Randall E. Hicks

Presence and growth of naturalized Escherichia coli in temperate soils from Lake Superior watersheds

ABSTRACT The presence of Escherichia coli in water is used as an indicator of fecal contamination, but recent reports indicate that soil populations can also be detected in tropical, subtropical, and some temperate environments. In this study, we report that viable E. coli populations were repeatedly isolated from northern temperate soils in three Lake Superior watersheds from October 2003 to October 2004. Seasonal variation in the population density of soilborne E. coli was observed; the greatest cell densities, up to 3 × 103 CFU/g soil, were found in the summer to fall (June to October), and the lowest numbers, ≤1 CFU/g soil, occurred during the winter to spring months (February to May). Horizontal, fluorophore-enhanced repetitive extragenic palindromic PCR (HFERP) DNA fingerprint analyses indicated that identical soilborne E. coli genotypes, those with ≥92% similarity values, overwintered in frozen soil and were present over time. Soilborne E. coli strains had HFERP DNA fingerprints that were unique to specific soils and locations, suggesting that these E. coli strains became naturalized, autochthonous members of the soil microbial community. In laboratory studies, naturalized E. coli strains had the ability to grow and replicate to high cell densities, up to 4.2 × 105 CFU/g soil, in nonsterile soils when incubated at 30 or 37°C and survived longer than 1 month when soil temperatures were ≤25°C. To our knowledge, this is the first report of the growth of naturalized E. coli in nonsterile, nonamended soils. The presence of significant populations of naturalized populations of E. coli in temperate soils may confound the use of this bacterium as an indicator of fecal contamination.

Presence and Sources of Fecal Coliform Bacteria in Epilithic Periphyton Communities of Lake Superior

ABSTRACT Epilithic periphyton communities were sampled at three sites on the Minnesota shoreline of Lake Superior from June 2004 to August 2005 to determine if fecal coliforms and Escherichia coli were present throughout the ice-free season. Fecal coliform densities increased up to 4 orders of magnitude in early summer, reached peaks of up to 1.4 × 105 CFU cm−2 by late July, and decreased during autumn. Horizontal, fluorophore-enhanced repetitive-PCR DNA fingerprint analyses indicated that the source for 2% to 44% of the E. coli bacteria isolated from these periphyton communities could be identified when compared with a library of E. coli fingerprints from animal hosts and sewage. Waterfowl were the major source (68 to 99%) of periphyton E. coli strains that could be identified. Several periphyton E. coli isolates were genotypically identical (≥92% similarity), repeatedly isolated over time, and unidentified when compared to the source library, suggesting that these strains were naturalized members of periphyton communities. If the unidentified E. coli strains from periphyton were added to the known source library, then 57% to 81% of E. coli strains from overlying waters could be identified, with waterfowl (15 to 67%), periphyton (6 to 28%), and sewage effluent (8 to 28%) being the major potential sources. Inoculated E. coli rapidly colonized natural periphyton in laboratory microcosms and persisted for several weeks, and some cells were released to the overlying water. Our results indicate that E. coli from periphyton released into waterways confounds the use of this bacterium as a reliable indicator of recent fecal pollution.

Archaeal Nucleic Acids in Picoplankton from Great Lakes on Three Continents

Phylogenetic analysis of PCR-amplified 16S rRNA genes revealed the presence of archaea in picoplankton collected from the Laurentian Great Lakes in North America, Africa’s Lake Victoria, and Lakes Ladoga and Onega in northeastern Eurasia. From 1 to 10% of the rRNA extracted from size-fractionated picoplankton (>0.2 µm but <1.2 µm) collected in the epilimnion and hypolimnion of these lakes was specific to the Archaea, whereas the majority of rRNA was derived from Bacteria. Analysis of the 16S rRNA genes cloned from these samples indicated they were closely related to crenarchaeal sequences that have been widely characterized from marine environments. The presence of nearly identical 16S rDNA clones in several of these geographically disparate lakes suggests a cosmopolitan distribution of specific subgroups of these Archaea in freshwater environments. Despite their abundance in the water column of freshwater lakes, we have no representatives of these crenarchaea in pure culture, and so their physiological characteristics and ecological role remain unknown.

Direct-count Estimates of Fungal and Bacterial Biovolume in Dead Leaves of Smooth Cordgrass (Spartina alterniflora Loisel.)

Two types of hyphal-extraction, direct-count methods of estimating fungal biovalume in standing-dead, autumn leaves of Spartina alterniflora were compared with a clearing+staining method which does not require homogenization. Bacterial biovolume also was estimated, by an acridine-orange direct-count method. Type of homogenization had little effect on measured fungal volume, but counts made using water-soluble-aniline-blue epifluorescence were consistently lower than those made using phase-contrast (by 6–10x). Clearing+staining could not be used to estimate hyphal lengths, but was of use in estimating total ascocarp volume (=0.06 mm3 per mm3 of leaf). Estimated fungal hyphal volume was approximately 0.27 mm3 per mm3 of leaf. Bacterial volume was <3% of fungal volume.

Deposition, resuspension, and decomposition of particulate organic matter in the sediments of Lake Itasca, Minnesota, USA

Sediment traps were used to investigate the settling, resuspension, and decomposition of particulate organic matter in Lake Itasca, MN (USA). Traps were deployed in the epilimnion and hypolimnion of the deepest basin during June, 1988, sampled twice during stratified conditions (August, September) and once after the lake had mixed (October). The downward flux of particulate material increased from summer to fall. The net sedimentation of organic matter ranged from 0.6 to 2.3 g m−2 d−1 at 4 m and increased to 2.1 to 3.2 g m−2 d−1 two meters above the bottom sediment indicating that resuspended sediment was at least 33% of the settling mass during all periods. The C:N ratios of captured particles (6.8–9.5) were between the ratios of plankton (5.8 to 6.8) and the sediments (9.9 to 10.2) but smaller than the ratios of terrestrial organic materials (13.5 to 222). The monosaccharide compositions of the entrapped particles were similar to plankton samples and different from the distinct composition of the sediments. Capture of rebound particles similar to the primary flux and not decomposition may have been responsible for this similarity. Total monosaccharide concentrations were lower in the sediments than in entrapped particles. Individual sugars exhibited different patterns of accumulation in the sediments. Glucose was lowest in sediments when the relative concentrations were compared to those in source materials and entrapped particles. In contrast, sediments had the highest rhamnose and fucose concentrations. Bacterial biomass could only account for small portions of these sugars in the sediment. The distinct monosaccharide composition of resuspended sediments was not strongly recorded in materials captured by the sediment traps even after the lake had mixed.

Loss and recycling of amino acids and protein from smooth cordgrass (Spartina alterniflora) litter

We investigated the source and composition of free and protein-bound amino acids during the decomposition ofSpartina alterniflora Loisel in laboratory percolators and in a field experiment in the Great Sippewissett Marsh (Falmouth, Massachusetts). In the percolator experiment, 50% of the nitrogen (N) could be extracted fromS. alterniflora litter in 16 d. This extract consisted of dissolved free amino acid N (28%), suspended protein amino acid N (16%), inorganic N (12%), and nitrogen from unidentified compounds (44%). Much of the free amino acid nitrogen was utilized by detrital microorganisms, resulting in a greater loss of suspended protein amino acid (SPAA) nitrogen from the biologically active percolator due to microbials biomass. Suspended microbial mass accounted for at least 50% of the SPAA washed out of the biologically active percolator. In the field study, 38% of the original litter nitrogen was leached fromS. alterniflora litter in litterbags during the first 13 d. After this initial leaching period, the concentration (41% to 69% of total nitrogen) and composition of most amino acids bound in the litter did not change over the 23-month period of the experiment. Increases in microbial protein did not account for increases in total nitrogen which occurred during the decomposition of the litter. Similarly, adsorbed ammonium did not appear to be responsible for this increase.

Distribution of genetic markers of fecal pollution on a freshwater sandy shoreline in proximity to wastewater effluent

Water, sand, and sediment from a Lake Superior harbor site continuously receiving wastewater effluent was sampled monthly for June to October 2010 and from May to September 2011. Understanding the dynamics of genetic markers of fecal bacteria in these matrices is essential to accurately characterizing health risks. Genetic markers for enterococci, total Bacteroides, and human-associated Bacteroides were measured in site-water, sand, and sediment and in final effluent by quantitative PCR. The similarity between the quantity of molecular markers in the water column and effluent indicated that the abundance of genetic markers in the water column was likely controlled by effluent inputs. Effluent turbidity was positively correlated (p ≤ 0.05) with AllBac and HF183 in final effluent and AllBac in the water column. In sand and sediment, Entero1 and AllBac were most abundant in the upper 1-3 cm depths, whereas HF183 was most abundant in the upper 1 cm of sand and at 7 cm in sediment. The AllBac and Entero1 markers were 1- and 2-orders of magnitude more abundant in sand and sediment relative to the water column per unit mass. These results indicate that sand and sediment may act as reservoirs for genetic markers of fecal pollution at some freshwater sites.

Escherichia coli Populations in Great Lakes Waterfowl Exhibit Spatial Stability and Temporal Shifting

ABSTRACT Populations of Escherichia coli from juvenile and adult ring-billed gulls, juvenile common terns, and adult Canada geese were sampled over 6 years at five locations on Lake Superior (Duluth, MN, and Wisconsin) and Lake Michigan (Wisconsin, Illinois, and Indiana) to determine the extent of spatial and temporal variability in E. coli strains. Strain identity was determined using horizontal fluorophore-enhanced repetitive element palindromic DNA fingerprinting. Multivariate statistics were used to determine if spatial or temporal changes in E. coli populations occurred in waterfowl species. Pairwise multivariate analyses of variance revealed that E. coli populations of adult gulls from three regions of Lake Michigan and the Wisconsin shore of Lake Superior were similar to one another but different from an E. coli population of gulls from the Duluth region of Lake Superior. Juvenile and adult gulls from the Duluth area harbored different E. coli populations. The E. coli strains from juvenile gulls, however, were similar to those found in juvenile terns obtained from the same island rookery. Temporal changes in E. coli populations from several waterfowl species were also demonstrated for this site. Although portions of source tracking databases might be successfully used in other geographic regions, it is clear that juvenile birds should not be the sole source of E. coli strains used for source tracking databases, and multiple-year libraries should be constructed in order to identify the potential sources of E. coli in the environment.

An improved gas chromatographic method for measuring glucosamine and muramic acid concentrations

A method of simultaneously measuring glucosamine and muramic acid concentrations in marsh grass litter was developed. Spartina alterniflora samples were preextracted with acetone to remove lipids containing amino sugars and then hydrolyzed in 6 N HCl (100 degrees C, 4.5 h). Amino sugars in the hydrolysates were isolated by ion-exchange chromatography, which gave good recoveries (greater than 90%) and reproducibility (CV less than 5%). Isolated amino sugars were converted to O-methyloxime acetates. beta-Phenylglucose and N-methylglucamine were added as internal standards. Sample derivatives were quantified by capillary column gas chromatography. OV-101 and SE-54 capillary columns completely separated glucosamine and muramic acid from other amino sugars. The detection limit of glucosamine and muramic acid during gas chromatographic analysis was below 30 pmol using splitless-mode injection (SE-54 column). Filamentous fungal and procaryotic biomasses may be estimated simultaneously by using glucosamine and muramic acid biomass conversion factors in conjunction with this method.

Annual and offshore changes in bacterioplankton communities in the western arm of lake superior during 1989 and 1990

A shallow site in the western arm of Lake Superior near Duluth, Minnesota was sampled bimonthly from May to October during 1989 and 1990 to identify seasonal and annual changes in bacterioplankton communities. The greatest change in bacterioplankton abundance was between 1989 (1.48 × 109/L ± 0.06 SE) and 1990 (1.14 × 109/L ± 0.06 SE). The majority of bacterial cells (65%) were cocci. Individual cells were larger during 1989 (0.067 μm3 ± 0.007 SE) than 1990 (0.025 μm3 ± 0.002 SE). Although the rate of thymidine incorporation varied from 0.2 to 47.0 pmol/L/h over both years (mean = 12.1 pmol/L/h ± 1.3 SE), no consistent temporal or spatial changes were detected. Bacteria were more abundant (∼2×) and productive (∼10×) at the mouth of the Lester River than offshore of this site. During July and August, a benthic nepheloid layer (BNL) formed at shallow offshore sites but bacterioplankton abundance and production in this BNL were usually similar to values measured in the hypolimnion. Three additional sites from the Duluth basin northeast to the Chefswet basin were sampled during late summer (Aug-Sept) 1990 to identify spatial differences in bacterioplankton communities. Although the number of bacteria was often greater at shallower sites compared to deeper sites further offshore, a strong gradient was not found and bacterial production was similar at all sites. These results may be due in part to the lake basin morphology in this region of Lake Superior, as well as the time when these additional offshore sites were investigated.