600-ns pulsed electric fields affect inactivation and antibiotic susceptibilities of Escherichia coli and Lactobacillus acidophilus.

Cell suspensions of Escherichia coli and Lactobacillus acidophilus were exposed to 600-ns pulsed electric fields (nsPEFs) at varying amplitudes (Low-13.5, Mid-18.5 or High-23.5 kV cm-1) and pulse numbers (0 (sham), 1, 5, 10, 100 or 1000) at a 1 hertz (Hz) repetition rate. The induced temperature rise generated at these exposure parameters, hereafter termed thermal gradient, was measured and applied independently to cell suspensions in order to differentiate inactivation triggered by electric field (E-field) from heating. Treated cell suspensions were plated and cellular inactivation was quantified by colony counts after a 24-hour (h) incubation period. Additionally, cells from both exposure conditions were incubated with various antibiotic-soaked discs to determine if nsPEF exposure would induce changes in antibiotic susceptibility. Results indicate that, for both species, the total delivered energy (amplitude, pulse number and pulse duration) determined the magnitude of cell inactivation. Specifically, for 18.5 and 23.5 kV cm-1 exposures, L. acidophilus was more sensitive to the inactivation effects of nsPEF than E. coli, however, for the 13.5 kV cm-1 exposures E. coli was more sensitive, suggesting that L. acidophilus may need to meet an E-field threshold before significant inactivation can occur. Results also indicate that antibiotic susceptibility was enhanced by multiple nsPEF exposures, as observed by increased zones of growth inhibition. Moreover, for both species, a temperature increase of ≤ 20 °C (89% of exposures) was not sufficient to significantly alter cell inactivation, whereas none of the thermal equivalent exposures were sufficient to change antibiotic susceptibility categories.

Occurrence and significance of pathogenicity and fitness islands in environmental vibrios.

Pathogenicity islands (PAIs) are large genomic regions that contain virulence genes, which aid pathogens in establishing infections. While PAIs in clinical strains (strains isolated from a human infection) are well-studied, less is known about the occurrence of PAIs in strains isolated from the environment. In this study we describe three PAIs found in environmental Vibrio vulnificus and Vibrio parahaemolyticus strains, as well as a genomic fitness island found in a Vibrio diabolicus strain. All four islands had markedly different GC profiles than the rest of the genome, indicating that all of these islands were acquired via lateral gene transfer. Genes on the PAIs and fitness island were characterized. The PAI found in V. parahaemolyticus contained the tdh gene, a collagenase gene, and genes involved in the type 3 secretion system II (T3SS2). A V. vulnificus environmental strain contained two PAIs, a small 25 kbp PAI and a larger 143 kbp PAI. Both PAIs contained virulence genes. Toxin-antitoxin (TA) genes were found in all three species: on the V. diabolicus fitness island, and on the V. parahaemolyticus and V. vulnificus PAIs.

Transposable elements: genome innovation, chromosome diversity, and centromere conflict.

Although it was nearly 70 years ago when transposable elements (TEs) were first discovered "jumping" from one genomic location to another, TEs are now recognized as contributors to genomic innovations as well as genome instability across a wide variety of species. In this review, we illustrate the ways in which active TEs, specifically retroelements, can create novel chromosome rearrangements and impact gene expression, leading to disease in some cases and species-specific diversity in others. We explore the ways in which eukaryotic genomes have evolved defense mechanisms to temper TE activity and the ways in which TEs continue to influence genome structure despite being rendered transpositionally inactive. Finally, we focus on the role of TEs in the establishment, maintenance, and stabilization of critical, yet rapidly evolving, chromosome features: eukaryotic centromeres. Across centromeres, specific types of TEs participate in genomic conflict, a balancing act wherein they are actively inserting into centromeric domains yet are harnessed for the recruitment of centromeric histones and potentially new centromere formation.

The hot oyster: levels of virulent Vibrio parahaemolyticus strains in individual oysters.

Vibrio parahaemolyticus is the leading cause of seafood-associated gastroenteritis and is most commonly transmitted by raw oysters. Consequently, detection of virulent strains of this organism in oysters is a primary concern for seafood safety. Vibrio parahaemolyticus levels were determined in 110 individual oysters harvested from two sampling sites in SC, USA. The majority of oysters (98%) contained low levels of presumptive V. parahaemolyticus However, two healthy oysters contained presumptive V. parahaemolyticus numbers that were unusually high. These two 'hot' oysters contained levels of presumptive V. parahaemolyticus within the gills that were ∼100-fold higher than the average for other oysters collected at the same date and location. Current V. parahaemolyticus detection practices require homogenizing a dozen oysters pooled together to determine V. parahaemolyticus numbers, a procedure that would dilute out V. parahaemolyticus in these 'hot' oysters. This study demonstrates the variability of V. parahaemolyticus densities taken from healthy, neighboring individual oysters in the environment. Additionally, environmental V parahaemolyticus isolates were screened for the virulence-related genes, tdh and trh, using improved polymerase chain reaction primers and protocols. We detected these genes, previously thought to be rare in environmental isolates, in approximately half of the oyster isolates.

Genes similar to the Vibrio parahaemolyticus virulence-related genes tdh, tlh, and vscC2 occur in other vibrionaceae species isolated from a pristine estuary.

Detection of the human pathogen Vibrio parahaemolyticus often relies on molecular biological analysis of species-specific virulence factor genes. These genes have been employed in determinations of V. parahaemolyticus population numbers and the prevalence of pathogenic V. parahaemolyticus strains. Strains of the Vibrionaceae species Photobacterium damselae, Vibrio diabolicus, Vibrio harveyi, and Vibrio natriegens, as well as strains similar to Vibrio tubiashii, were isolated from a pristine salt marsh estuary. These strains were examined for the V. parahaemolyticus hemolysin genes tdh, trh, and tlh and for the V. parahaemolyticus type III secretion system 2α gene vscC2 using established PCR primers and protocols. Virulence-related genes occurred at high frequencies in non-V. parahaemolyticus Vibrionaceae species. V. diabolicus was of particular interest, as several strains were recovered, and the large majority (>83%) contained virulence-related genes. It is clear that detection of these genes does not ensure correct identification of virulent V. parahaemolyticus. Further, the occurrence of V. parahaemolyticus-like virulence factors in other vibrios potentially complicates tracking of outbreaks of V. parahaemolyticus infections.

High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary.

Virulence factor genes encoding the thermostable direct hemolysin (tdh) and the thermostable direct hemolysin-related hemolysin (trh) are strongly correlated with virulence of the emergent human pathogen Vibrio parahaemolyticus. The gene encoding the thermolabile hemolysin (tlh) is also considered a signature molecular marker for the species. These genes are typically reported in very low percentages (1 to 2%) of nonclinical strains. V. parahaemolyticus strains were isolated from various niches within a pristine estuary (North Inlet, SC) and were screened for these genes using both newly designed PCR primers and more commonly used primers. DNA sequences of tdh and trh were recovered from 48% and 8.3%, respectively, of these North Inlet strains. The recovery of pathogenic V. parahaemolyticus strains in such high proportions from an estuarine ecosystem that is virtually free of anthropogenic influences indicates the potential for additional, perhaps environmental roles of the tdh and trh genes.