The Use of a Novel NanoLuc -Based Reporter Phage for the Detection of Escherichia coli O157:H7.

Rapid detection of the foodborne pathogen Escherichia coli O157:H7 is of vital importance for public health worldwide. Among detection methods, reporter phages represent unique and sensitive tools for the detection of E. coli O157:H7 from food as they are host-specific and able to differentiate live cells from dead ones. Upon infection, target bacteria become identifiable since reporter genes are expressed from the engineered phage genome. The E. coli O157:H7 bacteriophage ΦV10 was modified to express NanoLuc luciferase (Nluc) derived from the deep-sea shrimp Oplophorus gracilirostris. Once infected by the ΦV10 reporter phage, E. coli O157:H7 produces a strong bioluminescent signal upon addition of commercial luciferin (Nano-Glo(®)). Enrichment assays using E. coli O157:H7 grown in LB broth with a reporter phage concentration of 1.76 × 10(2) pfu ml(-1) are capable of detecting approximately 5 CFU in 7 hours. Comparable detection was achieved within 9 hours using 9.23 × 10(3) pfu ml(-1) of phage in selective culture enrichments of ground beef as a representative food matrix. Therefore we conclude that this NanoLuc reporter phage assay shows promise for detection of E. coli O157:H7 from food in a simple, fast and sensitive manner.

Sequence analysis of Escherichia coli O157:H7 bacteriophage PhiV10 and identification of a phage-encoded immunity protein that modifies the O157 antigen.

Bacteriophage PhiV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the PhiV10 genome is 39 104 bp long and contains 55 predicted genes. PhiV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage epsilon15 and a prophage from E. coli APEC O1. The attachment site of PhiV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. PhiV10 encodes an O-acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block PhiV10 superinfection, indicating that the O157 antigen is most likely the PhiV10 receptor.

Making it safer: a health centre's strategy for suicide prevention.

Developing and implementing consistent methodology for suicide assessment and intervention is challenging, particularly in a large community hospital which provides both inpatient care and a wide range of ambulatory and community based mental health programs. Patients, families, staff, and ongoing evaluation contributed to the development of an initiative to determine what is best practice and in effecting changes in clinical and organizational practices. The Suicide Assessment project resulted in improved clinical outcomes for patients and clients. Staff report that they feel more supported in probing for suicidal ideation and have the skills required to effectively intervene with the 'suicidal' patient/client. This project advanced our knowledge of suicide assessment and risk management and provided new insights to assist professionals in the aftermath of a client's suicide when, as Valente describes, clinicians report 'feeling overwhelmed with feelings of guilt, self-blame, and responsibility (Valente S: Journal of Psychosocial Nursing 40:7 22-23, 2002).

Cloning of a gene cluster involved in the catabolism of p-nitrophenol by Arthrobacter sp. strain JS443 and characterization of the p-nitrophenol monooxygenase.

The npd gene cluster, which encodes the enzymes of a p-nitrophenol catabolic pathway from Arthrobacter sp. strain JS443, was cloned and sequenced. Three genes, npdB, npdA1, and npdA2, were independently expressed in Escherichia coli in order to confirm the identities of their gene products. NpdA2 is a p-nitrophenol monooxygenase belonging to the two-component flavin-diffusible monooxygenase family of reduced flavin-dependent monooxygenases. NpdA1 is an NADH-dependent flavin reductase, and NpdB is a hydroxyquinol 1,2-dioxygenase. The npd gene cluster also includes a putative maleylacetate reductase gene, npdC. In an in vitro assay containing NpdA2, an E. coli lysate transforms p-nitrophenol stoichiometrically to hydroquinone and hydroxyquinol. It was concluded that the p-nitrophenol catabolic pathway in JS443 most likely begins with a two-step transformation of p-nitrophenol to hydroxy-1,4-benzoquinone, catalyzed by NpdA2. Hydroxy-1,4-benzoquinone is reduced to hydroxyquinol, which is degraded through the hydroxyquinol ortho cleavage pathway. The hydroquinone detected in vitro is a dead-end product most likely resulting from chemical or enzymatic reduction of the hypothetical intermediate 1,4-benzoquinone. NpdA2 hydroxylates a broad range of chloro- and nitro-substituted phenols, resorcinols, and catechols. Only p-nitro- or p-chloro-substituted phenols are hydroxylated twice. Other substrates are hydroxylated once, always at a position para to a hydroxyl group.

Molecular characterization of autoinduction of bioluminescence in the Microtox indicator strain Vibrio fischeri ATCC 49387.

Repeated attempts to clone the luxI from Vibrio fischeri ATCC 49387 failed to produce a clone carrying a functional LuxI. Sequence data from the clones revealed the presence of a polymorphism when compared with previously published luxI sequences, prompting further characterization of bioluminescence regulation in V. fischeri ATCC 49387. Further investigation of V. fischeri ATCC 49387 revealed that its LuxI protein lacks detectable LuxI activity due to the presence of a glutamine residue at position 125 in the deduced amino acid sequence. Specific bioluminescence in V. fischeri ATCC 49387 increases with increasing cell density, indicative of a typical autoinduction response. However, conditioned medium from this strain does not induce bioluminescence in an ATCC 49387 luxR-plux-based acyl homoserine lactone reporter strain, but does induce bioluminescence in ATCC 49387. It has been previously shown that a V. fischeri MJ-1 luxI mutant exhibits autoinduction of bioluminescence through N-octanoyl-L-homoserine lactone, the product of the AinS autoinducer synthase. However, a bioreporter based on luxR-plux from V. fischeri ATCC 49387 responded poorly to conditioned medium from V. fischeri ATCC 49387 and also responded poorly to authentic N-octanoyl-DL-homoserine lactone. A similar MJ-1-based bioreporter showed significant induction under the same conditions. A putative ainS gene cloned from ATCC 49387, unlike luxI from ATCC 49387, expresses V. fischeri autoinducer synthase activity in Escherichia coli. This study suggests that a regulatory mechanism independent of LuxR and LuxI but possibly involving AinS is responsible for the control of autoinduction of bioluminescence in V. fischeri ATCC 49387.