[Show abstract][Hide abstract] ABSTRACT: We investigated the prevalence and diversity of Escherichia coli strains isolated from surface waters from multiple watersheds within the South Nation River basin in eastern Ontario, Canada.
The basin is composed of mixed but primarily agricultural land uses. From March 2004 to November 2007, a total of 2,004 surface
water samples were collected from 24 sampling sites. E. coli densities ranged from undetectable to 1.64 × 105 CFU 100 ml−1 and were correlated with stream order and proximity to livestock production systems. The diversity of 21,307 E. coli isolates was characterized using repetitive extragenic palindromic PCR (rep-PCR), allowing for the identification of as many
as 7,325 distinct genotypes, without capturing all of the diversity. The community was temporally and spatially dominated
by a few dominant genotypes (clusters of more than 500 isolates) and several genotypes of intermediary abundance (clustering
between 10 and 499 isolates). Simpson diversity indices, assessed on a normalized number of isolates per sample, ranged from
0.050 to 0.668. Simpson indices could be statistically discriminated on the basis of year and stream order, but land use,
discharge, weather, and water physical-chemical properties were not statistically important discriminators. The detection
of Campylobacter species was associated with statistically lower Simpson indices (greater diversity; P < 0.05). Waterborne E. coli isolates from genotypes of dominant and intermediary abundance were clustered with isolates obtained from fecal samples collected
in the study area over the same period, and 90% of the isolates tested proved to share genotypes with fecal isolates. Overall,
our data indicated that the densities and distribution of E. coli in these mixed-use watersheds were linked to stream order and livestock-based land uses. Waterborne E. coli populations that were distinct from fecal isolates were detected and, on this basis, were possibly naturalized E. coli strains.
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli can be used to help identify sources of fecal contamination in the environment. Escherichia coli genotypic fecal libraries and pattern-matching algorithms were assessed for their effectiveness in correctly identifying sources. Fecal samples (n = 172) were collected from various sources from three agricultural landscapes in Canada. Escherichia coli isolates were fingerprinted using BOX- and enterobacterial repetitive intergenic consensus (ERIC) - polymerase chain reaction primers, revealing 769 and 1 057 distinct genotypes, respectively, for the 9 047 isolates collected in 2004 in Ontario. The average rate of correct classification (ARCC) was comparable for BOX- (48%) and ERIC-based (62%) libraries and between libraries with clones removed per sample (55%) and clones removed per unit (54%). ARCC increased with fewer classification units (from 44% to 65%). ARCC for k-nearest neighbour (64%) and maximum similarity (60%) algorithms were comparable, but maximum similarity had better sensitivity and specificity than k-nearest neighbour. Geographical and temporal shifts in community composition resulted in loss of accuracy. Several ERIC genotypes (n = 112) were common between sources and were removed from the library, improving ARCC (77%). The latter library proved to be more accurate, but its accuracy with respect to sourcing environmental isolates remains to be tested.
Canadian Journal of Microbiology 01/2010; 56(1):8-17. DOI:10.1139/w09-113 · 1.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Confined livestock production farms typically store their wastes prior to land application. Here, we employed three complementary approaches to evaluate changes in the population structure and stability of virulence genes in Escherichia coli during manure storage on a commercial farm that housed healthy swine. Isolates were genotyped by repetitive extragenic palindromic PCR using the BOXA1R primer and evaluated for the presence of selected virulence genes by PCR. Isolates obtained from the manure holding tank (n = 392) carried estB, fedA, stx(2e), astA, paa, aida-I, and sepA at lower frequencies than isolates obtained from fresh feces (n = 412). Fresh fecal material from the barn was added into diffusion chambers and immersed in the manure holding tank for 7 weeks. The fecal E. coli population was initially dominated by a single genotype, all isolates of which carried fedA and aida-I. After 7 weeks, a genotype that did not carry any virulence genes dominated the surviving population. In a second experiment, 48 fecal isolates of E. coli that varied in their genotypes and virulence gene complement were incubated in diffusion chambers in the manure holding tank for 3 weeks. Over 95% of the inoculum population carried at least one virulence gene, whereas after 3 weeks 90% of the recovered isolates carried no virulence genes. Taken together, these results indicate that during commercial manure storage, there was a significant reduction in the carriage of these virulence genes by E. coli. We propose that loss of virulence genes from enteric pathogens in the farm and in natural environments may, if generalized, contribute to the attenuation of a public health risk from contamination with agricultural wastes.
[Show abstract][Hide abstract] ABSTRACT: Repetitive extragenic palindromic PCR fingerprinting of Escherichia coli is one microbial source tracking approach for identifying the host source origin of fecal pollution in aquatic systems. The construction of robust known-source libraries is expensive and requires an informed sampling strategy. In many types of farming systems, waste is stored for several months before being released into the environment. In this study we analyzed, by means of repetitive extragenic palindromic PCR using the enterobacterial repetitive intergenic consensus primers and comparative analysis using the Bionumerics software, collections of E. coli obtained from a dairy farm and from a swine farm, both of which stored their waste as a slurry in holding tanks. In all fecal samples, obtained from either barns or holding tanks, the diversity of the E. coli populations was underrepresented by collections of 500 isolates. In both the dairy and the swine farms, the diversity of the E. coli community was greater in the manure holding tank than in the barn, when they were sampled on the same date. In both farms, a comparison of stored manure samples collected several months apart suggested that the community composition changed substantially in terms of the detected number, absolute identity, and relative abundance of genotypes. Comparison of E. coli populations obtained from 10 different locations in either holding tank suggested that spatial variability in the E. coli community should be accounted for when sampling. Overall, the diversity in E. coli populations in manure slurry storage facilities is significant and likely is problematic with respect to library construction for microbial source tracking applications.