Assessment of animal impacts on bacterial water quality in a South Carolina, USA tidal creek system.

Fecal pollution may adversely impact water quality in coastal ecosystems. The goal of this study was to determine whether cattle were a source of fecal pollution in a South Carolina watershed. Surface water samples were collected in June 2002 and February through March 2003 in closed shellfish harvesting waters of Toogoodoo Creek in Charleston County, SC. Fecal coliform concentrations in 70 % of the water samples taken for this study exceeded shellfish harvesting water standards. Ribotyping was performed in order to identify animal sources contributing to elevated fecal coliform levels. Escherichia coli isolates (n = 253) from surface water samples were ribotyped and compared to a ribotype library developed from known sources of fecal material. Ribotypes from water samples that matched library ribotypes with 90 % maximum similarity or better were assigned to that source. Less than half of the unknown isolates (38 %) matched with library isolates. About half (53 %) of the matched ribotypes were assigned to cattle isolates and 43 % to raccoon. Ribotyping almost exclusively identified animal sources. While these results indicate that runoff from cattle farms was a likely source of fecal pollution in the watershed, wildlife also contributed. Given the small size of the library, ribotyping was moderately useful for determining the impact of adjacent cattle farms on Toogoodoo Creek. Increasing the number and diversity of the wildlife sources from the area would likely increase the usefulness of the method.

A real-time qPCR assay for the detection of the nifH gene of Methanobrevibacter smithii, a potential indicator of sewage pollution.

AIMS: To develop a quantitative, real-time PCR assay to detect the nifH gene of Methanobrevibacter smithii. Methanobrevibacter smithii is a methanogenic archaea found in the intestinal tract of humans that may be a useful indicator of sewage pollution in water. METHODS AND RESULTS: Quantification standards were prepared from Meth. smithii genomic DNA dilutions, and a standard curve was used to quantify the target gene and calculate estimated genome equivalency units. A competitive internal positive control was designed and incorporated into the assay to assess inhibition in environmental extracts. Testing the assay against a panel of 23 closely related methanogen species demonstrated specificity of the assay for Meth. smithii. A set of 36 blind water samples was then used as a field test of the assay. The internal control identified varying levels of inhibition in 29 of 36 (81%) samples, and the Meth. smithii target was detected in all water samples with known sewage input. CONCLUSIONS: The quantitative PCR assay developed in this study is a sensitive and rapid method for the detection of the Meth. smithii nifH gene that includes an internal control to assess inhibition. Further research is required both to better evaluate host specificity of this assay and the correlation with human health risks. SIGNIFICANCE AND IMPACT OF THE STUDY: This research is the first description of the development of a rapid and sensitive quantitative assay for a methanogenic archaeal indicator of sewage pollution.

Growth and survival of Vibrio parahaemolyticus in postharvest American oysters.

Oysters at the retail stage of distribution generally contain greater densities of Vibrio parahaemolyticus than do oysters at harvest. The objective of this study was to determine the effects of postharvest storage at 26 and 3 degrees C on the growth and survival of naturally occurring V. parahaemolyticus in shellstock American oysters (Crassostrea virginica). Oysters were collected monthly from May 1998 through April 1999 from Mobile Bay, Alabama, and their V. parahaemolyticus densities were determined after 0, 5, 10, and 24 h of postharvest storage at 26 degrees C. After 24 h of storage at 26 degrees C, oysters were transferred to a refrigerator at 3 degrees C and analyzed 14 to 17 days later. V. parahaemolyticus numbers were determined by a direct plating method involving an alkaline-phosphatase-labeled DNA probe that targets the species-specific thermolabile hemolysin gene (tlh-AP) to identify suspect isolates. From April to December, when water temperatures at harvest were >20 degrees C, the geometric mean harvest density of V. parahaemolyticus was 130 CFU/g. When water temperatures were <20 degrees C, the geometric mean harvest density was 15 CFU/g. After harvest, V. parahaemolyticus multiplied rapidly in live oysters held at 26 degrees C, showing a 50-fold increase (1.7 log CFU/g) at 10 h and a 790-fold increase (2.9 log CFU/g) at 24 h (April through December). Average V. parahaemolyticus numbers showed a sixfold decrease (0.8 log CFU/g) after approximately 14 days of refrigeration. These results indicate that V. parahaemolyticus can grow rapidly in unrefrigerated oysters.

Heterogeneity of environmental, retail, and clinical isolates of Vibrio vulnificus as determined by lipopolysaccharide-specific monoclonal antibodies.

The opportunistic pathogen Vibrio vulnificus expresses lipopolysaccharide (LPS) antigens on its outer membrane surface. A serological typing system was developed for these antigens, utilizing the discriminatory recognition of monoclonal antibodies (MAb) by ELISA. MAb were used to recognize five unique types of LPS-associated antigens for examination of clinical. environmental, and retail isolates of V. vulnificus. The overall serotype profile of the clinical isolates was significantly different (P < 0.05) from that of the environmental and retail isolates. A higher percentage of clinical isolates were typable (61%) compared to the environmental isolates (41%) and retail isolates (44%). In particular, the percentage of serotype 1/5 among clinical isolates (33%), compared to that of environmental (9%) and retail (4%), was highly significant (P < 0.0001). Among the environmental Gulf Coast isolates, there were differences in the prevalence of serotypes 2 and 3 (P < 0.05), depending on whether isolates were obtained from Louisiana or Alabama harvest sites. There were no statistically significant differences between the serotype profiles of Gulf and Atlantic Coast retail isolates despite the absence of serotype 1/5 from the Atlantic Coast. While some serotype diversity was detected in V. vulnificus isolated during different seasons, from different geographic locations, and at retail versus at harvest, there was no apparent concordance between any of the serotype distributions obtained from oysters versus that isolated clinically. The heterogeneity of environmental isolates and relative homogeneity among clinical isolates suggest that human risk may not be predicted on quantitative exposure alone.

Evaluation of two direct plating methods using nonradioactive probes for enumeration of Vibrio parahaemolyticus in oysters.

Oysters (Crassostrea virginica) were collected monthly from May 1998 to April 1999 from Mobile Bay, Ala., and analyzed to determine Vibrio parahaemolyticus densities at zero time and after 5, 10, and 24 h of postharvest storage at 26 degrees C. After 24 h of storage at 26 degrees C, oysters were transferred to a refrigerator at 3 degrees C and then analyzed 14 to 17 days later. The V. parahaemolyticus numbers were determined by the most-probable-number procedure using alkaline phosphatase-labeled DNA probe VPAP, which targets the species-specific thermolabile hemolysin gene (tlh), to identify suspect isolates (MPN-VPAP procedure). Two direct plating methods, one using a VPAP probe (Direct-VPAP) and one using a digoxigenin-labeled probe (Direct-VPDig) to identify suspect colonies, were compared to the MPN-VPAP procedure. The results of the Direct-VPAP and Direct-VPDig techniques were highly correlated (r = 0.91), as were the results of the Direct-VPAP and MPN-VPAP procedures (r = 0.91). The correlation between the Direct-VPDig and MPN-VPAP results was 0.85. The two direct plating methods in which nonradioactive DNA probes were used were equivalent to the MPN-VPAP procedure for identification of total V. parahaemolyticus, and they were more rapid and less labor-intensive.