Transmission of Vibrio cholerae Is Antagonized by Lytic Phage and Entry into the Aquatic Environment

Cholera outbreaks are proposed to propagate in explosive cycles powered by hyperinfectious Vibrio cholerae and quenched by lytic vibriophage. However, studies to elucidate how these factors affect transmission are lacking because the field experiments are almost intractable. One reason for this is that V. cholerae loses the ability to culture upon transfer to pond water. This phenotype is called the active but non-culturable state (ABNC; an alternative term is viable but non-culturable) because these cells maintain the capacity for metabolic activity. ABNC bacteria may serve as the environmental reservoir for outbreaks but rigorous animal studies to test this hypothesis have not been conducted. In this project, we wanted to determine the relevance of ABNC cells to transmission as well as the impact lytic phage have on V. cholerae as the bacteria enter the ABNC state. Rice-water stool that naturally harbored lytic phage or in vitro derived V. cholerae were incubated in a pond microcosm, and the culturability, infectious dose, and transcriptome were assayed over 24 h. The data show that the major contributors to infection are culturable V. cholerae and not ABNC cells. Phage did not affect colonization immediately after shedding from the patients because the phage titer was too low. However, V. cholerae failed to colonize the small intestine after 24 h of incubation in pond water—the point when the phage and ABNC cell titers were highest. The transcriptional analysis traced the transformation into the non-infectious ABNC state and supports models for the adaptation to nutrient poor aquatic environments. Phage had an undetectable impact on this adaptation. Taken together, the rise of ABNC cells and lytic phage blocked transmission. Thus, there is a fitness advantage if V. cholerae can make a rapid transfer to the next host before these negative selective pressures compound in the aquatic environment

Long-Distance Delivery of Bacterial Virulence Factors by Pseudomonas aeruginosa Outer Membrane Vesicles

Bacteria use a variety of secreted virulence factors to manipulate host cells, thereby causing significant morbidity and mortality. We report a mechanism for the long-distance delivery of multiple bacterial virulence factors, simultaneously and directly into the host cell cytoplasm, thus obviating the need for direct interaction of the pathogen with the host cell to cause cytotoxicity. We show that outer membrane–derived vesicles (OMV) secreted by the opportunistic human pathogen Pseudomonas aeruginosa deliver multiple virulence factors, including β-lactamase, alkaline phosphatase, hemolytic phospholipase C, and Cif, directly into the host cytoplasm via fusion of OMV with lipid rafts in the host plasma membrane. These virulence factors enter the cytoplasm of the host cell via N-WASP–mediated actin trafficking, where they rapidly distribute to specific subcellular locations to affect host cell biology. We propose that secreted virulence factors are not released individually as naked proteins into the surrounding milieu where they may randomly contact the surface of the host cell, but instead bacterial derived OMV deliver multiple virulence factors simultaneously and directly into the host cell cytoplasm in a coordinated manner

Use of the Vectra Polyetherurethaneurea Graft for Dialysis Access in HIV-Positive Patients With End-Stage Renal Disease

The primary objective of this study was to establish the safety, efficacy, infection rate, and patency of the Vectra graft (polyetherurethaneurea) for dialysis access in patients diagnosed with human immunodeficiency virus (HIV) and end-stage renal disease. The Vectra graft has a unique self-sealing property; therefore we hypothesize that these patients will have fewer infections. A Vectra graft was implanted in 30 consecutive HIV-positive patients without sufficient veins for an autologous fistula. These surgeries were carried out over a 2.5-year period. Primary graft patency was 42% at 12 months and 3 (10%) of the grafts developed infection. This rate of graft infection was less (10% vs 45%) than both our prior experience and published reports using polytetrafluorothene bridge grafts. The unique self-sealing property of the Vectra graft minimizes the development of perigraft hematoma with repetitive needle cannulation and in the immunosuppressed HIV-positive patient, may account for the observed decrease in dialysis access infection

Early cannulation prosthetic graft (Flixene (TM)) for arteriovenous access

Purpose: Preferred hemodialysis (HD) access is an autologous fistula. Vascular grafts are used in patients with vessels unsuitable to accomplish an arteriovenous fistula (AVF). It is recommended that most current grafts mature in situ for 2-3 weeks before being accessed. Graft complications occur because the structure was not designed for the trauma of repeated cannulation. This study graft has a different structure that enables early use. Its design minimizes weeping through the graft walls and is meant to endure repeated access, thus minimizing the use of HD catheters. The purpose of this study was to show that the Flixene (TM) graft can be safely placed in patients where fistulas have failed and can be cannulated in 24-72 hr, while maintaining patency rates similar to other polytetrafluoroethylene (PTFE) grafts on the market. Flixene (TM) configuration should also reduce the incidence of pseudoaneurysms and seromas. Methods: A prospective two-center study placed 33 grafts in 33 patients; graft efficacy, post-operative complications, and patency were evaluated. Ease of cannulation and dialysis center complications related to early cannulation were documented. Six month follow-up data was analyzed. Results: Successful access was achieved in all 33 patients within 72 hr (29 patients within 24 hr). Overall primary patency at 6 months was 49%; primary-assisted patency at 6 months was 80%. No pseudoaneurysms or seromas were documented at 6 months. Complications were typical of graft access. Conclusion: Early cannulation was successful in all patients. Primary and secondary patency rates at 6-months were equivalent to other data reported on PTFE grafts. Flixene (TM) successfully prevented pseudoaneurysm and seroma formation at 6 months of prospective follow-up. This graft is a better last-resort option for patients who cannot receive a fistula, compared to double-lumen cuffed catheters

Hepatic DNA adduct dosimetry in rats fed tamoxifen:a comparison of methods

Liver homogenates from rats fed tamoxifen (TAM) in the diet were shared among four different laboratories. TAM-DNA adducts were assayed by high pressure liquid chromatography-electrospray tandem mass spectrometry (HPLC-ES-MS/MS), TAM-DNA chemiluminescence immunoassay (TAM-DNA CIA), and P-32-postlabeling with either thin layer (P-32-P-TLC) or liquid chromatography (P-32-P-HPLC) separation. In the first study, rats were fed a diet containing 500 p.p.m. TAM for 2 months, and the values for measurements of the (E)-alpha-(deoxyguanosin-N-2-yl)-tamoxifen (dG-N-2-TAM) adduct in replicate rat livers varied by 3.5-fold when quantified using ‘in house’ TAM-DNA standards, or other approaches where appropriate. In the second study, rats were fed 0, 50, 250 or 500 p.p.m. TAM for 2 months, and TAM-DNA values were quantified using both ‘in house’ approaches as well as a newly synthesized [N-methyl-H-3]TAM-DNA standard that was shared among all the participating groups. In the second study, the total TAM-DNA adduct values varied by 2-fold, while values for the dG-N-2-TAM varied by 2.5-fold. Ratios of dG-N-2-TAM:(E)-alpha-(deoxyguanosin-N-2-yl)-N-desmethyltamoxifen (dG-N-2-N-desmethyl-TAM) in the second study were similar to 1:1 over the range of doses examined. The study demonstrated a remarkably good agreement for TAM-DNA adduct measurements among the diverse methods employed

A multicenter study of permanent hemodialysis access patency: Beneficial effect of clipped vascular anastomotic technique.

Abstract Objective: There is an urgent and compelling need to reduce the morbidity and expense of maintaining hemodialysis vascular access patency. This large, long-term, retrospective, multicenter study, which compared access patency of autogenous arteriovenous fistulas (AVF) and synthetic bridge grafts (AVG) created with conventional sutures or nonpenetrating clips, was undertaken to resolve conflicting results from previous smaller studies. Design: Patency data for 1385 vascular access anastomoses (clipped or sutured) was obtained from 17 hospitals and dialysis centers (Appendix). Five hundred eighteen AVF (242 clip, 276 suture) and 827 AVG (440 clip, 384 suture) were analyzed. Statistical comparisons were made with Kaplan-Meier survival analysis, log-rank test, two-sample t test, and X 2 test. The Cox proportional hazards model was used to confirm Kaplan-Meier analysis. Results: Access patency (primary, secondary, overall, and intention to treat) was significantly improved in access anastomoses constructed with clips. In the intention-to-treat group, primary patency at 24 months was 0.54 for clipped AVF and 0.34 for sutured AVF, and was 0.36 for clipped AVG and 0.17 for sutured AVG. At 24 months, primary patency rate for AVF successfully used for dialysis was 0.67 for clips and 0.48 for sutures, and for AVG was 0.39 for clips and 0.19 for sutured constructs. Interventions necessary to maintain patency were significantly fewer in clipped anastomoses. Conclusion: Replacing conventional suture with clips significantly reduces morbidity associated with maintaining permanent hemodialysis vascular access. This beneficial effect may be due to the biologic superiority of interrupted, nonpenetrating vascular anastomoses