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Department of Infectious Diseases; Institute of Biomedicine, University of Gothenburg, Guldhedsgatan 10A, S-413 46 Gothenburg, Sweden
Department of Clinical Medicine; School of Health and Medical Sciences, rebro University, 70182 rebro, Sweden
a r t i c l e
i n f o
Article history:
Received 20 December 2012
Received in revised form 30 March 2013
Accepted 4 April 2013
Available online 11 April 2013
Keywords:
T-RFLP
Quantitative culture
Clone
Infantile gut
Microbiota diversity
16S RNA
a b s t r a c t
The infantile intestinal microbiota is a major stimulus for immune maturation. Both culture and DNA-based
methods can be used for microbiota characterization, but few studies have systematically compared their performance for analysis of the gut microbiota. Here, we examined fecal samples obtained on six occasions between
one week and 12 months of age from six vaginally delivered infants. After quantitative aerobic and anaerobic
culture of the samples on selective and non-selective media, DNA was extracted from the fecal samples and
analyzed regarding 16S rRNA gene polymorphism by terminal-restriction fragment length polymorphism
(T-RFLP). A database was constructed for direct identication of T-RFLP peaks by analysis of pure-culture bacteria
and analysis of a limited number of samples by 16S rRNA cloning and sequencing. Bacterial genera present at
>106 CFU/g feces, as determined by quantitative culture, were generally readily detected by T-RFLP, while
culture on selective media was more sensitive in detecting facultative anaerobes with lower population counts.
In contrast, T-RFLP more readily than culture detected several anaerobic species, also taxa that could not be
identied using the database. T-RFLP readily identied bacteria to the genus level and also provided some
sub-genus discrimination. Both T-RFLP and culture identied Bidobacterium, Clostridium and Bacteroides
spp. among the most common colonizers of the infantile microbiota throughout the rst year of life.
T-RFLP analysis showed that microbiota complexity was high in the rst weeks of life, declined to a minimum at 12 months of age, and thereafter increased again. Principal component analysis revealed that
early samples (1 week6 months) chiey differed between individual infants, while 12-month samples
were similar between children, but different from the early samples. Our results indicate that T-RFLP has
high sensitivity and adequate taxonomic discrimination capacity for analysis of gut microbiota composition,
but that both culture and molecular based analysis have limitations and both approaches may be needed to obtain a full picture of the complex gut microbiota.
2013 Elsevier B.V. All rights reserved.
1. Introduction
The human gastrointestinal tract harbors a complex microbial ecosystem, comprised mainly of strictly anaerobic species (Savage, 1977).
The intestinal microbiota is a major stimulus for the immune system (Crabbe et al., 1968) and we and others have shown that a low
38
2.1. Subjects
1511-1492) (Gutell, 1994). For T-RFLP, the forward primer was uorescently labeled with 6-FAM at 5. The PCR mixture contained 50 ng DNA
(25 and 75 ng were also tested; 25 ng DNA generated smaller T-RF
peaks), 25 l Hot Start Taq Master Mix (Qiagen), 0.3 M primer,
1 mM MgCl2 and water in a nal volume of 50 l (fecal samples) or
25 l (pure-culture strains and the reagents were cut in halves). The
PCR reaction was performed in an Eppendorf Mastercycler gradient
(Eppendorf, Hamburg, Germany) using the following program: an
initial activation of Taq polymerase at 95 C for 15 min, 25 cycles (28
and 32 were also tested) of 94 C for 1 min, 50 C for 45 s and 72 C
for 2 min, with a nal extension at 72 C for 7 min. Negative controls
containing all DNA extraction reagents, but without bacteria, were
included throughout the whole analysis to detect DNA contamination
from reagents or instruments.
2.5. T-RFLP analysis
2.5.1. Restriction enzyme digestion
Puried PCR products from fecal samples (100 ng) were digested
with 16 U (4, 10, and 20 U were also tested) of the MspI endonuclease
restriction enzyme (New England Biolab, Ipswich, USA) at 37 C for
5 h in a nal volume of 5 l. For pure-culture bacteria, 30 ng PCR
products were digested with 16 U MspI in a nal volume of 10 l. The
reaction was stopped by heating at 65 C for 20 min.
2.5.2. Fragment analysis
Fluorescently labeled T-RFs were detected using an ABI PRISM 310
genetic analyzer (Applied Biosystems, California, USA) with pop 4 gel.
Resolution (number and quality of peaks) was compared using different
volumes of digested PCR amplicons (0.5, 0.7, 1, 1.5, 2.5, 3.8 and 4.8 l),
run voltages (9.9, 12.2 kV and 15 kV), injection times (5 s and 10 s)
and size standards (ROX 500, ROX1000, combined ROX 500 and
ROX1000, LIZ 600 and LIZ 1200 Applied Biosystems). Reproducibility
was investigated by running samples in duplicate.
The fragment lengths were analyzed by GeneMapper, version 4
(Applied Biosystems) using Local Southern Method. True peaks
were separated from noise within a fragment length range between
28 and 1000 bp according to Abdo et al. (2006). Briey, the data were
standardized by dividing the area of each peak by the total peak area
of the particular sample. The standard deviation (SD) of the dataset
was then computed assuming that the true mean was zero. Peaks
with an area of 3 SD above the mean were considered as true signals.
They were collected and removed; whereafter the process was iterated
until no more true peaks were identied. T-RFs that differed by no
more than 1 bp in different analyses were considered as identical.
All fecal samples were analyzed in two independent PCRs and subsequent T-RFLP analyses. Only fragments represented in both runs were
considered and their peak sizes and areas were averaged.
2.5.3. In silico analysis
To obtain the expected T-RF size of different bacterial species,
in silico digestion was performed (RDP website: http://rdp8.cme.msu.
edu/html/TAP-trp.html). Cutting sites for the MspI enzyme in their
16S rRNA genes were detected and the size of the terminal fragments
were calculated and compared with observed T-RF size obtained from
T-RFLP analysis of the same type strains.
2.6. Cloning and sequencing
39
3. Results
40
It is crucially important that a single labeled fragment (T-RF) is produced after PCR and digestion of the 16S rRNA gene from a single
taxon. If more than one fragment is generated, each of these can be
interpreted as a unique bacterial species, i.e. a pseudo-T-RF. First
the PCR reaction was optimized using pure-culture strains. Up to
25 cycles, only 1500 nt amplicons, representing the full length 16S
rRNA gene, were produced, while after >25 cycles, fragments of other
sizes appeared that could be interpreted as T-RFs. Further, using b16 U
of the restriction enzyme MspI per 100 ng PCR amplicon led to appearance of pseudo-T-RFs as a result of incomplete digestion.
The optimal T-RFLP run conditions were found to be: a mixture of
1 l of digested 16S amplicon, 9 l of formamide and 0.5 l of LIZ 1200
being denatured at 95 C for 3 min and then placed immediately on
ice, a 5 s injection time and separation for 80 min at 15 kV. A run voltage of 15 kV gave more accurate fragment size and shorter run time
than 12 kV.
After optimization, T-RFLP reproducibility was investigated by
repeated analysis of the same samples. Between analyses performed
on the same day, T-RFs differed by 0.2 nucleotides, this increased to
1 nucleotides when the same sample was run on different days. We
decided to analyze all fecal samples in two independent PCRs and subsequent T-RFLP analyses and to include only T-RFs appearing in both
analyses. T-RFs differing no more than 1 nucleotides were considered
as a single taxon; their peak sizes and areas from the duplicates were
averaged.
3.1.2. Identication and exclusion of pseudo T-RFs
A troubling observation was that we repeatedly observed quite large
T-RF peaks representing nucleotide length 210(1) and 540(1)
(found 24 and 29 times, respectively, in the 36 fecal samples analyzed).
These fragments were neither generated when analyzing pure-culture
isolates, nor cloned DNA from fecal samples, suggesting that they
were pseudo-T-RFs. We found that these two peaks appeared only in
samples in which the Bacteroides-specic T-RF (size ~90) represented
>25% of the total peak area, i.e. samples in which Bacteroides spp.
were quantitatively dominant. In silico digestion analysis of the 16S
rRNA gene of Bacteroides type strains revealed three MspI enzyme
restriction sites (CCGG) situated at around 90, 210 and 540 nucleotides
in B. vulgatus and B. fragilis. B. ovatus and B. thetaiotaomicron only have
two sites (90 and 540) in. Complete digestion by MspI should generate
a single terminally labeled 90 nt fragment. If digestion at this site would
not be complete, digestion at one of the other MspI sites could generate
fragments of 210 or 540 nt, respectively. In samples with large quantities of Bacteroides DNA, such incomplete digests could be numerous
enough to pass the threshold and generate visible T-RFs. This hypothesis
was supported by a lack of the 210 nt fragment in the three fecal samples
that, according to culture, contained B. ovatus or B. thetaiotaomicron, but
no other Bacteroides species. Another source of variation that may explain
the occurrence of more than one peak per genome is that the 16S rrn operons may appear in several copies in the bacterial genome and that these
copies may differ slightly in sequence. If such nucleotide polymorphisms
affect restriction enzyme sites, T-RFs of different length may appear
(Crosby and Criddle, 2003). As the T-RFs of 210 and 540 nt were deemed
to represent pseudo-T-RFs, fragments of these sizes were excluded from
the analyses.
3.1.3. Discrepancies between true and observed T-RF size
We compared observed T-RF size from T-RFLP analysis of type strain
bacteria with the theoretical T-RF size obtained by in silico digestion
of the sequence of the same strain. Signicant discrepancies were
observed, i.e. the lengths of long fragments were underestimated as migration time increased non-linearly with size (Supplement Table S1).
Analysis of MW size standard markers conrmed that an increment in
size gave an increment in migration time that gradually diminished
with increasing MW (Supplement Fig. S1).
Table 1
Level of taxonomic resolution of culture and T-RFLP.
No of unique taxa
Genus
Facultative bacteria
Enterobacteriaceae
Enterococcus
Staphylococcus
Streptococcus
Obligate anaerobes
Bacteroides
Bidobacterium
Clostridium
Lactobacillus
Database
(species)
7
3
2a
9
10
9
13
9
T-RFLP
(T-RFs)
2
2
1
6
7
3
11
8
task that is very difcult even by sequencing the entire 16S rRNA gene
(Brugger et al., 2012).
30,31
41
129,130 149
T-RFno.(id)a
486
1w
25 (14)
2w
30 (19)
1mo
19 (12)
53 86
69 126
422
2mo
532
6mo
21 (11)
33 (19)
12mo
38 (21)
90,93
136
213
42
F6/E6
R2X[1] = 0.120029
R2X[2] = 0.0880433
Ellipse:Hotelling T2(0.95)
Fig. 2. Microbiota development over the rst year in six infants. Fecal samples from six infants (AF) obtained on six occasions over the rst year of life were analyzed by T-RFLP and
the T-RF pattern was analyzed by principal component analysis. (1 = 1 week, 2 = 2 weeks, 3 = 1 month, 4 = 2 months, 5 = 6 months and 6 = 12 months samples). The position of the sample is generated by the totality of T-RFs presented in the sample.
particular bacterial group was detected by culture or T-RFLP, respectively. As shown in Fig. 3, certain facultative bacteria were more
often detected by culture than that by T-RFLP. This was true of staphylococci and enterococci (Fig. 3). Enterobacteriaceae, were, however,
almost as often detected by T-RFLP as by culture. Aerobic streptococci
were readily detected by T-RFLP, but seldom by culture, which could
be explained by the fact that we did not include any selective culture
Enterococcus
Staphylococcus
Streptococcus
Enterobacteriaceae
Bacteroides
Bifidobacterium
12
C. difficile
Collinsella
Coprobacillus
Lactobacillus
Prevotella
Propionibacterium
T-RFLP
Culture
Ruminococcus
Clostridium
10
8
6
4
Detected
by T-RFLP
= No
= Yes
Sutterella
Veillonella
ro
nte
s
m
ile ides
us
ae
cu
riu
fic
ce
cill
oc
ro
dif
cte
ria
ba
a
e
o
loc
cte
t
m
t
b
y
c
c
u
Ba
ph
idi
ido
ba
La
f
r
a
i
o
t
t
r
s
B
S
te
Clo
En
12
24
36
us
cc
co
Number of samples
Fig. 3. Frequency of samples yielding a bacterial taxon by culture or T-RFLP. Altogether
36 fecal samples (6 samples each from 6 infants) were cultured quantitatively and
analyzed by T-RFLP. The bars indicate the number of samples in which a certain taxon
was detected by T-RFLP (lled bars) or culture (hatched bars), respectively. Facultative
bacterial taxa are indicated in bold.
Fig. 4. Fecal population levels of culturable bacteria detected and not detected by T-RFLP.
Population levels of different bacterial genera in 36 fecal samples were determined by
quantitative culture. The population levels of a particular genus were compared between
samples in which this genus was also detected by T-RFLP (lled symbols) and samples
which were negative for the genus in question by T-RFLP (open symbols). The bars indicate medians.
43
Table 2
Most prevalent bacterial groups in infantile fecal microbiota as determined by culture and T-RFLP.
Number of infants (n) yielding a specied bacterial group in feces
1 week
Culture
E. coli
Bidobacterium
Co-neg Staph
Bacteroides
Enterococcus
Clostridium
S. aureus
T-RFLP
Enterobacteriaceae
Bacteroides
Bidobacterium
Clostridium
Staphylococcus
Veillonella
Streptococcus
595#
702#
2 weeks
1 month
2 months
6 months
12 months
6
6
6
5
4
4
4
E. coli
Co-neg Staph
Bidobacterium
S. aureus
Bacteroides
Enterococcus
Clostridium
6
6
5
5
4
4
4
E. coli
Bidobacterium
S. aureus
Lactobacillus
Co-neg Staph
Bacteroides
Enterococcus
6
6
6
6
5
4
4
E. coli
Bidobacterium
Co-neg Staph
E. coli
Bidobacterium
Co-neg Staph
S. aureus
Bacteroides
Enterococcus
6
6
6
5
4
4
Bacteroides
Enterococcus
Clostridium
S. aureus
6
6
6
5
5
4
4
E. coli
Co-neg Staph
Bidobacterium
Bacteroides
Enterococcus
Clostridium
6
6
5
5
5
5
6
5b
5
5
4
4
4
4
4
Enterobacteriaceae
Bacteroides
Streptococcus
Bidobacterium
Barnesiella
Clostridium
595#
876#
6
5b
5
5
4
4
4
4
Bidobacterium
Enterobacteriaceae
Bacteroides
Veillonella
Lactobacillus
Streptococcus
5
5
4b
4
4
4
Enterobacteriaceae
Bidobacterium
Bacteroides
Bacteroides
Propionbacterium
Clostridium
Streptococcus
5
5
5b
4a
4
4
4
Enterobacteriaceae
Bacteroides
Bacteroides
Veillonella
Bidobacterium
Clostridium
Coprobacillus
Lactobacillus
495#
876#
6
6b
5a
5
5
5
4
4
4
4
Veillonella
Clostridium
Bacteroides
277#
Bidobacterium
Ruminococcus
Lactobacillus
Coprobacillus
Enterobacter
Selenomonas
136#
495#
516#
6
6
6ab
6
5
5
5
5
4
4
4
4
4
Fecal samples from six infants at six time points were analyzed by culture and T-RFLP. Bacterial taxa identied in at least 4/6 infants by culture or T-RFLP, respectively, are indicated
and n refers to the number of infants in whom the respective taxon was found. Italic = anaerobic bacteria, *Coagulase-negative Staphylococcus. #Unidentied T-RFs are presented
by their respective fragment sizes. a b = different Bacteroides with T-RF size around 80 and 90, respectively.
44
50
T-RFLP
Culture
40
No. of species
40
No.of T-RFs
50
30
20
10
30
20
10
A
143
69
B
91
73
C
136
63
D
144
63
E
169
76
F
162 Total
59 Id.(%)
A
68
B
58
D
68
C
41
E
55
F Infant
40 Total
Fig. 5. Microbiota complexity as a function of age in six infants. Fecal samples from infants AF were collected on six occasions over the rst year of life (1and 2 weeks of age and at
1, 2, 6 and 12 months of age), analyzed by T-RFLP and identied (when possible) by using a database. (A) The number of T-RFs detected in each sample is depicted by a black bar,
hence each infant is represented by six bars, one for each culture occasion. Below, the total number of T-RFs identied in that infant is indicated, as well as the proportion of T-RFs
that could be identied by using the database (Id %). Most infants showed a high complexity in the early samples, followed by a decrease in complexity around 12 months of age
(3rd4th bar), and again increased complexity in the last samples (6 and 12 months of age). (B) Analysis of the same samples by quantitative aerobic and anaerobic culture
revealed fewer bacterial taxa and did not reveal the biphasic complexity pattern seen with T-RFLP.
4. Discussion
In the present study, we optimized T-RFLP for analysis of the
microbiota of infants and examined its performance using fecal samples
from six infants who were followed from 1 week to 1 year of age.
In theory, the T-RF size may be calculated from knowledge of the
specic enzyme cleavage sites (in silico digestion). In practice, this
was impossible as migration time was not lineally related to T-RF size
across the size span, in accordance with earlier reports (Hahn et al.,
2001; Pandey et al., 2007). Therefore, we used T-RFLP analysis of
Table 3
Dominant bacterial groups as detected by cloning and sequencing.
Phylum
Dominant generaa
No. of clones
Infant C
Class
Bacteroidetes
Bacteroidia
Firmicutes
Clostridia
Negativicutes
Bacilli
Proteobacteria
Gamma
Alpha
Beta
Actinobacteria
Actinobacteria
Werrucomicrobia
Werrucomicrobiae
Total no. of clones
Infant D
Infant E
Infant F
53
43
61
30
101
17
2
14
14
6
9
6
Enterobacteriaceae
Koporiimonas
Sutterella
Bidobacterium
34
60
123
30
535
(64)
1
20
13
10
2
59
9
13
5
1
117
74
15
(14)
(9)
(2)
8
5
53
81
5
2
(10)
(0.006)
(0.002)
(0.002)
(0.004)
834
Akkermansia
58
69
61
69
124
Total % of
clones
57
3
101
244
51
Cloning was performed on nine fecal samples from four infant (C, D, E and F). Taxonomic classications of clones were achieved after sequencing analysis a Genera representing 15% of
the sequences b Ruminococcus, Clostridium, Eubacterium & Faecalibacterium.
45
46
Acknowledgments
This study is supported by the Swedish Medical Research Council
(grants K2009-57X-14455-08-3 and K2002-745X-14455-01A), the
European Commission (IMMUNOBIOTA, QLRT-2000-00538), the Medical faculty of Gothenburg University (LUA-ALFGBG-11006), the Torsten
and Ragnar Sderbergs Foundation, the Cancer and Allergy Foundation
and the Wilhelm and Martina Lundgrens Foundation.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.mimet.2013.04.002.
References
Abdo, Z., Schuette, U.M., Bent, S.J., Williams, C.J., Forney, L.J., Joyce, P., 2006. Statistical
methods for characterizing diversity of microbial communities by analysis of terminal
restriction fragment length polymorphisms of 16S rRNA genes. Environ. Microbiol. 8,
929938.
Abrahamsson, T.R., Jakobsson, H.E., Andersson, A.F., Bjorksten, B., Engstrand, L.,
Jenmalm, M.C., 2012. Low diversity of the gut microbiota in infants with atopic
eczema. J. Allergy Clin. Immunol. 129 (434440), e432.
Acinas, S.G., Marcelino, L.A., Klepac-Ceraj, V., Polz, M.F., 2004. Divergence and redundancy of 16S rRNA sequences in genomes with multiple rrn operons. J. Bacteriol.
186, 26292635.
Adlerberth, I., Wold, A.E., 2009. Establishment of the gut microbiota in Western infants.
Acta Paediatr. 98, 229238.
Adlerberth, I., Lindberg, E., Aberg, N., Hesselmar, B., Saalman, R., Strannegard, I.L., Wold,
A.E., 2006. Reduced enterobacterial and increased staphylococcal colonization of
the infantile bowel: an effect of hygienic lifestyle? Pediatr. Res. 59, 96101.
Adlerberth, I., Strachan, D.P., Matricardi, P.M., Ahrne, S., Orfei, L., Aberg, N., Perkin, M.R.,
Tripodi, S., Hesselmar, B., Saalman, R., Coates, A.R., Bonanno, C.L., Panetta, V., Wold,
A.E., 2007. Gut microbiota and development of atopic eczema in 3 European birth
cohorts. J. Allergy Clin. Immunol. 120, 343350.
Arumugam, M., Raes, J., Pelletier, E., Le Paslier, D., Yamada, T., Mende, D.R., Fernandes,
G.R., Tap, J., Bruls, T., Batto, J.M., Bertalan, M., Borruel, N., Casellas, F., Fernandez, L.,
Gautier, L., Hansen, T., Hattori, M., Hayashi, T., Kleerebezem, M., Kurokawa, K.,
Leclerc, M., Levenez, F., Manichanh, C., Nielsen, H.B., Nielsen, T., Pons, N., Poulain,
J., Qin, J., Sicheritz-Ponten, T., Tims, S., Torrents, D., Ugarte, E., Zoetendal, E.G.,
Wang, J., Guarner, F., Pedersen, O., de Vos, W.M., Brunak, S., Dore, J., Antolin, M.,
Artiguenave, F., Blottiere, H.M., Almeida, M., Brechot, C., Cara, C., Chervaux, C.,
Cultrone, A., Delorme, C., Denariaz, G., Dervyn, R., Foerstner, K.U., Friss, C., van de
Guchte, M., Guedon, E., Haimet, F., Huber, W., van Hylckama-Vlieg, J., Jamet, A.,
Juste, C., Kaci, G., Knol, J., Lakhdari, O., Layec, S., Le Roux, K., Maguin, E., Merieux, A.,
Melo Minardi, R., M'Rini, C., Muller, J., Oozeer, R., Parkhill, J., Renault, P., Rescigno,
M., Sanchez, N., Sunagawa, S., Torrejon, A., Turner, K., Vandemeulebrouck, G., Varela,
E., Winogradsky, Y., Zeller, G., Weissenbach, J., Ehrlich, S.D., Bork, P., 2011. Enterotypes
of the human gut microbiome. Nature 473, 174180.
Berg, R.D., 1999. Bacterial translocation from the gastrointestinal tract. Adv. Exp. Med.
Biol. 473, 1130.
Bisgaard, H., Li, N., Bonnelykke, K., Chawes, B.L., Skov, T., Paludan-Muller, G., Stokholm,
J., Smith, B., Krogfelt, K.A., 2011. Reduced diversity of the intestinal microbiota during
infancy is associated with increased risk of allergic disease at school age. J. Allergy Clin.
Immunol. 128 (646652), e641e645.
Brugger, S.D., Frei, L., Frey, P.M., Aebi, S., Muhlemann, K., Hilty, M., 2012. 16S rRNA
terminal restriction fragment length polymorphism for the characterization of
the nasopharyngeal microbiota. PLoS One 7, e52241.
Crabbe, P.A., Bazin, H., Eyssen, H., Heremans, J.F., 1968. The normal microbial ora as a
major stimulus for proliferation of plasma cells synthesizing IgA in the gut. The
germ-free intestinal tract. Int. Arch. Allergy Appl. Immunol. 34, 362375.
Crosby, L.D., Criddle, C.S., 2003. Understanding bias in microbial community analysis
techniques due to rrn operon copy number heterogeneity. Biotechniques 34, 790794
(796, 798 passim).
Dominguez-Bello, M.G., Costello, E.K., Contreras, M., Magris, M., Hidalgo, G., Fierer, N.,
Knight, R., 2010. Delivery mode shapes the acquisition and structure of the initial
microbiota across multiple body habitats in newborns. Proc. Natl. Acad. Sci. U. S. A.
107, 1197111975.
Ellis-Pegler, R.B., Crabtree, C., Lambert, H.P., 1975. The faecal ora of children in the
United Kingdom. J. Hyg. (Lond.) 75, 135142.
Engebretson, J.J., Moyer, C.L., 2003. Fidelity of select restriction endonucleases in determining microbial diversity by terminal-restriction fragment length polymorphism.
Appl. Environ. Microbiol. 69, 48234829.
Farris, M.H., Olson, J.B., 2007. Detection of Actinobacteria cultivated from environmental
samples reveals bias in universal primers. Lett. Appl. Microbiol. 45, 376381.
Ferrando, L., Fernandez Manay, J., Fernandez Scavino, A., 2012. Molecular and culturedependent analyses revealed similarities in the endophytic bacterial community
composition of leaves from three rice (Oryza sativa) varieties. FEMS Microbiol. Ecol.
80, 696708.
Gutell, R.R., 1994. Collection of small subunit (16S- and 16S-like) ribosomal RNA
structures: 1994. Nucleic Acids Res. 22, 35023507.
Hahn, M., Wilhelm, J., Pingoud, A., 2001. Inuence of uorophor dye labels on the
migration behavior of polymerase chain reactionamplied short tandem repeats
during denaturing capillary electrophoresis. Electrophoresis 22, 26912700.
Hayashi, H., Sakamoto, M., Benno, Y., 2002. Phylogenetic analysis of the human gut
microbiota using 16S rDNA clone libraries and strictly anaerobic culture-based
methods. Microbiol. Immunol. 46, 535548.
Kurokawa, K., Itoh, T., Kuwahara, T., Oshima, K., Toh, H., Toyoda, A., Takami, H., Morita,
H., Sharma, V.K., Srivastava, T.P., Taylor, T.D., Noguchi, H., Mori, H., Ogura, Y.,
Ehrlich, D.S., Itoh, K., Takagi, T., Sakaki, Y., Hayashi, T., Hattori, M., 2007. Comparative
metagenomics revealed commonly enriched gene sets in human gut microbiomes.
DNA Res. 14, 169181.
Lindberg, E., Adlerberth, I., Matricardi, P., Bonanno, C., Tripodi, S., Panetta, V., Hesselmar, B.,
Saalman, R., Aberg, N., Wold, A.E., 2011. Effect of lifestyle factors on Staphylococcus
aureus gut colonization in Swedish and Italian infants. Clin. Microbiol. Infect. 17,
12091215.
Liu, W.T., Marsh, T.L., Cheng, H., Forney, L.J., 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes
encoding 16S rRNA. Appl. Environ. Microbiol. 63, 45164522.
Matsumoto, M., Sakamoto, M., Hayashi, H., Benno, Y., 2005. Novel phylogenetic assignment database for terminal-restriction fragment length polymorphism analysis of
human colonic microbiota. J. Microbiol. Methods 61, 305319.
McOrist, A.L., Jackson, M., Bird, A.R., 2002. A comparison of ve methods for extraction
of bacterial DNA from human faecal samples. J. Microbiol. Methods 50, 131139.
Nieminen, T.T., Vihavainen, E., Paloranta, A., Lehto, J., Paulin, L., Auvinen, P., Solismaa,
M., Bjorkroth, K.J., 2011. Characterization of psychrotrophic bacterial communities
in modied atmosphere-packed meat with terminal restriction fragment length
polymorphism. Int. J. Food Microbiol. 144, 360366.
Palmer, C., Bik, E.M., DiGiulio, D.B., Relman, D.A., Brown, P.O., 2007. Development of the
human infant intestinal microbiota. PLoS Biol. 5, e177.
Pandey, J., Ganesan, K., Jain, R.K., 2007. Variations in T-RFLP proles with differing
chemistries of uorescent dyes used for labeling the PCR primers. J. Microbiol.
Methods 68, 633638.
Ramette, A., 2007. Multivariate analyses in microbial ecology. FEMS Microbiol. Ecol. 62,
142160.
Savage, D.C., 1977. Microbial ecology of the gastrointestinal tract. Annu. Rev. Microbiol.
31, 107133.
Steffen, E.K., Berg, R.D., Deitch, E.A., 1988. Comparison of translocation rates of various
indigenous bacteria from the gastrointestinal tract to the mesenteric lymph node.
J. Infect. Dis. 157, 10321038.
Wang, M., Ahrne, S., Antonsson, M., Molin, G., 2004. T-RFLP combined with principal
component analysis and 16S rRNA gene sequencing: an effective strategy for
comparison of fecal microbiota in infants of different ages. J. Microbiol. Methods
59, 5369.
Wang, Q., Garrity, G.M., Tiedje, J.M., Cole, J.R., 2007. Naive Bayesian classier for rapid
assignment of rRNA sequences into the new bacterial taxonomy. Appl. Environ.
Microbiol. 73, 52615267.
Wang, M., Karlsson, C., Olsson, C., Adlerberth, I., Wold, A.E., Strachan, D.P., Martricardi,
P.M., Aberg, N., Perkin, M.R., Tripodi, S., Coates, A.R., Hesselmar, B., Saalman, R.,
Molin, G., Ahrne, S., 2008. Reduced diversity in the early fecal microbiota of infants
with atopic eczema. J. Allergy Clin. Immunol. 121, 129134.
Westerhuis, J.A., van Velzen, E.J., Hoefsloot, H.C., Smilde, A.K., 2010. Multivariate paired
data analysis: multilevel PLSDA versus OPLSDA. Metabolomics 6, 119128.
Wilson, K.H., Blitchington, R.B., 1996. Human colonic biota studied by ribosomal DNA
sequence analysis. Appl. Environ. Microbiol. 62, 22732278.
Yatsunenko, T., Rey, F.E., Manary, M.J., Trehan, I., Dominguez-Bello, M.G., Contreras, M.,
Magris, M., Hidalgo, G., Baldassano, R.N., Anokhin, A.P., Heath, A.C., Warner, B.,
Reeder, J., Kuczynski, J., Caporaso, J.G., Lozupone, C.A., Lauber, C., Clemente, J.C.,
Knights, D., Knight, R., Gordon, J.I., 2012. Human gut microbiome viewed across
age and geography. Nature 486, 222227.