B. thetaiotaomicron in the Presence of Yogurt

The microbiome of humans contains an estimated 100 trillion microbial cells as well as an estimated quadrillion viruses. It is responsible for energy harvest, the breakdown of indigestible carbohydrates, the production of important biological molecules, and, most importantly, proper immune system development.

The microbiome of humans contains an estimated 100 trillion microbial cells as well as an estimated quadrillion viruses (3). It is responsible for energy harvest, the breakdown of indigestible carbohydrates, the production of important biological molecules, and, most importantly, proper immune system development.

Microbiome development begins at birth, and by eleven months of age, infants have their own unique microbiome. The microbiome can be influenced by genetics, as shown in a study conducted by Benson et al. (2). In their study, different breeds of mice were crossbred and 645 fecal profiles were sequenced, with results showing 64 preserved taxonomic phyla. Variation in the amounts of these bacterial groups were analyzed using SNP markers that identified 18 host quantitative trait loci (QTL) groups, which were attributed to the variation in the microbiomes of the mice. Microbiome colonization can also be affected by life events such as antibiotic usage, food consumption, and environmental exposures (3).

Most microbiome-host interaction occurs in the gut of humans, and as such, the gut is heavily colonized by a variety of microorganisms (7). Phylogenetic analysis has shown that bacterial organisms belonging to the Firmicutes and the Bacteroidetes phyla dominate the human gut. More importantly, these analyses have shown that a disruption of the microbiome is been linked to chronic diseases such as obesity, inflammatory bowel diseases, and diabetes (3). Studies have indicated that in the obese gut, the ratio of Bacteroidetes to Firmicutes is altered. Obesity is linked to higher levels of Lactobacilli (a Firmicute) species and lower levels of Bacteroidete species in the gut (6). A study performed by Armougom et al. showed the average number of Bacteroidetes and Firmicutes in twenty lean individuals was 1.35E+10 and 2.16e+10, respectively, whereas twenty obese individuals had 3.76E+09 and 1.37E+10, respectively (1), showing reduced numbers of Bacteroidetes in the obese gut. This led us to question whether the consumption of probiotic foods, such as yogurts, could adversely alter the gut microbiome, because many probiotic foods and supplements contain organisms that belong to the Firmicutes phylum (5). To explore this possibility, cultures of B. thetaiotaomicron, a member of the Bacteroidetes phylum and inhabitant of the human gut, were grown under two conditions: a) in the presence of Fage plain total 0% Greek yogurt with live and active bacterial cultures; and b) in the presence of re-pasteurized Fage plain total 0% Greek yogurt. We hypothesized that the thetaiotaomicron numbers would be reduced when grown with the Fage plain Greek yogurt containing live and active bacterial cultures. ANOVA statistical analysis revealed that there was no significant decrease (p = 0.90) in the numbers of B. thetaiotaomicron regardless of whether or not it was grown with Greek yogurt that contained bacteria. These findings are important because they indicate that eating yogurt that contains live and active bacterial cultures may not reduce the numbers of beneficial Bacteroidetes species in the gut.

Results

The number of colony-forming units per mL (CFUs/ mL), a measure of the number of viable bacteria cells, was calculated for B. thetaiotaomicron growing a) in thioglycolate broth; b) in the presence of Fage plain total 0% Greek yogurt containing live and active bacterial cultures; and c) in the presence of re-pasteurized presence of Fage plain total 0% Greek yogurt. Using the Real Statistics Data Analysis Resource Pack for Excel (8), the mean CFUs/mL, sample variance, and standard deviation were calculated (Table 1). The mean CFUs/mL of B. thetaiotaomicron growing in thioglycolate broth was 23,473,333 after 24 hours of incubation at 35°C starting from a 0.5 McFarland standard (Figure 1). The mean CFUs/mL of B. thetaiotaomicron growing with Fage plain total 0% Greek yogurt containing live and active bacterial cultures was 29,846,667 after 24 hours of incubation at 35°C starting from a 0.5 McFarland standard (Figure 1). The mean CFUs/mL of B. thetaiotaomicron growing with re-pasteurized Fage plain total 0% Greek yogurt was 37,233,333 after 24 hours of incubation at 35°C starting from a 0.5 McFarland standard (Figure 1).

Since there were three groups being tested, an analysis of variance (ANOVA) was the appropriate statistical test to use. ANOVA testing produced a p of 0.90, with α = 0.05, showing that there was no statistical difference among the mean CFUs/mL of B. thetaiotaomicron growing in the three conditions. ANOVA testing was verified by a Levene’s test. The Levene’s

Table 1. Data of B. thetaiotaomicron grown alone, in the presence of Fage plain total 0% Greek yogurt containing live and active cultures, and in the presence of sterile Fage plain total 0% Greek yogurt. The data show the mean CFUs/mL of bacterial growth of B. thetaiotaomicron in a 0.5 McFarland standard after 24 hours of growth, along with the sample variance and SD.

Figure 1. Mean CFUs/mL of Bacteroides thetaiotaomicronThis image shows the mean CFUs/mL of B. thetaiotaomicron growing alone in Thioglycolate broth, in the presence of FageTM plain total Greek yogurt containing live and active cultures, and in the presence of repasteurized FageTM plain total Greek yogurt. The error bar shows ±2 SD units.

other broths were inoculated with 1 mL of re-pasteurized Fage plain total 0% Greek yogurt. Re-pasteurization of the yogurt was achieved by placing Fage plain total 0% Greek yogurt in an Erlenmeyer flask with a stopper. Each flask was then placed in a 63°C water bath for 30 minutes prior to being used in the study. This method was chosen over membrane filtration because the thickness of the yogurt was too great to be pulled through a vacuum-pressure membrane filter. Additionally, previous studies exploring water bath pasteurization have been shown to be successful in killing microbial organisms in the substance being pasteurized (11). Furthermore, milk and milk products can be pasteurized at a temperature of 63°C for 30 minutes (12). Next, the 9 tubes were incubated at 35°C for 24 hours. After 24 hours of incubation, the thioglycolate broths containing the B. thetaiotaomicron alone or with the different yogurt cultures were serially diluted in sterile 0.85% saline to a 108 dilution. Saline was chosen because it is sterile, readily available, and the salt concentration was not presumed to adversely affect the growth of the microbe. From these dilution tubes, spot titers were performed on reduced BBL Bacteroides Bile Esculin agar, a selective agar for growing Bacteroides species (Fisher Science) (Figure 2). A spot titer was chosen as a way to reduce the number of agar plates used in this experiment, and this technique has been shown to be equally effective as both spread-plate and pour-plate procedures in achieving viable colony counts (4). The BBE plates were incubated anaerobically for 48 hours in a Brewer jar along with a BD GasPak EZ Gas generating system sachet (Fisher Science). The anaerobic system was placed directly into the 35°C incubator. After the plates were incubated for 48 hours, the CFUs/mL were calculated. The spot titer dilutionplate containing 25–250 colonies was chosen, as this numerical range is considered easily countable, and the colony count obtained was divided by 0.01, to account for pipetting 10 µL for the spot titre, and then multiplied by the dilution factor producing 25–250 colonies. This provided an estimate of the CFUs/mL.

References

  1. Armougom F, Henry M, Vialettes B, Raccah D, and Raoult D. 2009. Monitoring Bacterial Community of Human Gut Microbiota Reveals an Increase in Lactobacillus in Obese Patients and Methanogens in Anorexic Patients. PLoS ONE 4(9): e7125. From: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742902/
  2. Benson AK, Kelly SA, Legge R, Ma F, Low SJ, Kim J, Zhang M, Oh PL, Nehrenberg D, Hua K, Kachman SD, Moriyama EN, Walter J, Peterson DA, Pomp D, and Mackay TFC. 2010. Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proc Natl Acad Sci 107 (44):18933-18938
  3. Clemente JC, Ursell LK, Parfrey LW, Knight R. 2012. The Impact of the Gut Microbiota on Human Health: An Integrative View. Cell 148 (6):1258–1270. From: http://ac.els-cdn.com/ S0092867412001043/1-s2.0-S0092867412001043-main.pdf?_tid=1ed7be78-3c5b-11e4-811b-00000aab0f26&acdnat=1410732458_ bab34d9459918e87f7ab60f30bd937b1
  4. Gaudy AF, Abu-Niaaj, F, and Gaudy ET. 1962. Statistical Study of the Spot-Plate Technique for Viable-Cell Counts. Appl. Microbiol 11: 305-309.Larsen N, Vogensen FK, Gøbel RJ, Michaelsen KF, Forssten SD, Lahtinen SJ, Jakobsen M. 2013. Effect of Lactobacillus salivarius Ls-33 on fecal microbiota in obese adolescents. Clin Nutr 32 (6):935-40
  5. Larsen N, Vogensen FK, Gøbel RJ, Michaelsen KF, Forssten SD, Lahtinen SJ, Jakobsen M. 2013. Effect of Lactobacillus salivarius Ls-33 on fecal microbiota in obese adolescents. Clin Nutr 32 (6):935-40
  6. Million M, Maraninchi M, Henry M, Armougom F, Richet H, Carrieri P, Valero R, Raccah D, Vialettes B, and Raoult D. 2012. Obesity-associated gut microbiota is enriched in Lactobacillus reuteri and depleted in Bifidobacterium animalis and Methanobrevibacter smithii. International Journal of Obesity 36: 817–825.
  7. Sekirov I, Russell SL, Antunes LCM, and Finlay BB. 2010 Gut Microbiota in Health and Disease. Physiol Rev. 90: 859–904; doi:10.1152/physrev.00045.2009.
  8. Zaiontz C. No date. Real Statistics Data Analysis Resource Pack for Excel. From: http://www.real-statistics.com/free-download/real-statistics-resource-pack/
  9. Sutton, S. 2006. Measurement of Cell Concentration in Suspension by Optical Density. [Internet]. From: http://www.microbiol.org/resources/monographswhite-papers/measurement-of-cell-concentration-in-suspension-by-optical-density/
  10. Myers JA, Curtis BS, and Curtis WR. 2013. Improving accuracy of cell and chromophore concentration measurements using optical density. BMC Biophysics 6 (4).doi.org/10.1186/2046-1682-6-4.
  11. Worobo R and Padilla-Zakour O. 2016. The development of pasteurization methods for premium fermented apple cider without adding preservatives. Vivo Research and Expertise Across Cornell. From: http://vivo.cornell.edu/display/individual16419
  12. Alberta.ca. 2015. Food Safety How to Pasteurize Milk at Home. [Online]. 2016 October 25. From: https://myhealth.alberta.ca/Alberta/Pages/ how-to-pasteurize-milk.aspx

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B. thetaiotaomicron in the Presence of Yogurt

The microbiome of humans contains an estimated 100 trillion microbial cells as well as an estimated quadrillion viruses. It is responsible for energy harvest, the breakdown of indigestible carbohydrates, the production of important biological molecules, and, most importantly, proper immune system development.

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