Team:Paris Bettencourt/Project/Foot Odor

Aims and Achievement

Foot odor is generally perceived as socially awkward and negative. Although there are a many commercially available solutions for this problem, current products indiscriminately target bacteria on the foot skin microbiome. These type of products can have negative effects on skin health and microbiome dynamics. We aim to develop a targeted approach to prevent foot odor, by selectively killing microbes responsible for the biosynthesis of volatile compounds which compose the characteristic stinky feet smell, without destroying the beneficial microbes.

Figure 1. B. subtilis is gram positive bacterium which is commonly found in the human foot flora, which degrades the leucine aminoacid present in the sweat to produce isovaleric acid. Our strategy is to perturb the leucine degradation pathway by knocking out the vital enzymes in the pathway. The leucine dehydrogenase knock out mutants lacks the capability to produce leucine and prevents the conversion of leucine to 4-methyl 2-oxopentanoate. The isovaleryl-coA dehydrogenase α and β subunit knock mutants lacks the capability of conversion of 4-methyl 2-oxopentanoate to isovaleryl-coA.

Introduction

Foot odor (or bromhidrosis) is a condition in which a particular type of body odor, generally considered to be unpleasant, gets produced by bacteria during the catabolism of nutrients present in the sweat.

Although sweat is almost odorless, the microbial volatile compounds that are formed as by-products of bacterial metabolism are perceived as an unpleasant smell.
Bacillus subtilis, a common bacterium that lives on feet and it is thought to produce the malodorous molecules behind this socially awkward condition. One of the molecules produced by B. subtilis uses the leucine degradation pathway to produce isovaleric acid, which smells like parmesan cheese. It is considered to be to dominant odor tone of bromhidrosis.

Figure 3. The B. subtilis wt and mutant strains were grown overnight in M9 minimal medium. A double blind smell test was performed. The subjects were asked to rank the tubes in the order of 0 to 5. An analysis with 95% confidence interval was made. Though there is variation observed between the wt strain and the bcd knock out strain, it is not statistically significant. In the case of bkdaα knock out strain and the wt strain there is a statistically significant variation observed. This confirms that the bkdaα knock out strain produces less malodor compare to the wild type.

Results

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Methods

Synthetic sweat: The chemical constituents present in the human sweat were analyzed with the aid of gas chromatography and mass spectroscopy. Synthetic sweat was prepared by diluting the exact concentration of all the amino acids and salts found in human sweat, the pH of the synthetic sweat was adjusted to 6.5 with the aid of a pH meter. The synthetic sweat was then autoclaved and stored at 4°C to avoid bacterial contamination.

M9 minimal media: The minimal media was prepared by diluting 1ml of 50% glucose 1.1 ml of mgso4.3H20 2ml of 1% w/v casmino acids 2ml of 10mg/ml of tryptophan in 100 ml of distilled water The media is then filter sterilized under a laminar air flow cabinet.

Strain: The leucine dehydrogenase, isovaleryl coA alpha subunit and isovaleryl coA beta subunit. The knockout strains were obtained from Bacillus Genetic Stock Centre, where the knockout library of the B. subtilis was generated by replacing each gene with the erythromycin cassette. The mutant strains are trp- so they require supplementation of tryptophan in the growth media. The knockout strains were validated with the PCR reaction.

Growth curve: The growth kinetics of the wild type and the mutant strains of B. subtilis were studied with the aid of micro plate reader to have insights about the fitness advantage and disadvantage within the mutant and wild type strain.

Smell test: The presence of isovaleric acid in the bacterial culture was sensed with double blind smell test, where neither the subject nor the experimenter is aware of the tube labels. In order to optimize this smell test we grew the B. subtilis in odorless M9 minimal media. The media was supplemented with 0.1% of leucine to study the influence of leucine in production of foot odor.

Sock experiment: The bacterial cell culture is diluted in synthetic sweat till it reaches 0.1 OD. The sock was soaked in the synthetic sweat and hanged till it dry. After this step 2 cm2 of sock was cut and soaked in 5ml of 1% PBS. The tubes were stirred gently and supernatant is diluted in PBS to prepare 1X and 10X concentration of B. subtilis. The bacteria were then plated on LB agar.