Quantification of butyryl CoA:acetate CoA-transferase genes reveals different butyrate production capacity in individuals according to diet and age.

Abstract

The gastrointestinal microbiota produces short-chain fatty acids, especially butyrate, which affect colonic health, immune function and epigenetic regulation. To assess the effects of nutrition and aging on the production of butyrate, the butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium clusters lV and XlVa, the main butyrate producers, were analysed. Faecal samples of young healthy omnivores (24 ± 2.5 years), vegetarians (26 ± 5 years) and elderly (86 ± 8 years) omnivores were evaluated. Diet and lifestyle were assessed in questionnaire-based interviews. The elderly had significantly fewer copies of the butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014), while vegetarians showed the highest number of copies (P=0.048). The thermal denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting curve related to Roseburia/Eubacterium rectale spp. was significantly more variable in the vegetarians than in the elderly. The Clostridium cluster XIVa was more abundant in vegetarians (P=0.049) and in omnivores (P<0.01) than in the elderly group. Gastrointestinal microbiota of the elderly is characterized by decreased butyrate production capacity, reflecting increased risk of degenerative diseases. These results suggest that the butyryl-CoA:acetate CoA-transferase gene is a valuable marker for gastrointestinal microbiota function.

Full text

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R E S E A R C H L E T T E R
Quanti¢cationofbutyrylCoA:acetateCoA-transferasegenes
revealsdi¡erentbutyrateproductioncapacityinindividuals
accordingtodietandage
Berit Hippe1, Jutta Zwielehner1, Kathrin Liszt1, Cornelia Lassl1, Frank Unger2&
Alexander G. Haslberger1
1Department of Nutritional Sciences, University of Vienna, Vienna, Austria; and2Department of Pharmaceutical Technology
and Biopharmaceutics, Vienna, Austria
Correspondence: Alexander G. Haslberger,
Department of Nutritional Sciences,
University of Vienna, UZAII, Althanstrasse 2,
A-1090 Vienna, Austria. Tel.: 143 699 122
11212; fax: 143 1 879 5896; e-mail:
alexander.haslberger@univie.ac.at
Received 18 November 2010; revised 15
December 2010; accepted 15 December 2010.
DOI:10.1111/j.1574-6968.2010.02197.x
Editor: Rustam Aminov
Keywords
butyric acid; gut microbiota; qPCR; nutrition;
elderly; vegetarian.
Abstract
The gastrointestinal microbiota produces short-chain fatty acids, especially
butyrate, which affect colonic health, immune function and epigenetic regulation.
To assess the effects of nutrition and aging on the production of butyrate, the
butyryl-CoA:acetate CoA-transferase gene and population shifts of Clostridium
clusters lVand XlVa, the main butyrate producers,were analysed. Faecal samples of
young healthy omnivores (24?2.5 years), vegetarians (26?5 years) and elderly
(86?8 years) omnivores were evaluated. Diet and lifestyle were assessed in
questionnaire-based interviews. The elderly had significantly fewer copies of the
butyryl-CoA:acetate CoA-transferase gene than young omnivores (P=0.014),
while vegetarians showed the highest number of copies (P=0.048). The thermal
denaturation of the butyryl-CoA:acetate CoA-transferase gene variant melting
curve related to Roseburia/Eubacterium rectale spp. was significantly more variable
in the vegetarians than in the elderly. The Clostridium cluster XIVa was more
abundant in vegetarians (P=0.049) and in omnivores (Po0.01) than in the
elderly group. Gastrointestinal microbiota of the elderly is characterized by
decreased butyrate production capacity, reflecting increased risk of degenerative
diseases. These resultssuggest that the butyryl-CoA:acetate CoA-transferase gene is
a valuable marker for gastrointestinal microbiota function.
Introduction
Recent evidence suggests that 1000–1150 different species
are capable of living in the gut ecosystem. An individual
harbours at least 160 species (Qin et al., 2010), with high
interindividual variations in species diversity and evenness.
It has been reported that the microbiota composition is
influenced by diet (Larsen et al., 2010) and age (Mariat et al.,
2009), as well as genetic factors (Khachatryan et al., 2008).
The gastrointestinal microbiota produces short-chain fatty
acids (SCFAs). Butyrate is of particular interest due to
itsanticarcinogenicandanti-inflammatory
(Maslowski et al., 2009), its effects on the intestinal barrier
(Peng et al., 2007), satiety (Cani et al., 2009) and epigenetic
regulation (Rada-Iglesias et al., 2007). Two of the most
important groups of butyrate producers are Faecalibacterium
potential
prausnitzii from the Clostridium cluster IV, and the
Eubacterium rectale/Roseburia spp. from the Clostridium
cluster XIVa (Walker et al., 2010). Both clusters (now also
known as Ruminococcaceae and Lachnospiraceae) consist of
producers and nonproducers of butyrate (Pryde et al., 2002).
Isolated dietary compounds have been shown to promote
growth of butyrate producers (Hernot et al., 2009). For
example, the consumption of inulin significantly stimulated
growth of F. prausnitzii (Louis & Flint, 2009). In colonic in
vitro model systems, resistant starch stimulated the growth of
E. rectale (Leitch et al., 2007).
Butyrate is easily taken up by the gut mucosa and faecal
butyric acid levels give little information about the butyrate-
producing capacity of the gut microbiota. Therefore, a
function-based approachwas suggested for the enumeration
of butyrate-producing bacteria (Louis & Flint, 2007)
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MICROBIOLOGY LETTERS

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targeting the butyryl-CoA:acetate CoA-transferase gene.
Furthermore,the butyryl-CoA:acetate
route, using acetate as a cosubstrate, is suggested to be the
most important route for butyrate production in the gut
ecosystem (Duncan et al., 2004). Alternative routes are via
butyrate kinase and phosphotransbutyrylase, which are
found in a minority of bacteria (Louis et al., 2004) in the
human gastrointestinal tract.
To evaluate the relevance of the butyryl-CoA:acetate
CoA-transferase gene in butyrate-producing communities,
we compared the amount of butyryl-CoA:acetate CoA-
transferase gene in young omnivores, vegetarians and the
elderly by quantitative PCR (qPCR) with a 16S rRNA gene
quantification of Clostridium clusters IVand XIVa consider-
ing the effects of aging and nutrition.
CoA-transferase
Materials and methods
Study subjects
Study groups consisted of 15 institutionalized, mobile
elderly persons [age: 86?8 years, body mass index (BMI)
21.75?5.08], 15 young healthy vegetarians (age: 26?5
years, BMI 21.02?2.71) and 17 young healthy omnivores
(age: 24?2.5 years, BMI 22.68?3.4) consuming a central
European diet. All subjects were interviewed using a ques-
tionnaire assessing age, gender, body height, weight, indivi-
dual health status, lifestyle and dietary habits. Approval was
obtained from the Viennese Human Ethics committee (EK
07-153VK).
Sample material
Faeces was collected from each participant individually and
stored at ?181C until processed. DNA was extracted using
the DNA stool Mini Kit (Qiagen) following the manufac-
turer’s protocol with minor modifications and immediately
stored at ?201C. Efficiency and quality of extraction
was controlled by photometry (Nanodrop) and gel electro-
phoresis.
Quantification of specific metabolic genes and
16S rRNA genes by qPCR
The butyryl-CoA:acetate CoA-transferase genes were ampli-
fied with degenerated primers BCoATscrF/R as listed in
Table 1 (Louis & Flint, 2007) on a Rotor-Gene 3000A
(Qiagen) using the SensiMix SYBR Kit (Quantace). Ampli-
fication of one of the faecal samples with BCoATscrF/R leads
to a highly concentrated butyryl-CoA:acetate CoA-transfer-
ase gene mix. This purified PCR product was used for
quantification of samples. Amplification with primer pair
BcoATscr resulted in clearly distinguishable and assignable
melt peaks. Total bacteria (Yu & Morrison, 2004) and
Clostridium clusters IV and XIVa were quantified (Meier
et al., 1999; Matsuki et al., 2004) on a Rotor-Gene 3000A
(Qiagen) using the SensiMix Probe Kit (Quantace). The
primers and probes used in this study are listed in Table 1.
Samples were quantified using standards derived from one
clone [clone library CleptF/R; Promega Vector System,
specificity in library confirmed (Liszt et al., 2009) with
known concentration in the case of Clostridium cluster IV
and from a Blautia coccoidesTpure culture for cluster XIVa].
Thermal denaturation of butyryl-CoA:acetate
CoA-transferase amplicons
Melt curves from amplified PCR products were divided into
three areas (Fig. 1b), as described by Louis & Flint (2009).
These peaks were assigned to represent bacteria related to
Eubacterium hallii and Anaerostipes coli (82.5–85.01C),
Table 1. Primers and probes used for quantification of 16S rRNA genes and butyryl CoA:acetate CoA-transferase genes with qPCR
Target organismPrimer/probe Sequence (50–30) Size (bp) References
BCCT geneBCoATscrF GCIGAICATTTCACITGGAAY
WSITGGCAYATG
CCTGCCTTTGCAATRTCIACRAANGC
GCA CAA GCA GTG GAG T
CTT CCT CCG TTT TGT CAA
(FAM)-AGG GTT GCG CTC GTT-(BHQ)
GCA GTG GGG AATATT GCA
CTT TGA GTT TCATTC TTG CGA A
(FAM)-AAATGACGGTAC
CTGACTAA-(BHQ)
ACT CCTACG GGA GGC AG
GAC TAC CAG GGTATC TAATCC
(FAM)-TGC CAG CAG CCG CGG
TAATAC-(BHQ)
530Louis & Flint (2007)
BCoATscrR
sg-Clept-F
sg-Clept-R3
Clept-P
195-F
Ccocc-R
Ccocc-P
C. cluster IV 239 Matsuki et al. (2004)
C. cluster XIVaMeier et al. (1999)
Matsuki et al. (2004)
All bacteriaBAC-338-F
BAC-805-R
BAC-516-P
468Yu et al. (2005)
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B. Hippe et al.

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Roseburia/E. rectale spp. (85.5–89.01C) and F. prausnitzii
(89.5–92.51C) as illustrated in Fig. 1a and b.
Data analysis
All statistical analysis (Spearman’s rank, Kolmogorov–Smir-
nov, F- Kruskal–Wallis- and t-tests) was done using ORIGI-
NPRO 8G (http://www.originlab.com).
Results
Dietary analysis
Analysis of the dietary habits indicated similar consump-
tions of fruit and milk products in the individual groups.
Vegetarians stated significantly more frequent consumption
of vegetables (w2; Po0.03) but similar patterns of
consumption of carbohydrates, including whole-grain
products. The elderly stated significantly more frequent
consumption of meat and similar vegetable consumption
(w2test; Po0.04) compared with omnivores. The exercise
levels of vegetarians and omnivores were comparable.
Relative quantification
Vegetarians had 12?62% more and the elderly had
31?21% less 16S rRNA gene relative to absolute quantified
genes compared with omnivores (Fig. 2a). Many SCFA-
synthesizing bacteria belong to the Clostridium clusters lV
and XlVa. The Clostridium cluster lV (Fig. 2b) was signifi-
cantly more abundant in omnivores (36.3?11.2%) than in
the elderly (27?11.7%, P=0.04) quantified relative to total
bacterial16SrRNAgenes.
31.86?17.00% of Clostridium cluster IV. The Clostridium
cluster XlVa (Fig. 2c) was significantly more abundant in
omnivores (19.01?6.7%,
(14.52?5.6%,
(9.89?6.64%).
Vegetariansharboured
P40.01)
than
andvegetarians
theP=0.049) inelderly
Butyryl-CoA:acetate CoA-transferase genes in
faeces by qPCR
The elderly had significantly fewer copies (1.52?1011?
1.36?1010copiesg?1faeces) of the butyryl-CoA:acetate
CoA-transferase gene compared with the omnivores
(4.96?1011?3.22?1010copiesg?1faeces, P=0.01) and
the vegetarians (1.37?1012?1.47?1011copiesg?1faeces,
P=0.048) (Fig. 2d). The amount of the butyryl-CoA:acetate
CoA-transferase gene did not correlate significantly with the
amount of total bacteria.
Melt curve analysis
The E. hallii/A. coli melt peaks tend to be higher in
vegetarians (P=0.08) and omnivores (P=0.09) than in the
elderly. The abundance of E. rectale/Roseburia spp. melt peak
differed significantly between vegetarians and the elderly
(P=0.04). Melt peak attributed to F. prausnitzii was sig-
nificant lower in the elderly than in omnivores (P=0.049)
(Fig. 1a).
Spearman’s rank showed no significant correlation be-
tween the amount of the butyryl-CoA:acetate CoA-transfer-
ase gene and that of Clostridium clusters IVand XIVa at an
individual level.
Discussion
Analysis of the overall abundance of bacterial 16S rRNA
genes reveals that the vegetarians harboured more bacteria
than the omnivores. The low numbers of bacteria in the
elderly individuals (Fig. 2a) may reflect physiological altera-
tions such as prolonged colonic transit time, reduced dietary
Fig. 1 Thermal denaturation of butyryl CoA:acetate CoA-transferase
gene of vegetarians (v), omnivores (o) and the elderly (e). (a) Box plots
are grouped according to their melt curves representing bacteria related
to Eubacterium hallii and Anaerostipes coli (82.5–85.01C), Roseburia/
Eubacterium rectale spp. (85.5–89.01C) and Faecalibacterium prausnitzii
(89.5–92.51C). Rectangular marks represent mean levels, medians are
indicatedwithaline,whiskerrangesbetween5thand95thpercentilesand
box limits between the 25th and 75th percentile. The asterisk indicates a
significant difference (Po0.05). (b) Sample melt curves between 78 and
921C of two elderly individuals representing the peaks related to E. hallii
and A. coli (82.5–85.01C), Roseburia/E. rectale spp. (85.5–89.01C) and
F. prausnitzii (89.5–92.51C). dF/dT, rel. fluorescence units.
FEMS Microbiol Lett ]] (2011) 1–6
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Influence of diet and age on production of butyrate

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energy requirement and food uptake (Morley, 2007). Figure
2b illustrates the significantly higher abundance of Clostri-
dium cluster IV in omnivores. Mueller et al. (2006) detected
the highest levels of the Clostridium cluster IV in a Swedish
study population, whose dietary habits were characterized by
a high consumption of fish and meat (Mueller et al., 2006).
Despite high meat consumption in the elderly, the generally
smaller capacity for energy harvest from food may decrease
the abundance of Clostridium cluster IV (Li et al., 2008). The
elderly gut microbiota is also characterized by a significantly
lower relative contribution of Clostridium cluster XIVa
comparedwithyoung study participants(Fig.2c). Adecrease
of Clostridium cluster XIVa has already been described as an
effect of the ageing process (Bartosch et al., 2004). Elderly
study participants had lower physical activity levels and did
not consume whole-grain products, whereas the other
groups stated regular consumption of fibre-rich products.
Vegetarians and omnivores have significantly more copies
of the butyryl-CoA:acetate CoA-transferase genes compared
with the elderly (Fig. 2d). Although no clear correlation with
Clostridium cluster IVandXIVa levels were found, theelderly
tended to harbour fewer butyrate producers than did
young individuals. Melt curve analysis showed that the
butyryl-CoA:acetate CoA-transferase gene variant related to
E. rectale/Roseburia spp. is significantly more variable in
vegetarians than in the elderly (Fig. 1a). Correspondingly,
Clostridium cluster XIVa seems to be more abundant in
vegetarians. Biagi et al. (2010) found lower quantities of
Roseburia intestinalis, E. hallii and E. rectale in the elderly
(475 years) than in young adults using the HITchip
method. The abundance of the Clostridium cluster XIVa does
not show significant correlations with the abundance of the
butyrate gene variant as determined by melting curve
analysis related to Roseburia/E. rectale spp., as this cluster
also contains many nonbutyrigenic bacteria. As illustrated in
Fig. 1a, the level of the melt peak attributed to F. prausnitzii
was significantly lower in the elderly. This is of particular
interest as this species has been reported to influence gut
inflammation processes by exerting a butyrate-independent
anti-inflammatory effect (Sokol et al., 2009).
The vegetarian diet may have favoured growth of the
Roseburia/E. rectale spp. that carries the butyryl-CoA:acetate
Fig. 2 (a) Relative quantities (in %) of absolute
amplified 16S rRNA genes in vegetarians and the
elderly compared with omnivores. Absolute bac-
terial numbers of omnivores were set as 100%.
(b, c) Relative quantities of total bacterial 16S
rRNA genes (in %) of Clostridium cluster lV (b)
and Clostridium cluster XlVa (c) based on group-
specific qPCR of 16S rRNA genes in vegetarians
and the elderly compared with omnivores. (d)
qPCR quantification of the butyryl CoA:acetate
CoA-transferase gene in faecal samples of vege-
tarians and the elderly compared with omnivores.
Data are expressed in copy numbers of total DNA
per 1g faeces (d). Rectangular marks represent
mean levels, medians are indicated with a line,
whisker ranges between 5th and 95th percen-
tiles, and box limits between 25th and 75th
percentile. Asterisk indicates a significant differ-
ence (Po0.05) (b, c, d).
FEMS Microbiol Lett ]] (2011) 1–6
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B. Hippe et al.

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CoA-transferase gene, without causing an increase in the
abundance of butyrate producers in the entire Clostridium
cluster XIVa. Omnivores and vegetarians had a similar
potential to harbour butyryl-CoA:acetate CoA-transferase
genes and members of Clostridium clusters IV and XIVa,
possiblycaused by a similar intake of fruit and carbohydrates.
These results suggest that the elderly group in this study
harbours less total bacteria and an even lower abundance of
Clostridium clusters IV and XIVa. Together with a lower
abundance of the butyryl-CoA:acetate CoA-transferase gene,
the results indicate that in the elderly, microbiota may be
characterized by a low butyrate production capacity. In respect
oftheimportantnutritive,anti-inflammatoryandanticancero-
genic potential of butyrate in the human colon, these findings
demonstrate that these microbiota specificities may contribute
to the development of degenerative diseases (Guigoz et al.,
2008) and anorexia in advanced age (Donini et al., 2010).
Consideration of the results of the analysis must take into
account methodological limitations. Despite the extraction
controls discussed in Materials and methods, DNA extrac-
tion can be influenced by diet and the consistency of faeces.
Previous studies reported a higherabundanceof Clostridium
cluster XIVa than cluster IV, which is not seen in our results
(Fig. 2b and c). This points to the need to compare different
standards for these clusters. Amplification of the 16S rRNA
gene can be particularly biased due possible multiple
operons for this gene. The use of degenerated primers
carries a certain risk for unspecific amplification of non-
target DNA. To estimate the accuracy of our amplification,
we checked every PCR product in 2% agarose gels where all
PCR products gave bands of the expected size. Our melt
curve analysis assumes that the intensity of individual peaks
represents the initial proportion of the different butyryl-
CoA:acetate CoA-transferase gene variants.
In conclusion, the quantification of the butyryl-CoA:ace-
tate CoA-transferase gene may be a suitable biomarker for
butyrate production for an individualized assessment of
gastrointestinal health and microbiota function in addition
to analysis of gastrointestinal microbiota.
Acknowledgements
We thank all the study participants. We thank Dr Guadalupe
Pinar and Dr Katja Sterflinger for giving us access to DNA
quantification machinery. The Austrian Science Fund
(FWF) funded this study.
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SupportingInformation
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Dietary and activity levels of vegetarians (back),
omnivores (middle) and the elderly (front).
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