Team:Paris Bettencourt/Project/Old People Smell

Figure 1. Main idea of 'Teen spirit'. Samples were taken form 3 different sites of the body with a inoculating loop. Agar plates having as the main carbon source 2-nonenal (old people smell, were striked with the loop. Three different species of bacteria have been identified on the agar plates as candidaties to 2-nonenal degradation.

Aims and Achievement

‘Old people smell’ is a characteristic smell of people over 40 caused by the appearance of 2-nonenal in sweat. In this project we isolated and characterized natural bacteria that can metabolize 2-nonenal and therefore attenuate its smell. These bacteria could be used in a cosmetic product to reduce 2-nonenal in body odor.

Figure 4. Diffusion model of 2-nonenal through air.This animation shows the concentration of 2-nonenal diffusing through air from an old person over a time period of 0 to 1000 hours. From the graph on the left of the animation, you can see that the odor threshold is reached very early in the model, given that the concentration of 2-nonenal at the skin surface is kept at a constant concentration of 2.6 +/- 3.5 ppm.



Figure 5. Putative 2-nonenal degradation by Aldehyde Dehydrogenase into 2-nonoic acid by skin bacteria


Figure 6. Colonies of E.coli and M. luteus on LB plates (right) and M9 2-nonenal (left)



Box 1.Selected information about isolated bacterial strains groiwing in the presence of 2-nonenal as a major carbon source



Figure 7. Human detection thresholds for 2-nonenal. 12 human smellers were asked to identify pure 2-nonenal in water at the following fold dilutions: 102 10⁴ 10⁶ and 10⁸. All samples were solubilized with tween 80 at a concentration of 0.05%. All smell tests were double-blind. Samples are scored by the percentage of smellers who were able to positively distinguish them from tween-only controls.



Figure 8. Detection of toxicity threshold of 2-nonenal on M.luteus. The toxicity of different 2-nonenal concentrations ranging from 10000ppm to 312.5 ppm by a factor of 1/2 in LB-agar plates was tested on M.luteus. We observed an anti-reciprocal relationship between 2-nonal amount in the medium and number of colonies. The critical concentration is 1250ppm, above which no colonies have been observed.



Figure 9. Smell test results. 15 people smelled the samples of M.luteus , C. flaccumfaciens in LB with 625ppm 2-nonenal and LB with 625ppm 2-nonenal only (control), double-blind. The 2-nonenal smell was the least intense in the culture containing M.luteus (p-value: 1.04e-09, HSD turkey test). The culture of C.flaccumfaciens was confirmed also having less 2-nonenal smell (p-value: 9.72e-05, HSD turkey test). Error bars represent t-statistics 95% confidence intervals.

Results

Diffusion Model of 2-nonenal through air

A simple model was created for the diffusion of 2-nonenal into the air using COMSOL Multiphysics (a physics-based interface to solve partial differential equations). The model is shown in Fig.4. From literature, it was found that the odor threshold of 2-nonenal in air is approximately 10^-4 mg/kg air (Grosch, 2009). This odor threshold was reached approximately 7 hours in the model, at a distance of 5 cm from the skin surface.

Isolation of strains on 2-nonenal

We sought to isolate bacterial strains adapted to use 2-nonenal as a carbon source. Such strains could hypothetically be used to actively scavenge 2-nonenal from the skin, neutralizing the smell (Fig. 5). We chose to look for these strains within the natural human skin flora. Human sweat is rich in fatty acids (Callewaert et al., 2014), and lipophilic phenotypes are common among skin isolates. Natural skin bacteria are pre-adapted to the skin environment, so are more likely to be viable and metabolically active.

We prepared minimal 2-nonenal plates and inoculated them with human skin samples. M9 agar plates were prepared with 0.2% 2-nonenal solubilized with 0.05% tween 80. No other carbon sources were present. An inoculating loop was used to streak plates with samples collected from human forehead, hands, and outer nose. In total we sampled 3 body sites from 8 individuals.

We isolated three bacteria strains capable of growing on 2-nonenal (Fig. 6). Colonies appeared after 1 week. The strains were identified by 16S sequencing with universal primers (Box 1, information on each strain). We chose to focus further experiments on Micrococcus luteus as it performed the best in the M9 the 2-nonenal medium.

M. luteus is a natural human skin bacterium and a well-described oligotroph, or nutrient scavenger. Their genome sequence indicates a complete fatty acid degradation pathway, meaning they can plausibly degrade 2-nonenal.

We next sought to characterize the degradation of 2-nonenal by M. luteus. We first identified the smell detection threshold of 2-nonenal. Human smellers could consistently detect 2-nonenal at a concentration of 1000ppm (Fig. 7).

While this work was in progress, we learned that 2-nonenal is bactericidal for many species at concentrations as low as 1000 ppm (Cho et al., 2004) We therefore determined a threshold of toxicity of 2-nonenal for M. luteus. LB agar plates were prepared with varying concentrations of 2-nonenal. Growth of M. luteus was significantly inhibited at 2-nonenal concentrations above 1250ppm (Fig. 8).


2-nonenal smell attenuation

M.luteus in medium containing 625 ppm 2-nonenal lowers the intensity of the ‘old people smell’ significantly (p-value: 1.04e-09, HSD turkey test), while C. flaccumfaciens lowers the 2-nonenal smell mildly (p-value: 9.72e-05, HSD turkey test) in comparison to the medium with 625ppm 2-nonenal medium only (Fig. 9).

Methods

Diffusion model of 2-nonenal through air

The model is based on a 2D axisymmetric geometry, with the air modeled as the space 5 cm around the skin surface. Here, the skin surface is kept at a constant concentration, C0 = 2.6 +/- 3.5 ppm (in terms of concentration of skin surface lipids) (Haze et al., 2001). The diffusivity of 2-nonenal through the air was calculated using the Chapman-Enskog theory of gas diffusivity given by the following equation:

D = (1.858E-3 * T^(3/2) * (1/M1 + 1/M2)^(1/2)) / (p * s12^2 * w)

where D is the diffusivity of the 2-nonenal
T is the room temperature, 298 K
p is the air pressure, 1 atm
M1 is the mass of the 2-nonenal, 140.22 g/mol
M2 is the mass of the air, 29 g/mol
s12 is the average collision diameter, which is around 340 Angstroms for gas molecules in air, and w is the dimensionless temperature-dependent collision integral, usually on the order of 1.

Thus, the diffusion coefficient of 2-nonenal was tabulated to be 1.687E-9 m^2/s.

The odor threshold was a value found in literature for the diffusion of (E)-2-nonenal (Grosch, 2009).

Isolation of strains on 2-nonenal
M9 minimal agar with 0.2% 2-nonenal was used for selection of 2-nonenal metabolizing strains. 0.05% tween 80 was used to solubilize the 2-nonenal. No other carbon sources were present. An inoculating loop was used to streak plates with samples collected from human forehead, hands, outer nose and armpit. In total we sampled 3 body sites from 8 individuals. The plates were incubated for one week at 37 °C and then checked for the appearance of colonies.

Colony PCR and 16S sequencing
Colony PCR was performed with universal 16S rRNA specific primers 8F (5'-AGA GTT TGA TCC TGG CTC AG) and 1492R (5'-CGG TTA CCT TGT TAC GAC TT). Sanger sequencing was performed commercially by GATC Biotech (Germany). Strains were identified by aligning the sequenced DNA fragments to the greengenes database. Hits with the highest similarity were used to name isolates.

2-nonenal detection threshold
Serial dilutions of pure 2-nonenal were prepared in water at dilution factors of 10² 10⁴ 10⁶ and 10⁸. All samples were solubilized with tween 80 at a concentration of 0.05%. All smell tests were double-blind. Samples were scored by the percentage of smellers who were able to positively distinguish them from tween-only controls. Samples were evaluated by 12 volunteers 20-30 years old, without serious smell disability.

Toxicity threshold
Toxicity of 2-nonenal was evaluated at a range of concentrations (300 to 10000 ppm) on LB agar plates. M. luteus liquid cultures were grown to saturation in LB, serially diluted, and plated on LB - 2-nonenal. CFU counts were determined following overnight incubation.

Attenuation of 2-nonenal odor
2-nonenal was dissolved in LB at 625 ppm, near the sensitivity threshold we determined for the human nose. Samples were solubilized with tween 80 at 0.05%. LB-nonenal was innoculated with M. luteus, C. flaccumfaciens, or left as an untreated control. Samples were allowed to incubate overnight at 37 °C. 20 individuals scored all the three samples according to the scale 0 - no smell ; 1 - light smell ; 2 - medium smell, 3 - strong smell. All smell tests used randomized tube labels and opaque tubes to hide the appearance of the media. One-way anova was performed to compare mean scores of the three samples followed by HSD Tukey multiple comparisons of means with 95% family-wise confidence levels.

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