Have you tried the SPLITSTREE software? (http://www.splitstree.org/), it is very helpful to resolve phylogenetic relations between individuals inside the same group and it has various algorithms to construct different trees. Is super user-friendly, and due the size of your data, it would work very fast.
Escherichia coli genetics and evolution is especially interesting, not because of any property of the bacteria, but because of human interest in these bacteria such that we have complete genomes of thousands of isolates, we have long term evolution studies from cloned isolates, and humans have been intently studying them in many other ways for many decades. The classification an nomenclature of strains or lineages within Escherichia coli has several different levels, from serology (the O and H antigens for example) to many levels of genotyping. The most appropriate level to use for environmental isolates from soils, is not the most appropriate level to use for isolates from outbreaks of disease in humans.
I have attached a few papers from my collection of good genomics paper about these bacteria, but I have more, so write to me if you can provide more details about what level of classification you are most interested in.