William B. Nilsson

Sequence Polymorphism of the 16S rRNA Gene of Vibrio vulnificus Is a Possible Indicator of Strain Virulence

ABSTRACT Vibrio vulnificus exhibits considerable strain-to-strain variation in virulence. Attempts to associate phenotypic or genotypic characteristics with strain virulence have been largely unsuccessful. Based on a 17-nucleotide difference throughout the sequence of the small subunit 16S rRNA gene, there are two major groups of V. vulnificus designated types A and B. In a survey of the 16S rRNA genotype in 67 V. vulnificus human clinical and nonclinical strains, we determined that the majority of nonclinical isolates are type A (31 of 33) and that there is a statistically significant association between the type B genotype and human clinical strains (26 of 34).

Solubilities of methyl oleate, oleic acid, oleyl glycerols, and oleyl glycerol mixtures in supercritical carbon dioxide

Solubility isotherms for oleic acid and methyl oleate as well as mono-, di-, and trioleylglycerol (MO, DO, and TO) in supercritical fluid CO2 at 50 and 60°C are reported. Partition coefficients for quaternary (MO-DO-TO-CO2) mixtures were obtained at 60°C at pressures ranging from 172 to 309 bar. Data indicate that diolein, and especially monoolein, exhibit positive deviation from ideal behavior, possibly due to intermolecular hydrogen bonding. Supercritical fluid CO2 appears to be a good media for removal of mono- and diacylglycerol by-products from synthetic triglyceride reaction mixtures at moderate temperatures.

Population structure of clinical and environmental Vibrio parahaemolyticus from the Pacific Northwest coast of the United States.

Vibrio parahaemolyticus is a common marine bacterium and a leading cause of seafood-borne bacterial gastroenteritis worldwide. Although this bacterium has been the subject of much research, the population structure of cold-water populations remains largely undescribed. We present a broad phylogenetic analysis of clinical and environmental V. parahaemolyticus originating largely from the Pacific Northwest coast of the United States. Repetitive extragenic palindromic PCR (REP-PCR) separated 167 isolates into 39 groups and subsequent multilocus sequence typing (MLST) separated a subset of 77 isolates into 24 sequence types. The Pacific Northwest population exhibited a semi-clonal structure attributed to an environmental clade (ST3, N = 17 isolates) clonally related to the pandemic O3:K6 complex and a clinical clade (ST36, N = 20 isolates) genetically related to a regionally endemic O4:K12 complex. Further, the identification of at least five additional clinical sequence types (i.e., ST43, 50, 65, 135 and 417) demonstrates that V. parahaemolyticus gastroenteritis in the Pacific Northwest is polyphyletic in nature. Recombination was evident as a significant source of genetic diversity and in particular, the recA and dtdS alleles showed strong support for frequent recombination. Although pandemic-related illnesses were not documented during the study, the environmental occurrence of the pandemic clone may present a significant threat to human health and warrants continued monitoring. It is evident that V. parahaemolyticus population structure in the Pacific Northwest is semi-clonal and it would appear that multiple sequence types are contributing to the burden of disease in this region.

Partition Coefficients for Fatty Acid Esters in Supercritical Fluid CO2 with and Without Ethanol

The complex nature of fish oils was exploited to study the dependence of structural factors upon fatty acid ester solubility in supercritical fluid carbon dioxide (SCF-CO2). Partition coefficients were determined for a number of components present in two mixtures of fatty cid ethyl esters derived from menhaden oil in SCF-CO2 at 60°C and 125 bar. Analogous data also were obtained for SCF-CO2 with 5% (w/w) ethanol added. The addition of ethanol was found to increase partition coefficients for all species, but resulted in a decrease of fluid selectivity. Aside from the chain length of a component, both the degree and position of unsaturation were found to be structural factors that affect the value of the partition coefficient.

Solubility of simple and mixed triacylglycerols in supercritical CO2

A simple mixture of triacylglycerols (TAGs) was synthesized enzymatically from palmitic acid and oleic acid. The mixture contained tripalmitoylglycerol (PPP), trioleoylglycerol (OOO), and various isomers of palmitoyl-dioleoylglycerol (POO) and oleoyl-dipalmitoylglycerol (PPO). The behavior of this TAG mixture in equilibrium with supercritical carbon dioxide (SF-CO2) was studied at 40 and 60°C between 172 and 310 bar. Generally, the partition coefficients varied inversely with species molecular weight. The solubilities of PPP and OOO in SF-CO2 were determined at the same conditions. PPP was markedly more soluble in SF-CO2 when present in the TAG mixture than as a pure compound.

Environmental influences on the seasonal distribution of Vibrio parahaemolyticus in the Pacific Northwest of the USA.

Populations of Vibrio parahaemolyticus in the environment can be influenced by numerous factors. We assessed the correlation of total (tl+) and potentially virulent (tdh+) V. parahaemolyticus in water with three harmful algal bloom (HAB) genera (Pseudo-nitzschia, Alexandrium and Dinophysis), the abundance of diatoms and dinoflagellates, chlorophyll-a and temperature, salinity and macronutrients at five sites in Washington State from 2008-2009. The variability in V. parahaemolyticus density was explained predominantly by strong seasonal trends where maximum densities occurred in June, 2 months prior to the highest seasonal water temperature. In spite of large geographic differences in temperature, salinity and nutrients, there was little evidence of corresponding differences in V. parahaemolyticus density. In addition, there was no evident relationship between V. parahaemolyticus and indices of HAB genera, perhaps due to a lack of significant HAB events during the sampling period. The only nutrient significantly associated with V. parahaemolyticus density after accounting for the seasonal trend was silicate. This negative relationship may be caused by a shift in cell wall structure for some diatom species to a chitinous substrate preferred by V. parahaemolyticus. Results from our study differ from those in other regions corroborating previous findings that environmental factors that trigger vibrio and HAB events may differ depending on geographic locations. Therefore caution should be used when applying results from one region to another.

The use of polymerase chain reaction for the detection and speciation of bacterial bone and joint infection in children

We evaluated 36 consecutive patients presenting with signs and symptoms of bacterial bone and joint infection and 10 control patients using bacterial cultures of blood and the presumed site of infection compared with polymerase chain reaction (PCR) techniques using a universal primer and restriction endonuclease digestion. Of the 28 patients with definitive clinical and/or laboratory evidence of bacterial infection, 16 patients had positive bacterial cultures and 12 were PCR-positive. Twenty of 28 patients were either PCR- or culture-positive. Nine of the 16 subjects who had culture-positive samples also had PCR-positive samples (8 positive for the same organism and 1 with 2 organisms identified by culture, but only a single organism by PCR. Six culture positive patients were PCR-negative. Of the 12 patients who were culture-negative, 4 had bacterial genomic material present indicating infection. We conclude that current PCR methods are not superior to standard bacterial culture methods when applied to children with presumed bone or joint infections, but that PCR may complement existing microbiologic cultures for detection of bone and joint infections in children.

Pathogen update: Vibrio species

Abstract: Members of the genus Vibrio are common inhabitants of the marine environment, associated with vertebrate and invertebrate animals. Some species cause foodborne illness from seafood such as undercooked or raw fish and shellfish. This review focuses on the current state of knowledge for the human pathogenic Vibrio species. V. parahaemolyticus is the leading cause of bacterial seafood-borne illness worldwide, while V. vulnificus is the leading cause of seafood-related deaths. While much research has ascertained potential mechanisms of virulence and contributed to a variety of mitigation strategies, there is a need to improve methods to assess risk and to develop improved early warning systems for public health and resource managers.

Comparative Genomic Analysis of Vibrio diabolicus and Six Taxonomic Synonyms: A First Look at the Distribution and Diversity of the Expanded Species

Vibrio is a diverse genus of Gammaproteobacteria autochthonous to marine environments worldwide. Vibrio diabolicus and V. antiquarius were originally isolated from deep-sea hydrothermal fields in the East Pacific Rise. These species are closely related to members of the Harveyi clade (e.g., V. alginolyticus and V. parahaemolyticus) that are commonly isolated from coastal systems. This study reports the discovery and draft genome sequence of a novel isolate (Vibrio sp. 939) cultured from Pacific oysters (Crassostrea gigas). Questions surrounding the identity of Vibrio sp. 939 motivated a genome-scale taxonomic analysis of the Harveyi clade. A 49-genome phylogeny based on 1,109 conserved coding sequences and a comparison of average nucleotide identity (ANI) values revealed a clear case of synonymy between Vibrio sp. 939, V. diabolicus Art-Gut C1 and CNCM I-1629, V. antiquarius EX25 and four V. alginolyticus strains (E0666, FF273, TS13, and V2). This discovery expands the V. diabolicus species and makes available six additional genomes for comparative genomic analyses. The distribution of the expanded species is thought to be global given the range of isolation sources (horse mackerel, seawater, sediment, dentex, oyster, artemia and polycheate) and origins (China, India, Greece, United States, East Pacific Rise, and Chile). A subsequent comparative genomic analysis of this new eight-genome subclade revealed a high degree of individual genome plasticity and a large repertoire of genes related to virulence and defense. These findings represent a significant revision to the understanding of V. diabolicus and V. antiquarius as both have long been regarded as distinct species. This first look at the expanded V. diabolicus subclade suggests that the distribution and diversity of this species mirrors that of other Harveyi clade species, which are notable for their ubiquity and diversity.