference = 1.14, SD = 1.04). The low-density lipoprotein cholesterol level increased (mean difference = 0.23 mmol/L, SD = 0.49). CONCLUSIONS: The results suggest that a VLC dietary intervention has potential to promote both weight loss and glycemic control in overweight and obese adults with PCOS, two key components in the prevention of type 2 diabetes. TRIAL REGISTRATION NUMBER: NCT03987854.

The Virulence of Escherichia coli O157:H7 Isolates in Mice Depends on Shiga Toxin Type 2a (Stx2a)-Induction and High Levels of Stx2a in Stool.

In this study we compared nine Shiga toxin (Stx)-producing Escherichia coli O157:H7 patient isolates for Stx levels, stx-phage insertion site(s), and pathogenicity in a streptomycin (Str)-treated mouse model. The strains encoded stx2a, stx1a and stx2a, or stx2a and stx2c. All of the strains elaborated 105-106 cytotoxic doses 50% (CD50) into the supernatant after growth in vitro as measured on Vero cells, and showed variable levels of increased toxin production after growth with sub-inhibitory levels of ciprofloxacin (Cip). The stx2a+stx2c+ isolates were 90-100% lethal for Str-treated BALB/c mice, though one isolate, JH2013, had a delayed time-to-death. The stx2a+ isolate was avirulent. Both an stx2a and a recA deletion mutant of one of the stx2a+stx2c+ strains, JH2010, exhibited at least a three-log decrease in cytotoxicity in vitro and both were avirulent in the mice. Stool from Str-treated mice infected with the highly virulent isolates were 10- to 100-fold more cytotoxic than feces from mice infected with the clinical isolate, JH2012, that made only Stx2a. Taken together these findings demonstrate that the stx2a-phage from JH2010 induces to higher levels in vivo than does the phage from JH2012. The stx1a+stx2a+ clinical isolates were avirulent and neutralization of Stx1 in stool from mice infected with those strains indicated that the toxin produced in vivo was primarily Stx1a. Treatment of mice infected with Stx1a+Stx2a+ isolates with Cip resulted in an increase in Stx2a production in vivo and lethality in the mice. Our data suggest that high levels of Stx2a in stool are predictive of virulence in mice.

The Role of the AggR Regulon in the Virulence of the Shiga Toxin-Producing Enteroaggregative Escherichia coli Epidemic O104:H4 Strain in Mice.

An O104:H4 Shiga toxin (Stx)-producing enteroaggregative Escherichia coli (EAEC) strain caused a large outbreak of bloody diarrhea and the hemolytic uremic syndrome in 2011. We previously developed an ampicillin (Amp)-treated C57BL/6 mouse model to measure morbidity (weight loss) and mortality of mice orally infected with the prototype Stx-EAEC strain C227-11. Here, we hypothesized that mice fed C227-11 cured of the pAA plasmid or deleted for individual genes on that plasmid would display reduced virulence compared to animals given the wild-type (wt) strain. C227-11 cured of the pAA plasmid or deleted for the known pAA-encoded virulence genes aggR, aggA, sepA, or aar were fed to Amp-treated C57BL/6 mice at doses of 1010-1011CFU. Infected animals were then either monitored for morbidity and lethality for 28 days or euthanized to determine intestinal pathology and colonization levels at selected times. The pAA-cured, aggR, and aggA mutants of strain C227-11 all showed reduced colonization at various intestinal sites. However, the aggR mutant was the only mutant attenuated for virulence as it showed both reduced morbidity and mortality. The aar mutant showed increased expression of the aggregative adherence fimbriae (AAF) and caused greater systemic effects in infected mice when compared to the C227-11 wt strain. However, unexpectedly, both the aggA and aar mutants displayed increased weight loss compared to wt. The sepA mutant did not exhibit altered morbidity or mortality in the Amp-treated mouse model compared to wt. Our data suggest that the increased morbidity due to the aar mutant could possibly be via an effect on expression of an as yet unknown virulence-associated factor under AggR control.

Stroke Ready: a multi-level program that combines implementation science and community-based participatory research approaches to increase acute stroke treatment: protocol for a stepped wedge trial.

BACKGROUND: Post-stroke disability is common, costly, and projected to increase. Acute stroke treatments can substantially reduce post-stroke disability, but few patients take advantage of these cost-effective treatments. Practical, cost-efficient, and sustainable interventions to address underutilized acute stroke treatments are currently lacking. In this context, we present the Stroke Ready project, a stepped wedge design, multi-level intervention that combines implementation science and community-based participatory research approaches to increase acute stroke treatments in the predominately African American community of Flint, Michigan, USA. METHODS: Guided by the Tailored Implementation of Chronic Disease (TICD) framework, we begin with optimization of acute stroke care in emergency departments, with particular attention given to our safety-net hospital partners. Then, we move to a community-wide, multi-faceted, stroke preparedness intervention, with workshops led by peer educators, over 2 years. Measures of engagement of the safety-net hospital and the feasibility and sustainability of the implementation strategy as well as community intervention reach, dose delivered, and satisfaction will be collected. The primary outcome is acute stroke treatment rates, which includes both intravenous tissue plasminogen activator, and endovascular treatment. The co-secondary outcomes are intravenous tissue plasminogen activator treatment rates and the proportion of stroke patients who arrive by ambulance. DISCUSSION: If successful, Stroke Ready will increase acute stroke treatment rates through emergency department and community level interventions. The stepped wedge design and process evaluation will provide insight into how Stroke Ready works and where it might work best. By exploring the relative effectiveness of the emergency department optimization and the community intervention, we will inform hospitals and communities as they determine how best to use their resources to optimize acute stroke care. TRIAL REGISTRATION: ClinicalTrials.gov Trial Identifier NCT03645590 .

Shiga Toxin Type 1a (Stx1a) Reduces the Toxicity of the More Potent Stx2a In Vivo and In Vitro.

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes foodborne outbreaks of bloody diarrhea. There are two major types of immunologically distinct Stxs: Stx1a and Stx2a. Stx1a is more cytotoxic to Vero cells than Stx2a, but Stx2a has a lower 50% lethal dose (LD50) in mice. Epidemiological data suggest that infections by STEC strains that produce only Stx2a progress more often to a life-threatening sequela of infection called hemolytic-uremic syndrome (HUS) than isolates that make Stx1a only or produce both Stx1a and Stx2a. In this study, we found that an E. coli O26:H11 strain that produces both Stx1a and Stx2a was virulent in streptomycin- and ciprofloxacin-treated mice and that mice were protected by administration of an anti-Stx2 antibody. However, we discovered that in the absence of ciprofloxacin, neutralization of Stx1a enhanced the virulence of the strain, a result that corroborated our previous finding that Stx1a reduces the toxicity of Stx2a by the oral route. We further found that intraperitoneal administration of the purified Stx1a B subunit delayed the mean time to death of mice intoxicated with Stx2a and reduced the cytotoxic effect of Stx2a on Vero cells. Taken together, our data suggest that Stx1a reduces both the pathogenicity of Stx2 in vivo and cytotoxicity in vitro.

Expression and contribution to virulence of each polysaccharide capsule of Bacillus cereus strain G9241.

Bacillus cereus strain G9241 was isolated from a patient with pneumonia who had an anthrax-like illness. Like Bacillus anthracis, the virulence of G9241 is dependent on two large plasmids. In G9241 those plasmids are pBCXO1 and pBC210. There is a multi-gene capsule locus on each of these virulence plasmids, and both capsules are produced by G9241 in vitro and in mice. The hasACB operon on pBCXO1 is responsible for production of a hyaluronic acid (HA) capsule. The locus on pBC210 encodes a putative tetrasaccharide (TS) capsule that assembles in a Wzy-dependent manner. We found that the pBC210 capsule locus is transcribed as two operons and identified the promoter regions responsible for transcription. We constructed isogenic mutants to assess the role of genes in the two TS capsule operons in production of the capsule. Spores of strains deficient in production of either the HA or TS capsule were inoculated subcutaneously or intranasally into A/J and C57BL/6 mice to determine the lethal dose 50% of each bacterial mutant by each route of infection. The loss of the HA capsule attenuated G9241 more than the loss of the TS capsule for both infection routes in both mouse strains. Overall, our data further characterize the unique TS capsule on pBC210 and demonstrate that the two capsules do not have the same impact on virulence of G9241.

Certhrax Is an Antivirulence Factor for the Anthrax-Like Organism Bacillus cereus Strain G9241.

Bacillus cereus G9241 caused a life-threatening anthrax-like lung infection in a previously healthy human. This strain harbors two large virulence plasmids, pBCXO1 and pBC210, that are absent from typical B. cereus isolates. The pBCXO1 plasmid is nearly identical to pXO1 from Bacillus anthracis and carries genes (pagA1, lef, and cya) for anthrax toxin components (protective antigen [called PA1 in G9241], lethal factor [LF], and edema factor [EF], respectively). The plasmid also has an intact hyaluronic acid capsule locus. The pBC210 plasmid has a tetrasaccharide capsule locus, a gene for a PA1 homolog called PA2 (pagA2), and a gene (cer) for Certhrax, an ADP-ribosyltransferase toxin that inactivates vinculin. LF, EF, and Certhrax require PA for entry into cells. In this study, we asked what role PA1, PA2, LF, and Certhrax play in the pathogenicity of G9241. To answer this, we generated isogenic deletion mutations in the targeted toxin gene components and then assessed the strains for virulence in highly G9241-susceptible (A/J) and moderately G9241-sensitive (C57BL/6) mice. We found that full virulence of G9241 required PA1 and LF, while PA2 contributed minimally to pathogenesis of G9241 but could not functionally replace PA1 as a toxin-binding subunit in vivo Surprisingly, we discovered that Certhrax attenuated the virulence of G9241; i.e., a Δcer Δlef mutant strain was more virulent than a Δlef mutant strain following subcutaneous inoculation of A/J mice. Moreover, the enzymatic activity of Certhrax contributed to this phenotype. We concluded that Certhrax acts as an antivirulence factor in the anthrax-like organism B. cereus G9241.

Place of care: from referral to specialist palliative care until death.

BACKGROUND: While there are many poorly standardised studies focusing on place of death, there are limited data on place(s) of care during the final stages of disease. AIM: This study aims to identify where patients are cared for in the interval from referral to specialist palliative care until death. METHODS: All patients who died while under the care of a specialist palliative care service over a 6-month period were considered. RESULTS: Of the 507 patients included, 255 (50.3%) were men and 428 (84.4%) had a malignant diagnosis. The mean referral-to-death interval was 70 days (SD 113, Range 1-838). The majority (n=281, 55.4%) received care in a single care setting-hospital (28.4%), home (21.5%), nursing home/community hospital (4.1%), hospice (1.4%)-and had a shorter mean referral-to-death interval. Most patients with more than one care setting spent three-quarters of their time in their normal place of residence. A total of 199 (39.3%) died in hospital, 131 (25.8%) in hospice, 131 (25.8%) at home (25.8%) and 46 (9.1%) in a nursing home/community hospital. Patients referred by a general practitioner (n=80 patients, 15.8%) were more likely to be cared for at home (p<0.001), and die at home (p<0.001). CONCLUSIONS: A significant number of patients received specialist palliative care across multiple care settings. Late referral is associated with a single domain of care. General practitioner involvement supports patient care and death at home. Place of care and ease of transfer between care settings may be better indicators of the quality of care we provide.

The roles of AtxA orthologs in virulence of anthrax-like Bacillus cereus G9241.

AtxA is a critical transcriptional regulator of plasmid-encoded virulence genes in Bacillus anthracis. Bacillus cereus G9241, which caused an anthrax-like infection, has two virulence plasmids, pBCXO1 and pBC210, that each harbor toxin genes and a capsule locus. G9241 also produces two orthologs of AtxA: AtxA1, encoded on pBCXO1, and AtxA2, encoded on pBC210. The amino acid sequence of AtxA1 is identical to that of AtxA from B. anthracis, while the sequences of AtxA1 and AtxA2 are 79% identical and 91% similar to one another. We found by qRT-PCR that AtxA1 and AtxA2 function as positive regulators of toxin (AtxA1) and capsule operon (both) transcription in G9241 and that a ΔatxA1 mutant produced lower levels of the anthrax toxins and no hyaluronic acid capsule. Deletion of atxA1 or atxA2 decreased the virulence of spores administered intranasally or subcutaneously to C57BL/6 mice but not to A/J mice, and deletion of both genes rendered spores avirulent in A/J mice. In addition, unlike AtxA1, AtxA2 did not form stable homomultimers in vitro, although AtxA1 and AtxA2 formed heterodimers. Our data show that AtxA1 is the primary regulator of G9241 virulence factor expression and that AtxA1 and AtxA2 are both required for full virulence.

Shiga Toxin (Stx) Type 1a Reduces the Oral Toxicity of Stx Type 2a.

BACKGROUND: Shiga toxin (Stx) is the primary virulence factor of Stx-producing Escherichia coli (STEC). STEC can produce Stx1a and/or Stx2a, which are antigenically distinct. However, Stx2a-producing STEC are associated with more severe disease than strains producing both Stx1a and Stx2a. METHODS AND RESULTS: To address the hypothesis that the reason for the association of Stx2a with more severe disease is because Stx2a crosses the intestinal barrier with greater efficiency that Stx1a, we covalently labeled Stx1a and Stx2a with Alexa Fluor 750 and determined the ex vivo fluorescent intensity of murine systemic organs after oral intoxication. Surprisingly, both Stxs exhibited similar dissemination patterns and accumulated in the kidneys. We next cointoxicated mice to determine whether Stx1a could impede Stx2a. Cointoxication resulted in increased survival and an extended mean time to death, compared with intoxication with Stx2a only. The survival benefit was dose dependent, with the greatest effect observed when 5 times more Stx1a than Stx2a was delivered, and was amplified when Stx1a was delivered 3 hours prior to Stx2a. Cointoxication with an Stx1a active site toxoid also reduced Stx2a toxicity. CONCLUSIONS: These studies suggest that Stx1a reduces Stx2a-mediated toxicity, a finding that may explain why STEC that produce only Stx2a are associated with more severe disease than strains producing Stx1a and Stx2a.

Virulence Potential of Activatable Shiga Toxin 2d-Producing Escherichia coli Isolates from Fresh Produce.

Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named "activation." Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice.

Mapping of genetic loci that modulate differential colonization by Escherichia coli O157:H7 TUV86-2 in advanced recombinant inbred BXD mice.

BACKGROUND: Shiga toxin (Stx)-producing E. coli (STEC) are responsible for foodborne outbreaks that can result in severe human disease. During an outbreak, differential disease outcomes are observed after infection with the same STEC strain. One question of particular interest is why some infected people resolve infection after hemorrhagic colitis whereas others progress to the hemolytic uremic syndrome (HUS). Host age and infection dose have been implicated; however, these parameters do not appear to fully account for all of the observed variation in disease severity. Therefore, we hypothesized that additional host genetic factors may play a role in progression to HUS. METHODS AND RESULTS: To mimic the genetic diversity in the human response to infection by STEC, we measured the capacity of an O157:H7 outbreak isolate to colonize mouse strains from the advanced recombinant inbred (ARI) BXD panel. We first infected the BXD parental strains C57BL/6 J (B6) and DBA/2 J (D2) with either 86-24 (Stx2a+) or TUV86-2, an Stx2a-negative isogenic mutant. Colonization levels were determined in an intact commensal flora (ICF) infection model. We found a significant difference in colonization levels between the parental B6 and D2 strains after infection with TUV86-2 but not with 86-24. This observation suggested that a host factor that may be masked by Stx2a affects O157:H7 colonization in some genetic backgrounds. We then determined the TUV86-2 colonization levels of 24 BXD strains in the ICF model. We identified several quantitative trait loci (QTL) associated with variation in colonization by correlation analyses. We found a highly significant QTL on proximal chromosome 9 (12.5-26.7 Mb) that strongly predicts variation in colonization levels and accounts for 15-20 % of variance. Linkage, polymorphism and co-citation analyses of the mapped region revealed 36 candidate genes within the QTL, and we identified five genes that are most likely responsible for the differential colonization. CONCLUSIONS: The identification of the QTL on chromosome 9 supports our hypothesis that individual genetic makeup affects the level of colonization after infection with STEC O157:H7.

Reduced Toxicity of Shiga Toxin (Stx) Type 2c in Mice Compared to Stx2d Is Associated with Instability of Stx2c Holotoxin.

Shiga toxin (Stx) is an AB5 ribotoxin made by Stx-producing Escherichia coli (STEC). These organisms cause diarrhea, hemorrhagic colitis and the hemolytic uremic syndrome. STEC make two types of Stxs, Stx1 and/or Stx2. Stx2 has one prototype (a) and six subtypes (b-g), but only STEC that make Stx2a, and/or Stx2c, or Stx2d are associated with severe disease. However, Stx2c is about 10-fold less toxic than Stx2d in vivo despite only two amino acid differences in the A subunit at positions 291 and 297. We made mutations at these two sites to create intermediate toxins between Stx2c and Stx2d, and determined the 50% cytotoxic dose on Vero cells before and after heat treatment, and the 50% lethal dose in mice of the toxins. We found that serine 291 was associated with increased toxicity in vivo and that either amino acid change from that in Stx2c to that in Stx2d increased heat stability. We also assessed the secondary structure of Stx2c and Stx2d by circular dichroism (CD) spectroscopy. The CD studies suggest that Stx2c has a less-ordered secondary structure than Stx2d. We conclude that both amino acids at positions 291 and 297 in Stx2c contribute to its decreased stability and in vivo toxicity compared to Stx2d.

Shiga toxin 2a and Enteroaggregative Escherichia coli--a deadly combination.

In 2011, a Shiga toxin (Stx) type 2a-producing enteroaggregative E. coli (EAEC) strain of serotype O104:H4 caused a large lethal outbreak in Northern Europe. Until recently, the pathogenic mechanisms explaining the high virulence of the strain have remained unclear. Our laboratories have shown that EAEC genes encoded on the pAA virulence plasmid, particularly the AggR-regulated AAF/I fimbriae, enhance inflammation and enable the outbreak strain to both adhere to epithelial cells and translocate Stx2a across the intestinal epithelium, possibly explaining the high incidence of the life threatening post-diarrheal sequelae of hemolytic uremic syndrome. Epidemiologic evidence supports a model of EAEC pathogenesis comprising the concerted action of multiple virulence factors along with induction of inflammation. Here, we suggest a model for the pathogenesis of the O104:H4 outbreak strain that includes contributions from EAEC alone, but incorporating additional injury induced by Stx2a.