Draft Genome Sequences of Histamine-Producing Photobacterium kishitanii and Photobacterium angustum, Isolated from Albacore (Thunnus alalunga) and Yellowfin (Thunnus albacares) Tuna.

Histamine-producing bacteria are responsible for scombrotoxin (histamine) fish poisoning, a leading cause of fish poisoning in the United States. We report here the draft genome sequences of four histamine-producing (HP) Photobacterium kishitanii strains and nine HP Photobacterium angustum strains isolated from tuna.

Defining and using microbial spectral databases.

This work shows how fingerprints of mass spectral patterns from microbial isolates are affected by variations in instrumental condition, by sample environment, and by sample handling factors. It describes a novel method by which pattern distortions can be mathematically corrected for variations in factors not amenable to experimental control. One uncontrollable variable is "between-batch" differences in culture media. Another, relevant for determination of noncultured extracts, is differences between the cells' environmental experience (e.g., starved environmental extracts versus cultured standards). The method suggests that, after a single growth cycle on a solid medium (perhaps, a selective one), pyrolysis MS spectra of microbial isolates can be algorithmically compensated and an unknown isolate identified using a spectral database defined by culture on a different (perhaps, nonselective) medium. This reduces identification time to as few as 24 h from sample collection. The concept also proposes a possible way to compensate certain noncultured, nonisolated samples (e.g., cells concentrated from urine or impacted from aerosol or semi-selectively extracted by immunoaffinity methods from heavily contaminated matrices) for identification within half an hour. Using the method, microbial mass spectra from different labs can be assembled into coherent databases similar to those routinely used to identify pure compounds. This type of data treatment is applicable for rapid detection in biowarfare and bioterror events as well as in forensic, research, and clinical laboratory contexts.

Metabolic reprogramming of the immune response in the tumor microenvironment.

A Division of Cancer Biology, NCI sponsored workshop, Metabolic Reprogramming of the Immune Response in the Tumor Microenvironment, was held October 2nd in Bethesda, MD. The purpose of the workshop was to bring together cancer cell biologists and immunologists to explore the mechanistic relationships between the metabolic pathways used by cancer cells and anti-tumor immune cells and how this information could be used to improve cancer immunotherapy. At the conclusion of the workshop a general discussion focused on defining the major challenges and opportunities concerning the impact of metabolism on anti-tumor immunity and cancer immunotherapy as well as what tools, technologies, resources or community efforts are required to accelerate research in this area. Overall, future studies need to consider how cancer cell metabolic pathways differ from activated lymphocytes in order to define a therapeutic window for cancer therapy. Further, studies aimed at reprogramming the metabolic qualities of T cells with the goal of improving immunotherapy were considered a promising avenue.

Rapid phenotypic characterization of Vibrio isolates by pyrolysis metastable atom bombardment mass spectrometry.

Pyrolysis mass spectrometry was investigated for rapid characterization of food-borne bacterial pathogens. Nine isolates of Vibrio parahaemolyticus and one isolate each of Vibrio fluvialis, Vibrio hollisae, and Vibrio vulnificus were analyzed. Pyrolysis mass spectra, generated via an alternative ionization method, metastable atom bombardment, were subject to principal component-discriminant analysis. The spectral patterns were used to distinguish Vibrio isolates differing in species, serotype and expression of the thermostable direct hemolysin gene. The patterns of similarity and dissimilarity amongst spectra in the Vibrio test set generally reflected those associated with species, serotype or hemolysin-producing genes, though the combined influence of these and other variables in the multi-dimensional data did not produce a simple clustering with respect to any one of these characteristics. These results suggested that with enough examples to model the most common combinations, the method should be able to characterize Vibrio isolates according to their phenotypic characteristics. Pyrolysis-mass spectrometry with metastable atom bombardment and pattern recognition appeared suitable for rapid infraspecific comparison of Vibrio isolates. This integrated analytical, pattern-recognition system should be examined further for potential utility in clinical and public health diagnostic contexts.

Evaluation of the Listertest™ Method for Quantitation of Listeria monocytogenes in Seafoods.

The U.S. Food and Drug Administration most probable number (MPN) method and the Listertest™ (immunomagnetic capture) were used to enumerate Listeria monocytogenes on laboratory-inoculated cooked crabmeat ( Callinectus sapidus ) and cold-smoked salmon ( Salmo salar ). The products were inoculated with <1.0 to 4.0 log CFU of L. monocytogenes per g and analyzed immediately. In the analyses of crabmeat, the results obtained by the MPN method were significantly lower (P < 0.05) than counts produced by the Listertest™. There was no significant difference (P < 0.05) between results produced by MPN and the Listertest™ in analyses of smoked salmon. The methods had the same variance in the quantitation of L. monocytogenes in these seafood products.

Incidence and Survival of Listeria monocytogenes in Ready-To-Eat Seafood Products.

The effects of processing and postprocess storage conditions on the incidence and survival of Listeria monocytogenes on crawfish ( Procambaris sp.), crabmeat ( Callinectus sapidus ), and smoked salmon ( Salmo salar ) were evaluated. L. monocytogenes was recovered from 3% of whole boiled market crawfish samples and 17% of frozen vacuum-packaged partially cooked crawfish tail meat, but not from boiled crabmeat or smoked salmon. Contamination was most likely due to postprocess handling as commonly used methods of cooking (5 min boil or 20 min steep) reduced L. monocytogenes to nondetectable levels in laboratory-contaminated crawfish. In postprocess storage temperature abuse studies, cooked whole crawfish were inoculated internally and externally with 3.0 log CFU of L. monocytogenes per g and incubated at 22 or 30°C for 6 h. The greatest increase in numbers of cells, 1.9 log CFU/g (determined by standard plate count), occurred at 30°C on externally contaminated crawfish. There was little change in numbers of L. monocytogenes during cold storage (6°C, 5 days; -20°C, 15 days). There was little change in cell numbers associated with products stored at 22 or -20°C. At 6°C, numbers of cells associated with crabmeat increased by 3.8 log MPN/g after 6 days; however, there was no increase in numbers of cells associated with salmon. The results show that the survival and growth characteristics of L. monocytogenes are dependent on storage time and temperature and the nature of the seafood product.

Effect of Three Biocides on Latin American and Gulf Coast Strains of Toxigenic Vibrio cholerae O1.

The survivability of toxigenic Vibrio cholerae O1 aboard cargo ships in ballast water taken from cholera-contaminated waterways may be enhanced by its attachment to particles. This study examined the effects of three biocides on attached and suspended cells of toxigenic V. cholerae isolate "J" (ballast water isolate) recovered from ballast water; strain C6707 (Latin American epidemic strain) involved in the Latin American epidemic and strain VRL 1984 (the toxigenic strain endemic to the Gulf Coast). Chitin was the substrate used for attachment. Attached cells of isolate "J" were reduced by 4 logs and those of strains C6707 and VRL 1984 were reduced by 3 logs after exposure to 100 ppm Povidone-iodine for 20 min. Attached isolate "J" cells were reduced by 5 logs, and attached C6707 cells were reduced to <1 CFU/ml (6 log decrease) after exposure to 800 ppm chlorine after 20 min. Although numbers of VRL 1984 were reduced to < 1 CFU/ml after exposure to 800 ppm chlorine for 5 min, counts rose to 101 CFU/ml in 20 min. Numbers of isolate "J" were reduced to <1 CFU/ml and those of C6707 were reduced by 6 logs after exposure to 200 ppm Roccal II (QAC) for 10 min. No VRL 1984 cells were recovered after 5 min exposure to 400 ppm and 20 min exposure to 200 ppm QAC. Suspended cells were reduced to <1 CFU/ml after exposure to 25 ppm iodine, 100 ppm chlorine or 50 ppm QAC for 2 min; however, intact nonculturable cells were detected by polymerase chain reaction in iodine-treated suspensions. Latin American and Gulf Coast strains were equally susceptible to disinfection.

Recovery of Heat-Stressed Listeria monocytogenes from Experimentally and Naturally Contaminated Shrimp.

A method was developed to enhance recovery of thermally stressed Listeria monocytogenes from internally contaminated shrimp. Shrimp tail meat was inoculated with 105 L. monocytogenes cells/g and boiled for 1-5 min. Thermally stressed L. monocytogenes cells were recovered following cold enrichment for 3 d without broth. Methods of the Food and Drug Administration and the U.S. Department of Agriculture for isolating L. monocytogenes permitted recovery of the organism from shrimp boiled for 1 min: however, with the new method, L. monocytogenes cells were recovered from shrimp boiled up to 5 min. No Listeria spp. were recovered from naturally contaminated, frozen, imported shrimp after 1 min of boiling.

Autocrine activation by interleukin 1alpha induces the fibrogenic phenotype of systemic sclerosis fibroblasts.

OBJECTIVE: To explore the cellular localization of interleukin 1alpha (IL-1alpha) in cultured fibroblasts from lesional skin of patients with systemic sclerosis (SSc) and to study the role of intracellular IL-1alpha in the activation of fibroblasts. METHODS: Dermal fibroblasts were derived from 12 patients with SSc. Expression of IL-1alpha mRNA was examined using reverse transcriptase-polymerase chain reaction (RT-PCR). The cellular distribution of IL-1alpha was examined by subcellular fractionation, flow cytometry, and immunocytochemistry. A full-length IL-1alpha cDNA was subcloned into the pcDNA3 vector to create sense and antisense-encoding constructs. Normal and SSc fibroblasts were stably transfected with the sense and antisense-encoding constructs, respectively. Stably transfected fibroblast clones were analyzed for the production of procollagen and IL-6 protein by ELISA, alpha1(I) procollagen mRNA by Northern blot hybridization, and proliferation by [3H]thymidine incorporation. RESULTS: SSc-affected fibroblasts constitutively expressed intracellular IL-1alpha, which was predominantly located in the nucleus. Inhibition of IL-1alpha expression in SSc-affected fibroblasts using antisense constructs resulted in decreased proliferation, IL-6 production, and procollagen synthesis. Conversely, overexpression of IL-1alpha in normal fibroblasts resulted in development of the SSc fibroblast phenotype. CONCLUSION: IL-1alpha is an important autocrine fibrogenic factor in SSc, suggesting that inhibition of intracellular IL-1alpha may be a novel strategy for the treatment of SSc.