Vibrio parahaemolyticus in Rhode Island coastal ponds and the estuarine environment of narragansett bay.

Quantification of the abundance of Vibrio parahaemolyticus in water and oysters from Rhode Island showed the presence of environmental strains and low levels of potentially pathogenic strains when water temperatures were ≥18°C, with peak levels in late July to early August. A higher abundance of the trh gene than of the tdh gene was observed.

Sample preparation methods for quantitative detection of DNA by molecular assays and marine biosensors.

The need for quantitative molecular methods is growing in environmental, food, and medical fields but is hindered by low and variable DNA extraction and by co-extraction of PCR inhibitors. DNA extracts from Enterococcus faecium, seawater, and seawater spiked with E. faecium and Vibrio parahaemolyticus were tested by qPCR for target recovery and inhibition. Conventional and novel methods were tested, including Synchronous Coefficient of Drag Alteration (SCODA) and lysis and purification systems used on an automated genetic sensor (the Environmental Sample Processor, ESP). Variable qPCR target recovery and inhibition were measured, significantly affecting target quantification. An aggressive lysis method that utilized chemical, enzymatic, and mechanical disruption enhanced target recovery compared to commercial kit protocols. SCODA purification did not show marked improvement over commercial spin columns. Overall, data suggested a general need to improve sample preparation and to accurately assess and account for DNA recovery and inhibition in qPCR applications.

Chlorine dioxide as a treatment for ballast water to control invasive species: shipboard testing.

The efficacy of chlorine dioxide (ClO2) in eliminating organisms present in estuarine ballast water of a containership was determined under actual operating conditions by comparing the survival of planktonic communities present in waters of treated and control ballast tanks. Sampling was via ballast-tank hatches. The treatment (5 mg L(-1)ClO2 without pre-filtration) delivered by a prototype ClO2-generating system was generally effective against planktonic assemblages, although bacterial communities rebounded after a few days. Regardless of temperature, ClO2 was very effective against phytoplankton; the effect was immediate, without resurgence. Some zooplankters in the ≥ 50-µm fraction may survive the biocide, especially those able to find refuge within a protective coating (e.g., cysts, resting eggs, and shells) or in sediment. In order to boost efficacy, a pre-filtration step is recommended (now installed as standard equipment) to lower the intake of the ≥ 50-µm fraction and lessen the challenge posed by this size class.

Performance evaluation of canine-associated Bacteroidales assays in a multi-laboratory comparison study.

The contribution of fecal pollution from dogs in urbanized areas can be significant and is an often underestimated problem. Microbial source tracking methods (MST) utilizing quantitative PCR of dog-associated gene sequences encoding 16S rRNA of Bacteroidales are a useful tool to estimate these contributions. However, data about the performance of available assays are scarce. The results of a multi-laboratory study testing two assays for the determination of dog-associated Bacteroidales (DogBact and BacCan-UCD) on 64 single and mixed fecal source samples created from pooled fecal samples collected in California are presented here. Standardization of qPCR data treatment lowered inter-laboratory variability of sensitivity and specificity results. Both assays exhibited 100% sensitivity. Normalization methods are presented that eliminated random and confirmed non-target responses. The combination of standardized qPCR data treatment, use of normalization via a non-target specific Bacteroidales assay (GenBac3), and application of threshold criteria improved the calculated specificity significantly for both assays. Such measures would reasonably improve MST data interpretation not only for canine-associated assays, but for all qPCR assays used in identifying and monitoring fecal pollution in the environment.