A multi-tiered approach to assess fecal pollution in an urban watershed: Bacterial and viral indicators and sediment microbial communities.

Development of effective pollution mitigation strategies require an understanding of the pollution sources and factors influencing fecal pollution loading. Fecal contamination of Turkey Creek in Gulfport, Mississippi, one of the nation's most endangered creeks, was studied through a multi-tiered approach. Over a period of approximately two years, four stations across the watershed were analyzed for nutrients, enumeration of E. coli, male-specific coliphages and bioinformatic analysis of sediment microbial communities. The results demonstrated that two stations, one adjacent to a lift station and one just upstream from the wastewater-treatment plant, were the most impacted. The station adjacent to land containing a few livestock was the least impaired. While genotyping of male-specific coliphage viruses generally revealed a mixed viral signature (human and other animals), fecal contamination at the station near the wastewater treatment plant exhibited predominant impact by municipal sewage. Fecal indicator loadings were positively associated with antecedent rainfall for three of four stations. No associations were noted between fecal indicator loadings and any of the nutrients. Taxonomic signatures of creek sediment were unique to each sample station, but the sediment microbial community did overlap somewhat following major rain events. No presence of Escherichia coli (E. coli) or enterococci were found in the sediment. At some of the stations it was evident that rainfall was not always the primary driver of fecal transport. Repeated monitoring and analysis of a variety of parameters presented in this study determined that point and non-point sources of fecal pollution varied spatially in association with treated and/or untreated sewage.

Gene mapping and phylogenetic analysis of the complete genome from 30 single-stranded RNA male-specific coliphages (family Leviviridae).

Male-specific single-stranded RNA (FRNA) coliphages belong to the family Leviviridae. They are classified into two genera (Levivirus and Allolevivirus), which can be subdivided into four genogroups (genogroups I and II in Levivirus and genogroups III and IV in Allolevivirus). Relatively few strains have been completely characterized, and hence, a detailed knowledge of this virus family is lacking. In this study, we sequenced and characterized the complete genomes of 19 FRNA strains (10 Levivirus strains and 9 Allolevivirus strains) and compared them to the 11 complete genome sequences available in GenBank. Nucleotide similarities among strains of Levivirus genogroups I and II were 75% to 99% and 83 to 94%, respectively, whereas similarities among strains of Allolevivirus genogroups III and IV ranged from 70 to 96% and 75 to 95%, respectively. Although genogroup I strain fr and genogroup III strains MX1 and M11 share only 70 to 78% sequence identity with strains in their respective genogroups, phylogenetic analyses of the complete genome and the individual genes suggest that strain fr should be grouped in Levivirus genogroup I and that the MX1 and M11 strains belong in Allolevivirus genogroup III. Strains within each genus share >50% sequence identity, whereas between the two genera, strains have <40% nucleotide sequence identity. Overall, amino acid composition, nucleotide similarities, and replicase catalytic domain location contributed to phylogenetic assignments. A conserved eight-nucleotide signature at the 3' end of the genome distinguishes leviviruses (5' ACCACCCA 3') from alloleviviruses (5' TCCTCCCA 3').

Quantifying stream periphyton assemblage responses to nutrient amendments with a molecular approach.

Nutrient (nitrogen [N] and phosphorus [P]) pollution is a pervasive water quality issue in the USA for small streams and rivers. The effect of nutrients on the biotic condition of streams is often evaluated with biological indicators such as macroinvertebrate assemblages or periphyton assemblages, particularly diatoms. Molecular approaches facilitate the use of periphyton assemblages as bioindicators because periphyton is diverse and its composition as a whole, rather than just diatoms, soft-bodied algae, or any single group, may convey additional information about responses to nutrients. To further develop the concept that a taxonomically-broad evaluation of periphyton assemblages could be useful for developing stream bioindicators, we examined microbial assemblage composition with both 16S and 18S rRNA genes, enabling us to evaluate composition in 3 domains. We measured otherwise unknown nutrient responses of different periphyton groups in situ with experiments that used glass fiber filters to allow diffusion of amended nutrients into a stream. We deployed these experimental setups in 2 streams that differ in the extent of agricultural land-use in their catchments in the southeastern USA. Experiments consisted of controls, N amendments, P amendments, and both N and P amendments. Periphyton assemblages that grew on the filters differed significantly by stream, date or season, and nutrient treatment. Assemblage differences across treatments were more consistent among Bacteria and Archaea than among eukaryotes. Effects of nutrient amendments were more pronounced in the stream with less agricultural land use and, therefore, lower nutrient loading than in the stream with more agricultural land use and higher nutrient loading. Combined N and P amendments decreased species richness and evenness for Bacteria and Archaea by ∼36 and ∼9%, respectively, compared with controls. Indicator species analysis revealed that specific clades varied in their response to treatments. Indicators based on the responses of these indicator clades were related to nutrient treatments across sites and seasons. Analyses that included all the taxa in a domain did not resolve differences in responses to N vs P. Instead, better resolution was achieved with an analysis focused on diatoms, which responded more strongly to P than N. Overall, our results showed that in situ nutrient-diffusing substrate experiments are a useful approach for describing assemblage responses to nutrients in streams. This type of molecular approach may be useful to environmental agencies and stakeholders responsible for assessing and managing stream water quality and biotic condition.

Use of composite data sets for source-tracking enterococci in the water column and shoreline interstitial waters on Pensacola Beach, Florida.

Sources of Enterococcus faecalis isolates from Pensacola Beach, FL. were identified using a library-based approach by applying the statistical method of average similarity to single and composite data sets generated from separate analyses. Data sets included antibiotic resistance analysis (ARA), rep-fingerprints, and fatty acid methyl ester (FAME) profiles. Use of a composite data set composed of ARA and rep-fingerprints, added to the confidence of the identifications. The addition of FAME data to composite data sets did not add to the confidence of identifications. Source identification was performed to better understand risk associated with higher densities of enterococci found in swash zone interstitial water (SZIW) as compared to adjacent bathing water on Pensacola Beach, FL. The "swash zone" is that area of the beach continually washed over by waves. As the potential sources of enterococci were limited in this environment, only two library units, sea gull and human, were constructed. Identification of the beach isolates using a composite data set indicated a sea gull origin. The clonality of the beach isolates suggested that the beach environment selects certain subspecies of E. faecalis.

Vitellogenin mRNA regulation and plasma clearance in male sheepshead minnows, (Cyprinodon variegatus) after cessation of exposure to 17 beta-estradiol and p-nonylphenol.

Research was conducted to determine the kinetics of hepatic vitellogenin (VTG) mRNA regulation and plasma VTG accumulation and clearance in male sheepshead minnows (Cyprinodon variegatus) during and after cessation of exposure to either 17 beta-estradiol (E2) or para-nonylphenol (NP). Adult fish were continuously exposed to aqueous measured concentrations of 0.089 and 0.71 microg E2 per l, and 5.6 and 59.6 microg NP per l for 16 days using an intermittent flow-through dosing apparatus. Fish were sampled on days 8 and 16 of exposure followed by sampling at discrete intervals for up to 96 days post-exposure. At each interval five fish were randomly sampled from each concentration and hepatic VTG mRNA and serum VTG levels for individual fish determined by slot blot and direct enzyme-linked immunosorbent assay (ELISA), respectively. Exposure to E2 and NP resulted in a dose dependent increase in hepatic VTG mRNA and plasma VTG over the course of the 16-day exposure period. Mean plasma VTG levels at day 16 were >100 mg/ml for both high doses of E2 and NP, and >20 mg/ml for the low exposure treatments. Within 8 days post-exposure, hepatic VTG mRNA levels returned to baseline in both high and low E2 treatments but remained elevated 2-4 fold in the NP treatments. Due to a shortened sampling period, a clearance rate for plasma VTG in the 5.6 microg NP per l treatment could not determined. In the 0.089, 0.71 microg E2 per l, and 59.6 microg NP per l treatments, VTG levels began decreasing within 4 days after exposure cessation and exhibited an exponential rate of elimination from plasma. Clearance rates for 0.71 microg E2 per l and 59.6 microg NP per l were not significantly different (P=0.47), however, both demonstrated significantly higher rates of clearance (P<0.02) than observed in the 0.089 microg E2 per l treatment. Our results indicate that hepatic VTG mRNA rapidly diminishes after cessation of estrogenic exposure in sheepshead minnows, but plasma VTG clearance is concentration and time dependent and may be detected at measurable levels for months after initial exposure to an estrogenic compound.

Genomic sequences of two novel levivirus single-stranded RNA coliphages (family Leviviridae): evidence for recombinationin environmental strains.

Bacteriophages are likely the most abundant entities in the aquatic environment, yet knowledge of their ecology is limited. During a fecal source-tracking study, two genetically novel Leviviridae strains were discovered. Although the novel strains were isolated from coastal waters 1130 km apart (North Carolina and Rhode Island, USA), these strains shared 97% nucleotide similarity and 97-100% amino acid similarity. When the novel strains were compared to nine Levivirus genogroup I strains, they shared 95-100% similarity among the maturation, capsid and lysis proteins, but only 84-85% in the RNA-dependent RNA polymerase gene. Further bioinformatic analyses suggested a recombination event occurred. To the best of our knowledge, this is the first description of viral recombinants in environmental Leviviridae ssRNA bacteriophages.

Design and assessment of a real time reverse transcription-PCR method to genotype single-stranded RNA male-specific coliphages (Family Leviviridae).

A real-time, reverse transcription-PCR (RT-qPCR) assay was developed to differentiate the four genogroups of male-specific ssRNA coliphages (FRNA) (family Leviviridae). As FRNA display a trend of source-specificity (human sewage or animal waste) at the genogroup level, this assay provides a tool to help identify the origin of fecal contamination. Primers and probes were designed using complete genomic sequences from 29 FRNA phages. The final selection of primer/probe sets were based on (i) ability to amplify a single, specific product, (ii) genogroup specificity, (iii) lack of cross-reactivity, and (iv) experimental reproducibility and sensitivity over a range of target concentrations. Assay time was reduced by using heat-released viral RNA rather than purified RNA. For quality assurance, a custom RNA molecule was employed as an internal, non-competitive control. The usefulness of this method to identify sources of fecal contamination was tested on a total of 49 FRNA phages isolated from various warm-blooded animals, sewage and combined sewage overflow. FRNA phages from animal wastes were genotyped as 86% I, 4% III Q-like and 9% IV. Two sewage isolates typed to genogroup I and combined sewage overflow isolates genotyped as 40% II and 52% III. Primer specificity designed from this comprehensive sequence database may better discriminate FRNA from different sources.

A reverse transcription-PCR assay to distinguish the four genogroups of male-specific (F+) RNA coliphages.

Goals of reducing fecal contamination in recreational, drinking, shellfishing and other waters and accurately assessing risk from exposure can best be attained if tools to distinguish between sources of pollution are available. The male-specific RNA coliphage (FRNA) genogroups display a trend of source specificity. Reverse transcription-PCR (RT-PCR) can be effectively used for genotyping if specific primer sets are designed to be capable of identifying all members within each genogroup. In this study genogroup-specific primer sets were designed using a minimum of 5 to a maximum of 10 complete phage genome sequences from strains in each genogroup. With these primers and employing a heat-release procedure that eliminated the need for RNA purification an RT-PCR method for genotype identification of FRNA phages was developed. The four genogroup-specific primer sets generated discrete PCR amplicon sizes from a variety of environmental FRNA phage strains. Limits of detection, cross-reactivity and/or non-specific binding to strains from other genogroups were evaluated.