Abstract
The characterization of low complexity (only a few species present) bacterial communities or commercial consortia products in terms of microbial composition can require a variety of molecular techniques for supporting forensic investigations. We examined a low complexity commercial consortium productfor water treatment application as a model for a tiered molecular approach to studying microbial communities. PCR amplification of 16S rDNA and cpn60 genes were performed on total genomic DNA extracted from the consortium. First, the PCR amplicons were cloned, sequenced and subjected to both DGGE and RFLP analysis, or they were fluorescently labeled and hybridized to a dual backbone taxonomic DNA microarray. Secondly, total genomic DNA from the commercial consortium was subjected to quantitative PCR to determine the concentration of the different components. The data showed that the dual backbone DNA microarray is extremely useful as a first step to identify the major members of the consortium, including lot-to-lot variation of the commercial product, as validated by independent analyses. More importantly, the DNA microarray proved to be a useful screening tool to detect unexpected and potentially pathogenic microbes in the commercial product. This tiered approach using a DNA microarray screen can be a useful guide for application of more rapid and targeted molecular tools in forensic investigations of microbial communities.
Highlights
A serious difficulty facing both federal regulatory agencies and those involved in environmental risk assessments is the lack of information concerning the specific composition of commercial microbial bioproducts or consortia
Two Enterococcal 16S rRNA (E. saccharolyticus and E. faecium) probes were positive for the expired lot of Product A Figure 1A whereas only the E. saccharolyticus probe was positive in the new lot of Product A (Figure 1B)
2) The Quantitative real time Polymerase Chain Reaction (Q-PCR) approach provides an excellent means of quantifying the levels of bacteria in consortia in order to identify the major bacterial species
Summary
A serious difficulty facing both federal regulatory agencies and those involved in environmental risk assessments is the lack of information concerning the specific composition of commercial microbial bioproducts or consortia. This is partially due to the lack of standardized methodologies for characterizing microbial communities or commercial microbial consortia even though there are numerous biochemical, microbiological and molecular biology techniques [1]. For bacterial communities of lower complexity, molecular techniques like DGGE or T-RFLP on taxonomic amplicons can provide a rapid partial index of biodiversity using total extracted DNA [2]. Laborious and time consuming, small subunit rRNAgene or metagenomic sequencing remains one of most accurate means of determining microbial community content and diversity
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