Optimization and validation of rep-PCR genotypic libraries for microbial source tracking of environmental Escherichia coli isolates.

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.

Population structure of two beetle-associated yeasts: comparison of a New World asexual and an endemic Nearctic sexual species in the Metschnikowia clade.

The genetic structure of two related yeast species, one sexual and one asexual, was compared using polymorphic DNA markers. Although both yeasts propagate by asexual budding of haploid cells, Metschnikowia borealis reproduces sexually when compatible strains come in contact. To what extent this has occurred in nature was not known. As Candida ipomoeae is a closely related, asexual species, the two yeasts provide an excellent model system to assess the role of sexual reproduction in a biogeographic context. Natural isolates of the two species were characterized using several polymorphic DNA markers. As predicted for an organism whose reproduction is strictly clonal, C. ipomoeae exhibited low haplotype diversity, high linkage disequilibrium, and high population differentiation. In contrast, M. borealis had unique haplotypes in most isolates, lower population differentiation, and little linkage disequilibrium, demonstrating that sexual recombination is prevalent. Geographic gradients were identified in both species, indicating that historical and climatic factors both play a role in shaping the populations. The spatial structure is also thought to be influenced by the ecology of the small floricolous beetles (family Nitidulidae) that vector the yeasts. For example, Hawaiian strains of C. ipomoeae show evidence of having undergone a genetic bottleneck, most likely when the vector was introduced to the islands. The two haplotypes found in Hawaii were nearly identical and were also found in North and Central America. M. borealis had a more continuous distribution where the genetic markers follow latitudinal and longitudinal gradients.