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News

The Unculturables


 

Unculturable bacteria are not necessarily uncharacterizable. Microbiological techniques, like PCR and DNA sequencing of "housekeeping" genes, has allowed scientists to continue to gain understand about microbes, whether or not they are culturable. However, the ability to fight human infections and develop cures requires these unculturables to become cultured, so the microorganisms can be grown and studied in laboratory settings. 

The question remains, why are these organisms 'unculturable' ?

"The Unculturables"

Culturing bacteria has always been a challenge. At first, it was a struggle to find the right media; eventually shifting from broth to agar, which is still used today. The change to solid media helped physically separate bacterial colonies, so that it was possible to avoid mixtures of microorganisms.

However, these organisms, although physically separated, did not necessarily grow on this agar. In fact, it is estimated that only 2% of all bacteria can be cultured in lab. When focusing on oral bacteria, about 50% are culturable. In contrast, almost all colonic microflora are unculturable. Because of the inability to culture microorganisms, there can be weak understanding of diseases. For example, the causative agent of syphilis (Trephonema pallidum). has never been cultured

Instead of continuing to struggle separating and culturing the organisms, molecular biology has made it possible to study mixed bacterial samples. A common method is using DNA sequences of genes (or even proteins) to create phylogenetic trees showing evolutionary relationships. For these types of technologies, “housekeeping” genes are used because they typically do not evolve and are widespread throughout different species.  An example of a very easy-to-match gene throughout different species is the 16S ribosomal RNA gene.  In addition to DNA sequencing, another method called PCR can be used to amplify DNA and maximize the amount of data recovered from the sample. These genes, once cloned, can be sequenced separately. Note, the microorganisms themselves are not required to be physically separated to determine this information!

Many oral bacteria have been studied using the above techniques. Because of these molecular technologies, it has been discovered that the majority of novel bacteria species isolated from oral infections can be categorized as either Cytophagas or low G+C Gram-positive bacteria.

There are many reasons why bacteria may not grow in a sample. It could be that the required nutrient is simply not present in the medium, or it could be that the medium itself is toxic to the microorganism. In addition, other bacteria growing in the culture could produce substances that target the desired organism, preventing its growth. In general, bacteria are extremely interdependent; especially for growth. For example, oral bacteria have evolved into a community, or biofilm, which grow togethers. Lastly, the use of solid agar may disrupt the cytokine networks between bacteria signaling. These communication networks make it possible for biofilms, like dental plague, to thrive. Without it, the bacteria cannot communicate amongst themselves, which prevents growth.

In conclusion, the inability of some microorganisms to be cultured is a setback, but it is one that science has been able to sidestep past. It is still possible to characterize and individually study bacteria through different forms of molecular biology without growing the organisms on agar.  However, the more organisms that are able to be cultured, the better, because new discoveries for human infections can be made when the microflora can be reproduced and tested in labs. The future of science stresses that the “unculturables” become cultured.

Reference: Unculturable bacteria - the uncharacterized organisms that cause oral infections, can be found at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1279316/