Summer Undergraduate Research Program - Fred Hutchinson ...

Gut Microbiota Comparison in Women with High and Low
Body Mass Index Using Quantitative PCR Analysis.
B. Hammons1 , M.A. J. Hullar2 , F. Li2,3 , J.W. Lampe2,3 .
1 New Mexico State University, 2Fred Hutchinson Cancer Res. Ctr., Seattle,
WA, 3 Univ. of Washington, Seattle, WA.
2009 Best Poster Presentation Award
RESULTS:
Linear regression showed a positive association between
Bacteroidetes ratio and BMI (P=0.001). No association between
BMI and Clostridial cluster XIVa or Archaea.
ANOVA test showed a significance difference of Bacteroidetes
ratio among three BMI groups (P=0.0003). Scheffe post hoc test
showed significant differences between groups of BMI >30 to
BMI 30 and 22‐30
(P = 0.014).
There were no significance differences in % of Archaea and
Clostridial cluster XIVa between BMI groups. (Figure 2)
AIM:
To estimate the gut composition of selected microbial groups in a
population of women with a broad range of BMI.
INTRODUCTION
Gut microbial community is populated by 10 Eubacteria phyla and 1
Archaea. The majority of bacteria are Bacteroidetes and Firmicutes.
METHODS:
Study population Fecal samples were collected previously from
120 premenopausal women aged 40‐45y. Women were identified
from the Group Health Breast Cancer Screening Program.
Exclusion criteria were women taking exogenous hormones or
antibiotics 3 months prior to study participation [5].
Dietary fiber is fermented by gut microbiota, producing short chain fatty
acids (SCFA) propionate, acetate, butyrate, and lactate.
SCFA are estimated to provide 10% of total dietary energy supply in
humans.
Stool Collection and DNA extraction. Fecal samples from
participants were initially stored in RNAlater. For DNA extraction,
samples were homogenized in RNAlater. DNA was extracted using
the optimized method of Li et al [6].
Increased hydrogen‐oxidizing methanogenesis promotes fermentation
and produces more SCFA. Propionate, lactate and butyrate can be
converted to acetate, which in combination with hydrogen, is used by
Methanogens (Figure 1). Methanogens are the only Archaea found in
human gut. It is hypothesized that an increase in Methanogen
populations would increase the fermentation of dietary fiber.
QPCR – Quantitative PCR (QPCR) with variable primers was used
to quantify total microbial 16S rRNA gene copy numbers as well as
Bacteroidetes, Clostridial cluster XIVa and Methanogens (Table 1).
Total DNA preparations from gut microbiota were used as
templates for PCR amplification in an ABI Prism 7900HT Sequence
Detection System (Applied Biosystems, Foster City, CA) using SYBR
green QPCR kit (Invitrogen, Carlsbad CA). Optimized QPCR
conditions were used according to Zhang, et al [1]; Rekha et al [7];
Matsuki, et al [8]; Ahmend et al [9].
The presence of Methanogens in obese individuals is hypothesized to
increase energy uptake of SCFA by consuming hydrogen, which increases
fermentation efficiency of carbohydrates and proteins [1].
164
Studies of Firmicutes and Bacteroidetes ratio in obese populations have
shown conflicting data. Ley et al showed that there were more Firmicutes
and fewer Bacteroidetes in obese individuals than lean counterparts [2].
In contrast, others have found no difference in Bacteroidetes group
between obese and lean individuals [3, 4].
Figure 2. Percentage of Bacteria and Archaea among BMI groups.
SUMMARY:
Ratio of Bacteroidetes to total Eubacteria (assessed by 16S
rRNA gene copy number) was positively associated with BMI,
whereas there was no association between those of Clostridial
group XIVa and Archaea and BMI.
Other studies showed an increase in Bacteroidetes with
weight loss [2], no change in Bacteroides or Clostridial clusters
XIVa [3], and positive association between Methanogens and
Prevotellaceae and BMI [1].
Ours is the largest study to date of a well‐defined, relatively
homogeneous sample of women. The difference in the trends
of the gut microbiota with obesity across studies could reflect
the different human populations sampled.
Further evaluation of these data is needed.
Table 1. QPCR primers of selected microbial groups
References:
1. Zhang, H.S., et al., Human gut microbiota in obesity and after gastric bypass. Proc Natl Acad Sci USA, 2009. 106(7): 2365‐2370.
2. Ley, R.E., et al., Microbial ecology: human gut microbes associated with obesity. Nature, 2006. 444(7122): 1022‐3.
3. Duncan, S.H., et al., Reduced dietary intake of carbohydrates by obese subjects results in decreased concentrations of butyrate
and butyrate‐producing bacteria in feces. Appl Environ Microbiol, 2007. 73(4): 1073‐8.
4. Armougom, F. and D. Raoult, Use of pyrosequencing and DNA barcodes to monitor variations in Firmicutes and Bacteroidetes
communities in the gut microbiota of obese humans. Bmc Genomics, 2008. 9.
5. Atkinson, C., et al., Demographic, anthropometric, and lifestyle factors and dietary intakes in relation to daidzein‐metabolizing
phenotypes among premenopausal women in the United States. Am J Clin Nutr, 2008. 87(3): 679‐87.
6. Li, F., M.A. Hullar, and J.W. Lampe, Optimization of terminal restriction fragment polymorphism (TRFLP) analysis of human gut
microbiota. J Microbiol Methods, 2007. 68(2): 303‐11.
7. Rekha, R., et al., Designing and validation of genus‐specific primers for human gut flora study. J of Biotechnology, 2006.
9(5):505‐11.
8. Matsuki, T., et al., Development of 16S rRNA‐Gene‐Targeted Group‐Specific Primers for the Dectection and Identification of
Predominant Bacteria in Human Feces. Appl Environ Microbiol, 2002. 68(11); 5445‐51.
9. Ahmed, S., et al., Mucosal‐Associated Bacterial Diversity associated with Human Terminal Ileum and Colonic Biopsy Samples.
Appl Environ Microbiol, 2007. 73(22); 7435‐42.
Data analysis – Linear Regression was performed between BMI
and transformed 16S rRNA gene copy number ratios of selected
bacterial groups or Archaea to total Eubacteria. BMI was also
categorized into tertile groups (30
kg/m2 ). We used an ANOVA with post hoc testing (Scheffe test)
on transformed Bacterial or Archaeal 16S rRNA gene copy
number ratio to determine any significant difference between
BMI groups.
Figure 1. Anaerobic degradation of organic matter showing relationship
between gut bacteria fermentation and hydrogen use by Archaea.
We hypothesized that increased body mass index (BMI) would be
positively associated with Firmicutes (Clostridial cluster XIVa) and
Methanogens and inversely associated with Bacteroidetes.
This work is supported in part by NCI grants: 5 U54 CA132381 (FHCRC), 5 U54 CA132383 (NMSU) and R01
CA97366.