Relative Abundance of Bacteroides spp. in Stools and Wastewaters as Determined by Hierarchical

Relative Abundance of Bacteroides spp. in Stools and Wastewaters as Determined

by Hierarchical Oligonucleotide Primer Extension (HOPE)

Pei-Ying Hong1, Jer-Horng Wu

2 and Wen-Tso Liu

1*

1Division of Environmental Science and Engineering, National University of Singapore,

Singapore 117576

2Sustainable Environment Research Center, National Cheng Kung University, Taiwan,

701

*corresponding author: Wen-Tso LIU

E-mail: [email protected]

phone: +65-6516 1315, fax: +65-6774 4202

Division of Environmental Science and Engineering

National University of Singapore

E2-04-07, 1 Engineering Drive 2

Singapore 117576

Running title: HOPE measuring abundance of Bacteroides spp.

[Key words]: HOPE, determination of relative abundance, Bacteroides spp., fecal

microbiota, microbial source tracking, disease-related bacterial populations

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Copyright © 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02568-07 AEM Accepts, published online ahead of print on 14 March 2008

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ABSTRACT

A molecular method, termed hierarchical oligonucleotide primer extension (HOPE), is

used to determine the relative abundance of predominant Bacteroides spp. present in

fecal microbiota and wastewaters. For this, genomic DNA in feces of healthy human

adults, bovines and swines, and in wastewaters were extracted, and total bacterial 16S 5

rRNA genes were PCR amplified and used as the DNA template of HOPE. Nineteen

oligonucleotide primers were designed to detect 14 Bacteroides spp. at different

hierarchical levels (domain, order, cluster, and species), arranged and used in six

multiplexing HOPE reactions. Results showed that species like B. vulgatus, B.

thetaiotaomicron, B. caccae, B. uniformis, B. fragilis, B. eggerthii and B. massiliensis 10

could be individually detected in human feces at abundances down to 0.1 % of PCR-

amplified 16S rRNA genes. Minor ones like B. pyogenes, B. salyersiae and B. nordii

were only detected collectively using a primer that targets the B. fragilis subgroup (~ 0.2

% of PCR-amplified 16S rRNA genes). Furthermore, Bac303-related targets (i.e., most

Bacteroidales) were observed to account for 28-44 % of PCR-amplified 16S rRNA genes 15

in human fecal microbiota, and their abundances were higher than that observed in the

bovine and swine fecal microbiota, and in wastewaters by a factor of five and two,

respectively. These results were comparable to that obtained by quantitative PCR or

reported previously using whole cell fluorescence hybridization and 16S rRNA clone

library methods, supporting that HOPE can be a sensitive, specific and rapid method to 20

determine relative abundance of Bacteroides spp. predominant in fecal samples.

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INTRODUCTION

Approximately 1014

microbial cells reside in the human intestine and form close

symbiotic associations with the host (18). Among them, genera Bacteroides and

Parabacteroides in the order Bacteroidales are known to make up 14-40 % of cultivable

microorganisms in human feces (21). Species including B. caccae, B. fragilis, B. 5

eggerthii, B. uniformis, B. thetaiotaomicron, B. vulgatus, B. nordii, B. salyersiae, B.

coprocola, B. massiliensis, B. intestinalis, P. distasonis, P. merdae and P. goldsteinii

have been successfully isolated from gut or feces of humans (3, 12, 23, 26, 38),

representing up to 80% of the fecal bacterial isolates from the genus Bacteroides

documented in Ribosomal Database Project II (http://rdp.cme.msu.edu). 10

Due to their high prevalence in the gut/fecal microbiota, Bacteroides spp. and

Parabacteroides spp. have been considered as clinically important anaerobes that are

related to the gastrointestinal well-being (13). For example, species like B. vulgatus and B.

caccae can colonize on the surface area of intestinal mucosa and inhibit the adherence of

enteroinvasive pathogens (8, 18). Others like B. thetaiotaomicron and B. ovatus can break 15

down a wide variety of indigestible polysaccharides to supply its host with 10-15 % of

the daily metabolic needs (35, 42). Besides relating to gastrointestinal functioning,

certain Bacteroides spp. and Parabacteroides spp. are also suspected to be human gut-

specific, and can act as biomarkers in identifying fecal pollutions of human and non-

human sources (9, 27, 29). For instance, abundances of P. distasonis, B. fragilis, B. 20

thetaiotaomicron and B. vulgatus are reported to be 100-1000 fold higher in human feces

than in animal feces (27), while species in uncultivated animal-specific Bacteroidales

clusters (Fig. 1, labeled clades) are seldom detected in human feces. Given the

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importance of Bacteroides spp. and Parabacteroides spp., determining the abundance of

all or specific strains in the microbiota has become important for establishing a

correlation to the health-status of the host, and for performing microbial source tracking.

At present, qualitative and quantitative detections of Bacteroidales in feces-

related samples are mainly achieved through cell cultivation and the use of conventional 5

molecular tools (2, 4, 10, 19, 21, 33). Both approaches however have their strengths and

weaknesses. The former can be time consuming and favor the growth of selective

species (40). The latter methods [e.g., whole cell fluorescence in-situ hybridization

(FISH), quantitative-PCR (Q-PCR), and 16S rRNA gene clone libraries] can circumvent

the issues related to cultivability, but may also encounter other technical challenges. For 10

example, FISH can be affected by the in-situ accessibility of labeled probes through the

cell membranes and the copies of rRNA inside inactive cells (7, 17). Clone library

construction, with its resolution dependent on the total number of clones picked for

analysis, can be time-consuming (15). Q-PCR can provide excellent quantitative

detection sensitivity (down to a few copies of target fragments) but require standard 15

curves to be established for individual targets (24). As such, there is still a need to

develop a direct and simple-to-use technique which can complement the existing

molecular methods to rapidly evaluate the relative abundance of Bacteroides spp. and

Parabacteroides spp. present in complex microbial communities.

Recently, a molecular method termed hierarchical oligonucleotide primer 20

extension (HOPE) has been developed to measure the relative abundance of multiple

bacterial 16S rRNA gene among total PCR amplified 16S rRNA genes (41). In this

method, multiple oligonucleotide primers that are designed with different lengths by the

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addition of poly-A(s) are used to target specific 16S rRNA gene sequences at different

taxonomical levels (i.e. domain, phylum, order … and species). During primer extension

reaction, these primers anneal to their complementary target sequences, and are

subsequently extended with a fluorescently labeled dideoxynucleoside triphosphate

(ddNTP) by DNA polymerase. The extended primers are identified by DNA sequencer 5

based on their fragment sizes and the type of the extended fluorescent ddNTP. Based on

the fluorescence intensity detected, peak areas of extended specific primers are then

quantified, and the ratio relative to that of a common reference primer is calculated and

used to determine the relative abundance of interested bacterial targets (Fig. 2).

In the previous study, a 10-plexing HOPE reaction targeting Bacteroides at 10

domain, group and species level were developed and applied to detect the relative

abundance of Bacteroides spp. in the influent and effluent of domestic wastewater

treatment plant. It was successfully shown that HOPE could achieve single mismatch

discrimination, and had a detection sensitivity of targets down to 0.01-0.05 % of total

PCR-amplified 16S rRNA genes (41). These promising results have further encouraged 15

us to develop HOPE as a robust method to rapidly detect and measure the relative

abundance of a total of 14 functionally important Bacteroides-Parabacterioides spp. at

species, group and domain levels in feces and wastewaters. To do so, we have added 12

more species-specific primers in the genera Bacteroides and Parabacterioides (Fig. 1 and

Table 1). To further validate the ability of HOPE to determine relative abundance of 20

bacterial targets, we have compared the HOPE results with conventional molecular

methods like Q-PCR.

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MATERIALS AND METHODS

Bacterial strains and environmental samples Nineteen bacterial reference strains

obtained from Japan Collection of Microorganisms (Wako, Japan) or Bioresources

Collection and Research Centre (Hsinchu, Taiwan) were used (Table 1). These bacterial

strains are classified into the B. fragilis group (i.e., B. uniformis, B. helcogenes, B. 5

stercoris, B. caccae, B. acidifaciens, B. fragilis, B. thetaiotaomicron, B. vulgatus, B.

coprocola, B. massiliensis, B. eggerthii, B. nordii, B. salyersiae, B. intestinalis, and B.

ovatus) and the Parabacteroides distasonis group (i.e., P. distasonis, P. merdae and P.

goldsteinii). Within the B. fragilis subgroup, bacterial strains can be further classified into

Bth274 [T]-related subcluster (i.e., B. pyogenes, B. tectus, B. nordii, B. salyersiae and B. 10

thetaiotaomicron) and Bth274 [C]-related subcluster (i.e., B. uniformis and B. fragilis).

Fecal samples were collected from (i) three healthy donors of age 26 - 32 years old, (ii)

three healthy pigs, and (iii) three healthy cows. Environmental samples were collected

from the influent of a municipal treatment plant located in Singapore and a swine

wastewater treatment plant in Tainan, Taiwan. Samples were collected and kept at -20 oC 15

prior to DNA extraction.

DNA extraction Total DNA of pure cultures and influent samples was extracted

according to a previously described protocol with minor modifications (36). Total DNA

of fecal samples was extracted using QIAamp DNA stool mini kit (Qiagen) (32).

PCR amplification of 16S rRNA genes Each PCR reaction (50 µl in volume) 20

contained 50 to 100 ng of genomic DNA in 1X Takara Ex-Taq buffer, 200 nM of forward

primer [11F, 5’-GTT TGA TCC TGG CTC AG-3’] (25) and reverse primer [1512R,

GGC TAC CTT GTT ACG ACT T-3’] (28), 200 mM of dNTP and 0.5 U of Ex-Taq

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DNA polymerase (Takara). The optimal number of thermal cycles required to obtain a

proportional amplification of the microbial community was examined by varying the

cycle number of thermal program (denaturation, 95 oC for 30 s; annealing, 55

oC for 45 s;

and extension, 72 oC for 60 s) from 10 to 35 cycles at an increment of 5 cycles.

Amplicons obtained at each cycle were concentrated and purified using QIAquick PCR 5

purification kit (Qiagen), and the concentrations quantified by UV absorbance

measurement using DU730 spectrophotometer (Beckman Coulter).

Cloning and sequencing To ensure culture purity, 16S rRNA genes of reference

bacterial strains were separately cloned into the pCRII vector using the TA cloning kit

(Invitrogen Corporation, Carlsbad, CA, USA). For each clone library, 20 colonies were 10

selected, and the presence of DNA insertion was confirmed by direct PCR amplification

with M13R and M13F primers. The inserted DNA fragment was then sequenced using

ABI PRISM 3130 genetic analyzer (Applied Biosystems) and Bigdye Sequencing kit

(Applied Biosystems). These sequences were compared against the 16S rRNA gene

database using BLAST software (1), and deposited in GenBank database under the 15

accession number of EU136678-97.

Species-specific primers design A total of 12 new primers specifically targeting

different human-specific Bacteroides spp. was designed using the probe design function

of ARB software (30). An updated ssu_jan04.arb database (http://www.arb-home.de),

which contained 28289 nearly complete 16S rRNA sequences (length >1450 nt), was 20

used together with 189 aligned sequences of cultivated Bacteroides spp. obtained from

the Ribosomal Database Project II (RDP) and nearly complete 16S rRNA sequences of

the 19 reference bacterial strains. To enhance the specificity of HOPE primers, each

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primer was designed with mismatch positions of non-targets located at the 3’-terminus.

The specificity of designed primers was first verified in-silico against RDP database, and

subsequently in HOPE reactions against all reference strains to ensure that primers do not

extend when non-target bacterial strains were used as DNA template.

Hierarchical primer arrangement Table 1 listed the name, sequences and 5

specificities of those19 different HOPE primers used in this study. These primers were

assigned into six different multiplexing HOPE reactions based on the phylogenetic

affiliation of those bacterial targets (Table 1 and Fig. 1). Reactions 1-5 contained two

higher ranking primers that target both B. fragilis group and its subgroup (i.e. primer

Bfrg602 or Bth274) together with 14 species-specific primers. Thus, the abundance of 10

individual bacterial targets targeted by each species-specific primer relative to those

targeted by higher ranking primers can be determined. In reaction 6, the relative

abundance of Bacteroidales, B. fragilis group and its subgroup, and the Parabacteroides

group as represented by Bac303, Bfrg602, Bth274, and Bdts_gp980 can be determined

against the total 16S rRNA gene amplicons. To clearly differentiate extended primers in 15

the same HOPE reaction, primers that extend with the same ddNTP were further

modified with different lengths of poly-A tails (5-24 nt) at the 5’ end.

Internal standard oligonucleotide mixture The mixture contained four

oligonucleotides of different lengths [5’- (GT) x -3’; where x = 18, 20, 22 and 24], which

were synthesized and end-labeled at the 5’ end with four different fluorophores (i.e., 20

dR110, dR6G, dTAMRA and dROX) (Applied Biosystems). The concentration of

individual oligonucleotides was measured at 260 nm, and subsequently diluted to 3 nM

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for 5’-dR110- (GT) 20 -3’, 15 nM for 5’-dTAMRA-(GT) 22 -3’, 18 nM for 5’-dROX- (GT)

24 -3’, and 5 nM for 5’-dR6G-(GT)18-3’.

HOPE reactions Unless stated otherwise, each HOPE reaction (in total, 5 µl)

contained 2.5 µl of SNaPshot premix, 5-30 fmol of DNA template, 10-60 pmole of

oligonucleotide primers (Sigma-Proligo, Singapore and Mission Biotech, Taiwan), and 5

various amount of deionized water. The SNaPshot premix consisted of DNA polymerase,

ionic buffer and fluorescently labeled dideoxynucleotides (dROXTM

-ddTTP,

dTAMRATM

-ddCTP, dR110-ddGTP and dR6G-ddATP). HOPE thermal program

consisted of 20 cycles of denaturation (96 oC, 10 s), annealing (64

oC, 30 s) and extension

(72 oC, 15 s). After primer extension reaction, 1U of shrimp alkaline phosphatase (Roche 10

Applied Science, Penzberg, Germany) was added and incubated at 37 oC for 60 min in

order to remove unincorporated dye-terminators. This reaction was terminated by heating

at 75 oC for 10 min.

Capillary electrophoresis 0.5-1 µl of HOPE products were mixed with 0.125 µl of

GeneScan Liz 120 standard (Applied Biosystems), 0.25 µl of the standard 15

oligonucleotide mixture, and 12 µl of Hi-Di formamide (Applied Biosystems).

Electrophoresis program included a denaturation step (60 oC), an injection step with an

applied voltage of 1.0-2.1 kV for 12-40 s and a separation step. Fluorescence data were

subsequently analyzed by the fragment analysis software GeneMapper, where the

fragment sizes and peak areas of the extended primers and internal standard 20

oligonucleotides were recorded.

Calculation of relative abundance of HOPE products The concentration of

extended primers was quantified as follow:

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* +* + * +* +oligo internal of volume

oligo internal associated of areaPeak

sample injected of volume

primer extended of areaPeak

stdp CC? (i)

Where Cp represents the molar concentration of a specific primer extended in the HOPE

reactions for the samples; Cstd represents the molar concentration of the associated

standard oligonucleotide.

Calibration factors (CF) for each specific primer with respect to a higher ranked primer 5

can be obtained using the M13 amplicons of associated reference strains as template, and

calculated as follow:

b

a

C B,primer extended ofion Concentrat

C A,primer extended ofion Concentrat?/BACF (ii)

Where primer B is targeting at a higher hierarchical level compared to primer A.

The relative abundance of 16S rRNA gene amplicons targeted by primer A with respect 10

to those targeted by primer B can then be calculated as follow:

Relative abundance of target (%) = %100C

C

b

a ·· /BACF

(iii)

Quantification of Bacteroides spp. with Q-PCR Standard curves used for the

detection of Bacteroides spp. at different taxonomical levels were obtained in replicates

with 40 nmol of forward and reverse primers (Table 2), 1X iQ Sybr Green mastermix 15

(BioRad), and varying concentrations (1-10000 picograms per 25 µl of reaction mixture)

of two independent reference strains (B. fragilis BCRC10619 and B. uniformis JCM5825).

Forty cycles of thermal program (denaturation at 96 oC for 10 s, annealing at 64

oC for 30

s, extension at 72 oC for 30 s) was performed with iCycler (BioRad). Standard curves

were generated by plotting the concentration of genomic DNA template against mean 20

threshold cycles (CT). Similarly, real time quantitative measurement of Bacteroides spp.

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that were present in human fecal samples was subsequently performed in replicates with

1000 pg of genomic DNA.

RESULTS

PCR amplification PCR amplification was first examined at various cycle intervals 5

to determine the appropriate number of thermal cycles needed to generate amplicons that

are representative of the actual microbial community. With 2 ng/µl of genomic DNA as

the starting template in PCR, it was observed that after 15 to 20 thermal cycles, the

amplicons obtained were proportionally amplified, and the use of these amplicons for

downstream analyses could minimize the effect of PCR bias. After 20 to 35 cycles of 10

PCR, the amplification curve deviated from the exponential shape and underwent a

quasilinear phase. Amplification eventually reached plateau beyond 35 cycles as the

amount of accumulated amplicons could not be increased further (Fig. 3). Thus,

amplicons that underwent 20 thermal amplification cycles were subsequently used as the

DNA template for all HOPE reactions in this study. 15

Specificity of primer extension The specificities of primer extension were validated

against reference strains. Results showed that all species-specific primers correctly

extended with the same nucleotide as in-silico predicted, when their targeted species were

used as DNA template. Likewise, all species-specific primers were not extended when

non-targets were used. Group-specific primer Bth274 extended with nucleotide “C” in the 20

presence of B. uniformis and B. fragilis, or “T” in the presence of B. tectus, B. pyogenes,

B. nordii, B. salyersiae and B. thetaiotaomicron. Bfrg602 that was designed to target

Bacteroides spp. in the B. fragilis cluster extended with “C”, and Bdts_gp980 that was

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designed to target Parabacteroides distasonis group correctly extended with “G”.

Bac303 and U1390 were designed to target all reference bacterial strains at the order and

domain level respectively, and extended with “C” and “A” respectively (Table 1).

However, minimal extension for Bac303 primer was observed in the presence of P.

goldsteinii JCM13446. Re-sequencing the 16S rRNA gene sequence of P. goldstenii 5

revealed that the target sequence region had a single mismatch with Bac303 at the fifth

position from the 3’-end of the primer, and this mismatch could significantly reduce the

primer extension efficiency in comparison to the perfect match sequence (37). Since P.

goldsteinii is not a predominant member in the fecal microbiota, its mismatch to Bac303

should not affect the measurement of the relative abundance of Bacteroidales to the total 10

PCR-amplified 16S rRNA genes in fecal microbiota.

Calculation of CF values CF values were established in the presence of 16S RNA

gene amplicons derived from the bacterial reference clone. For instance, when B.

thetaiotaomicron 16S rRNA gene amplicon was used as HOPE template, only primers in

reactions 1 and 6 were extended and they were correctly identified by size and the type of 15

the extended fluorescence (Fig. 4A). Peak areas of individual primers Bth584 and

Bth274_[T] in reaction 1, and Bth274, Bfrg602, Bac303 and U1390 in reaction 6 were

noted, and the individual extended concentrations were calculated. The extended

concentration of B. thetaiotaomicron-specific primer (e.g. primer Bth584) was

normalized against that of the higher ranked primer in the same reaction (e.g. 20

Bth274_[T] ) to obtain CF values (e.g. CFBth584-Bth274_[T]). By repeating this for all tested

reference bacterial strains, CF values were obtained for all primers (Table 3, legend).

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These values were subsequently used for calculating the relative abundance of bacterial

targets present in the fecal and wastewater samples.

Electropherograms of tested samples Figure 4B shows the electropherograms

obtained after HOPE was carried out in the presence of total 16S rRNA gene amplicons

derived from human stool H3. As higher ranking primers had more targets to prime to, 5

their peak heights and areas were considerably larger than those representing species-

specific primers. Furthermore, as primers should be extended at a specific fragment size,

all other peaks that did not coincide with the anticipated size can be treated as

background noise and removed from subsequent analyses.

Relative abundance of Bacteroides spp. in feces as determined by HOPE Table 10

3 and Figure 5 describe the relative abundances of the 14 tested Bacteroides spp. present

in the three tested human feces at various taxonomical levels. At the higher taxonomical

levels, relative abundance of the Bacteroidales group targeted by Bac303 primer ranged

from 28.1- 44.4 % of PCR-amplified 16S rRNA genes. B. fragilis subgroup that

extended with “C” (including B. uniformis and B. fragilis) account for 0.8-1.3 %, while 15

the B. fragilis subgroup that extended with “T” (including B. thetaiotaomicron, B. tectus,

B. caccae, B. pyogenes, B. nordii and B. salyersiae) account for 0.2-1.1 %. The

abundance of B. fragilis cluster, which varied across the host, ranged from 5.0-12.1 % of

PCR-amplified 16S rRNA genes.

Within the B. fragilis group, B. vulgatus was observed as the predominant species 20

(14.8-49.8 % of amplified 16S rRNA gene from the B. fragilis group), and B. caccae and

B. uniformis were present at varying relative abundances (1.3-8.9 %) in different human

fecal microbiota. Other species like B. fragilis, B. eggerthii, B. intestinalis and B.

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massiliensis were detected to be between 0.6 and 3.1 % in volunteer H2 and H3, but were

too low to be detected in volunteer H1. In total, these targeted Bacteroides spp. accounted

for 26.6-62.6 % of amplified 16S rRNA gene from the B. fragilis group. These

observations further suggest that other important and unknown Bacteroides spp. from this

group were present in the human fecal microbiota (Figure 5B). Within B. fragilis 5

subgroup that extended with “T”, B. thetaiotaomicron was detected as the predominant

and major species (58.9-97.5 %) in this group (Table 3).

In contrast, in the swine and bovine fecal microbiota, none of the 14 species-

specific primers extended, suggesting that Bacteroides spp. examined in this study were

present at abundances below the detection sensitivity of HOPE. These results further 10

suggest that the Bacteroides spp. examined in this study are probably host-specific to

human fecal microbiota. In the animal fecal microbiota, relative abundance of most

Bacteroidales could only account for 4.6-5.2 % and 1.8-2.9 % of PCR-amplified 16S

rRNA genes present in the swine fecal microbiota and the bovine feces, respectively.

These abundances were lower than those in human fecal microbiota by a factor of 5 to 25, 15

and were clustered significantly apart from the latter. Furthermore, the inter-individual

differences among the animal fecal microbiota were less apparent than the human fecal

microbiota (Figure 5C).

Abundance of Bacteroides spp. as quantified by Q-PCR To validate the relative

abundance of Bacteroides spp. as determined by HOPE, Q-PCR was also deployed to 20

determine the abundance of Bacteroidales, the B. fragilis group and its subgroup, and the

Parabacteroides group that were present in the feces of human volunteers H1 to H3. As

the 16S rRNA operon copy number of different Bacteroides spp. are not well defined yet,

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quantitative expression of Q-PCR calculation was based on genomic mass (picograms).

Standard curves generated showed linear relation over the range of one to 10000

picograms, and had an amplification efficiency of 90.8-100.7 % at an annealing

temperature of 64 oC. Relative abundance of most Bacteroidales as targeted by primer

Bac303 accounted for 30.1-50.9 % in the feces of all three human volunteers. Likewise, B. 5

fragilis group, Parabacteroides group and B. fragilis subgroup comprised 4.7-14.8 %,

3.1-5.4 % and 1.7-2.0 % respectively (Table 4). These results were in close proximity to

those obtained using HOPE.

Relative abundance of Bacteroides spp. in wastewaters Table 3 and Figure 5

indicate the relative abundances of Bacteroides spp. detected in the influent of a pig and 10

municipal wastewater treatment plant. Bacteroides spp. that were previously detected in

the human feces were also present in municipal wastewater, with B. fragilis, B. caccae, B.

uniformis, B. vulgatus and B. massiliensis accounting for 3.6 %, 2.7 %, 18.3 %, 31.5 %,

and 5.2 % of the B. fragilis group, respectively. B. thetaiotaomicron remained as the

predominant species in the B. fragilis subgroup that extended with “T” (95.2 %). The 15

relative abundance of these Bacteroides spp. in the municipal wastewater therefore

approximates to those found in human fecal microbiota. However, the relative

abundance of most Bacteroidales decreased by more than two-fold, possibly due to the

die-off effect of these Bacteroides spp. and the proliferation of other microbial

populations during the transportation in the sewage system. 20

In the influent of swine wastewater treatment plant, none of the primers targeting

Bacteroides spp. at the species-level could be detected. Relative abundance of targets in

the order Bacteroidales was also about two-fold lower than in municipal wastewater.

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DISCUSSION

In this study, HOPE with its features in detection sensitivity, specificity, rapidity and

throughput, has been successfully demonstrated as a method that can improve

understanding on the relative abundance of Bacteroides-Parabacteroides spp. present in

fecal microbiota. Findings from the measurement of human feces suggested that the fecal 5

microbiota contain a relatively stable profile of Bacteroides spp. with major species like

B. vulgatus, B. fragilis, B. thetaiotaomicron, B. uniformis, and B. caccae consistently

detected in all tested samples. Clear inter-individual differences in the diversity of

Bacteroides spp. were observed, presumably due to differences in living habits, age and

diet (20, 22). The total abundance of these Bacteroides spp. targeted by the species-10

specific primers within the B. fragilis cluster represented 27-63 %, further suggesting the

existence of other Bacteroides spp. in human fecal microbiota. It is possible that a small

fraction of these unidentified Bacteroides spp. could be represented by B. ovatus and B.

stercoris, which were not determined in this study. However, these two species are

unlikely to be predominant as previous FISH studies have reported a low detection 15

frequency with them (34). The remaining largely unidentified fraction could be

represented by the yet-to-be-cultured species in the B. fragilis cluster as reported by

Eckburg and coworkers (11). Based on 16S rRNA gene clone library, they detected 65

Bacteroidetes-related phylotypes and as much as 65 % are classified as novel.

Likewise, when HOPE was applied to animal feces and wastewaters, all of the 20

species-specific primers were not detected, and a reduction in Bacteroidales was

observed in the wastewater treatment influent. These reconfirmed previous reports that

those predominant Bacteroides spp. in human fecal microbiota are present in much lower

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abundance in animal stools (27). Therefore, certain Bacteroides 16S rRNA genes have

been suggested and used as effective biomarkers to differentiate the origin and duration

of fecal contamination, thereby achieving microbial source tracking (5, 9, 14).

We further compared the HOPE results on the diversity and abundance of those

fecal bacterial populations with that obtained using 16S rRNA clone libraries and FISH 5

(11, 16, 19, 34, 39) (Table 4). Though 16S rRNA clone libraries and FISH expressed

abundance of bacterial targets based on different normalization units (i.e., percentage of

total number of clones picked, and percentage of total number of cells hybridized with

domain Bacteria-specific probe) and inter-individual variation in the composition of the

Bacteroides flora exists, both molecular methods have shown a relatively similar range of 10

abundance of Bacteroides-Prevotella (14.5-48 %) in the human fecal microbiota as

HOPE (28.1- 44.4 %). FISH results showed that both B. fragilis cluster and P. distasonis

group added up to 4.3-21 % of the total Bacteria, while B. vulgatus accounted for 4.2 %

of the total Bacteria respectively (16, 19, 34). Similarly, after picking and sequencing

300-700 clones to construct a clone library, B. fragilis group and P. distasonis group 15

were found to account for 13.3 % and 2.3 % of total Bacteria respectively, while

predominant species like B. vulgatus, B. thetaiotaomicron, B. uniformis, and B. caccae

accounted for 2.1-15 %, 1.4-6.2 %, 4.9 %, 1.1 % of total Bacteria (11, 39).

To further validate the reliability of HOPE results obtained in this study, we used

Q-PCR to determine the abundance of selected Bacteroides spp. on the same set of 20

human fecal samples. The relative abundance of Bacteroidales, B. fragilis cluster and its

subcluster, and Parabacteroides distasonis cluster as determined by both Q-PCR and

HOPE results were in close approximation (Table 4). This suggests that when inter-

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sample differences in the composition of the Bacteroides spp. are minimal, the two PCR-

based molecular methods complement each other in determining the abundance of

specific bacterial targets.

It is clear through this comparison that HOPE, with its advantages in the detection

sensitivity, specificity and throughput, can complement other available molecular 5

methods and aid understanding of microbial studies. It has been demonstrated that HOPE

was able to detect bacterial targets at different phylogenetic specificity (i.e. species,

genus, …domain), and at sensitivity down to 0.1 % of the total PCR-amplified bacterial

targets (41). This sensitivity approximates to constructing a 300-clones library (39), and

as a result, can easily detect bacterial targets like Bacteroides spp. in the human feces at 10

the species level. Unlike clone libraries, in which abundance of a particular bacterial

target is dependent on the ultimate number of clones picked, HOPE examines the entire

PCR-amplified microbial community and can better report relative abundances of

bacterial targets. In comparison to other molecular tools, the comparable results obtained

by HOPE (Table 4) suggest that the specificity and reliability of HOPE were also not 15

compromised. The entire HOPE procedure for the identification and quantification of

those 14 Bacteroides-Parabacteroides spp. after primary DNA extraction and PCR

amplification is less than 120 min, and is significantly shorter than that required for FISH

or clone library construction. Furthermore, the total number of bacterial targets can be

easily increased simply by adding additional HOPE reactions or additional primer(s) into 20

individual reactions (Fig. 1).

At present, HOPE has only been demonstrated to determine the relative

abundance of known bacterial targets and therefore do not aid in further understanding

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the diversity of the microbial community. The complexity of making multiplexing tube

arrangements and analyzing the electropherograms can also increase with the increase in

the number of applied primers. Thus, there is a strong need to automate both processes.

It should be noted that the determination of the relative abundance of bacterial targets by

HOPE can be subjected to PCR bias. To minimize this bias, one can apply amplicons 5

obtained at the exponential phase of PCR amplification as DNA templates in HOPE

reactions. The sensitivity of the method may also be compromised by an excessively long

poly-A tail (REF) and by the possible interferences due to the non-targeted DNA in

complex environmental samples. To circumvent these effects, it is therefore essential to

perform a systematic study that looks into an optimal poly-A tail length, and to perform a 10

thorough purification of the HOPE products prior to capillary electrophoresis.

In summary, this study has demonstrated the potential of HOPE as a rapid and

high-throughput detection method that is able to examine the relative abundances of

bacterial targets at various taxonomical levels. It can be used to identify disease-related

bacterial populations that are present in gut or feces over a temporal and spatial change. 15

Likewise, the current set of human-specific Bacteroides HOPE primers can be improved

to include primers targeting animal-specific uncultivated Bacteroidales (5, 6, 9, 10, 14,

29), and be used to detect the presence of host-specific Bacteroidales in water bodies that

may be contaminated with multiple fecal sources. The simplicity and versatility of this

method can further facilitate the attempts to understand microbial diversity in different 20

ecologies by complementing it with other existing molecular tools. For example, one can

use fingerprinting methods to quickly evaluate the microbial diversity of an

environmental sample. Once the key microbial populations are identified using the

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fingerprinting methods, HOPE can be subsequently applied to effectively monitor the

dynamics of those key populations at a much refined resolution.

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Table 1. HOPE primers were designed to target human-specific Bacteroides spp. at various hierarchical levels (species, group, order and domain level).

Primer Target Sequence (5’-3’) Poly-A tail

(nt)

Type of

ddNTP added

HOPE reaction

tube no.

Reference

Bnd 136 B. nordii (JCM12987) AGC CTA TCC CCG AGT AAA A 0 G 1 This study

Bsls 1016 B. salyersiae (JCM12988) GCC TTG CGG CTA TGC CG 10 T 1 This study

Pg_dtc 732 B. pyogenes (JCM6294) GTT CCG GCC CGG TGA GCT 20 G 1 This study

Bth 584 B. thetaiotaomicron

(BCRC10624) CAA CTG ACT TAA CTG TCC AC 16 C 1 (41)

Bfrg 1026 B. fragilis (BCRC10619) TCA CAG CGG TGA TTG CTC 20 A 2 This study

Bcc 1066 B. caccae (JCM9498) CGT ATG GGT TTC CCC ATA A 15 T 3 This study

Badf 1009 B. acidifaciens (JCM10556) CGG CTA ACA TGT TTC CAC 0 A 3 This study

Bitt 141 B. intestinalis (JCM13266) CGA AAG GCT ATC CCG GAA 0 T 3 (41)

Begt 999 B. eggerthii (JCM12986) GTT TCC ACT ACA TTC CGC 0 T 4 This study

Bhcg 171 B. helcogenes (JCM6297) TTT CAG TGC CAT CGG GCA T 8 T 4 This study

Bufm 1018 B. uniformis (JCM5828) CTG CCT TGC GGC TGA CA 20 T 4 (41)

Bvg 1016 B. vulgatus (BCRC12903) ATG CCT TGC GGC TTA CGG C 0 T 5 This study

Bcpc 1015 B. coprocola (JCM12979) CGC CTT GCG GCT TAC AAG T 24 T 5 This study

Bmsl 1000 B. massiliensis (JCM12982) GCG TTT CCG CCA TAT TCG G 19 T 5 This study

Bdts_gp980 P. distasonis (JCM5825)

P. merdae (JCM9497)

P. goldsteinii (JCM13446)

CGT TCA AAC CCG GGT AA 12 G 6 This study

Bth 274 B. fragilis sub-group CCC CTA TCC ATC GAA GG 15 C/T 6, 2, 1 (41)

Bfrg 602 B. fragilis cluster GAG CCG CAA ACT TTC ACA A 19 C 6, 5, 4, 3, 2 (19)

Bac 303 Most Bacteroidales CCA ATG TGG GGG ACC TT 5 C 6 (31)

U1390 Most Bacteria YGA CGG GCG GTG TGT 17 A 6 (43)

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Table 2. Primers targeting Bacteroides spp. at various taxonomical levels were used in Q-PCR to validate

against HOPE results. Appropriate reverse primers were chosen to result in an average amplicon size of

less than 250 bp.

Target Primer pair Primer sequence (5’-3’) Standard curve Amplification

efficiency (%),

E

1390F ACA CAC CGC CCG TCR Total Bacteria

1512R GGC TAC CTT GTT ACG ACT T

y= -3.309x + 26.069

100.5

Bac303F AAG GTC CCC CAC ATT GG Bacteroides-Prevotella

530R CCG CGG CKG CTG GCA C

y= -3.306x + 24.345

100.7

Bfrg602F TTG TGA AAG TTT GCG GCT C Bacteroides fragilis cluster

797R GGA CTA CCA GGG TAT CTA ATC CTG TT

y= -3.4043x + 25.999

96.7

Bdts_gp980F TTA CCC GGG TTT GAA CG Parabacteroides distasonis

cluster 1055R CAR CCA TGC AGC ACC

y= -3.375x + 29.742

97.8

Bth274F CCT TCG ATG GAT AGG GG Bth274[C]- and Bth274[T]-

related subcluster 530R CCG CGG CKG CTG GCA C

y= -3.565x + 28.925

90.8

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Table 3. Relative abundance of bacterial targets in the human (H1 to H3), pig (P1 to P3) and cow (C1 to C3) feces, together with those present in the influent of a

municipal and swine wastewater treatment plant (Municipal WW and Pig WW respectively), were determined individually using multiplexing HOPE method.

H1 H2 H3 P1 P2 P3 C1 C2 C3 Municipal

WW

Pig

WW

Target wrt Relative abundance (%) 4

B. fragilis ND3 3.1± 0.1 2.9± 0.2 3.6± 0.3

B. caccae 8.9± 1.5 1.3± 0.8 1.6± 0.2 2.7± 0.4

B. acidifaciens ND ND ND ND

B. intestinalis ND ND 1.2± 0.3 ND

B. uniformis 7.5± 0.5 3.9± 0.4 7.1± 0.3 18.3± 0.7

B. eggerthii ND 0.6± 0.1 ND ND

B. helcogenes ND ND ND ND

B. vulgatus 28.4± 0.9 14.8± 0.3 49.8± 2.2 31.5± 1.2

B. massiliensis ND 2.9± 1.0 ND 5.2± 0.2

B. coprocola

ND ND ND

ND

B. nordii ND ND ND ND

B. salyersiae ND ND ND ND

B. pyogenes ND ND ND ND

B.

thetaiotaomicron

72.2± 0.3 58.9± 0.7 97.5± 0.4

95.2± 2.0

Bac303-related 28.1± 0.3 44.4± 2.0 29.9± 3.0 5.2± 0.4 4.6± 0.9 5.1± 0.7 2.3± 0.3 2.9± 0.2 1.8± 0.2 10.4± 1.6 4.6± 0.6

Bfrg602-related 5.0± 0.9 11.8± 1.0 12.1± 1.2 ND ND ND ND ND ND 6.2± 0.2 ND

Bdts_gp980-related 2.4± 0.1 2.5± 0.2 4.1± 0.2 1.3± 0.1 1.4± 0.1 1.7± 0.1 0.7± 0.2 0.7± 0.2 0.5± 0.1 5.1± 0.2 1.8± 0.1

Bth274[C]-related1 0.8± 0.1 1.2± 0.2 1.3± 0.2 ND ND ND ND ND ND 1.5± 0.8 ND

Bth274[T]-related2

1.1± 0.3 0.5± 0.1 0.2± 0.1 ND ND ND ND ND ND 0.3± 0.2 ND

ND

ND

ND

ND

ND

ND

Bfrg

602-related

gro

up

Bth

27

4 [T

]-

related g

roup

U139

0-related

dom

ain

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1 Primer Bth274 [C]_15dA extends by ddCTP when annealed to bacterial targets in this group. 2 Primer Bth274 [T]_15dA extends by ddTTP when annealed to

bacterial targets in this group. 3 ND, not detected. 4 Relative abundance of all bacterial targets were averaged from triplicates (n=3).

Calibration factors (CF) obtained for primers in multiplexing tube one, Bth274 [T]_15dA: Bnd136: Bsls1016_10dA: Pg_dtc732_20dA: Bth584_16dA = 1: 2.05: 0.39:

0.36: 0.19. CF values were obtained using clones from bacterial strains that include B. nordii, B. salyersiae, B. pyogenes and B. thetaiotaomicron.

CF obtained for primers in multiplexing tube two to five, Bfrg602_19dA: Bfrg1026_20dA: Bcc1066_15dA: Badf1009: Bitt141: Bufm1018_20dA: Begt999:

Bhcg171_8dA: Bvg1016: Bmsl1000_19dA: Bcpc1015_24dA = 1: 0.17: 2.96: 0.36: 16.51: 4.61: 8.17: 13.86: 8.37: 3.13: 3.06. CF values were obtained using clones

from bacterial strains that include B. fragilis, B. caccae, B. acidifaciens, B. uniformis, B. eggerthii, B. helcogenes, B. vulgatus, B. massiliensis and B. coprocola.

CF obtained for primers in multiplexing tube six, U1390_17dA: Bac303_5dA: Bfrg602_19dA: Bdts_gp980_12dA: Bth274 [C]_15dA: Bth274 [T]_15dA = 1: 1.206:

0.091: 1.998: 0.158: 0.432. CF values were obtained using clones from bacterial strains that include B. fragilis, B. thetaiotaomicron, B. tectus, B. uniformis, B. nordii,

P. distasonis and P. merdae.

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Table 4. Comparison of the abundance of Bacteroides at different hierarchical levels, obtained from various published literature.

Method Sampling size Bacteroides

/Prevotella

B. fragilis

subgroup

P. distasonis

subgroup

Bth274-

related

subgroup

B.

vulgatus

B. thetaiotao-

micron

B.

uniformis

B. caccae

Franks et

al. (16)

FISH1 Nine volunteers,

fecal samples

NA 21 %

(Bfra602- and Bdis656-related)

ND 4

Harmsen

et al. (19)

FISH Eleven

volunteers, fecal

samples

27.7 %

(Bac303-

related)

ND

ND

Rigottier-

Gois et

al. (34)

FISH Twenty

volunteers, fecal

samples

14.5 %

(Bac303-

related)

3.9 %

(Bfra998-

related)

0.4 %

(Bdist1025-

related)

ND 4.2 %

(Bvg1017

-related)

ND ND ND

Suau et

al. (39)

16S rRNA

clone

libraries2

One volunteer,

fecal samples,

284 clones

31 %

13.3 %

2.3 %

NA

2.1 %

1.4 %

4.9 %

1.1 %

Eckburg

et al. (11)

16S rRNA

clone

libraries

Three volunteers,

mucosa and fecal

samples, total of

395 bacterial

phylotypes

48%

NA4

NA

15 %

6.2 %

NA

This

study

HOPE3 Three volunteers,

fecal samples

28.1-44.4 %

(Bac303-

related)

5.0 -12.1

%

(Bfrg602-

related)

2.4 - 4.1 %

(Bdts_gp980-

related)

1.5 -1.9 %

(Bth274-

related)

1.4 -6.0 %

(Bvg1016

-related)

0.2 -0.8 %

(Bth584-

related)

0.4 -0.9

%

(Bufm101

8-related)

0.2-0.4 %

(Bcc1066-

related)

This

study

Q-PCR3 Three volunteers,

fecal samples

30.1-50.9 %

(Bac303-

related)

4.7 -14.8

%

(Bfrg602-

related)

3.1 -5.4 %

(Bdts_gp980-

related)

1.7 -2.0 %

(Bth274-

related)

ND

1 Results denoted in FISH are expressed in terms of hybridized cell counts normalized against Eub338 2 Results denoted in clone libraries are expressed in terms of total number of clones picked 3 Results denoted in HOPE and Q-PCR are expressed in terms of total PCR-amplified 16S rRNA genes 4 ND, not determined; NA, information not available from the literature.

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29

Legend

Fig. 1. A phylogenetic representation of different cultivated and uncultivated

Bacteroides spp.. In this study, a total of 19 primers were designed to target 14

functionally important Bacteroides spp. and Parabacteroides spp. at various

taxonomical levels. The associated coverage of each primer was marked on the

right. Primers were then arranged into six reaction tubes, with those targeting at

the species-level strewn across reaction tube one to five. Within reaction tube

one to five, an associated higher ranking primer, usually targeting not beyond

the family level, was placed alongside. In the last reaction tube, all higher

ranking primers that were used in reaction tube one to five (i.e. Bth274, Bfrg602,

Bdts_gp980) were placed along with primers targeting at an even higher

taxonomical level like Bac303 and U1390.

Fig. 2. A schematic illustration of HOPE. PCR amplicons, fluorophore-labeled

ddNTPs, DNA polymerase and oligonucleotide primers were mixed together to

form HOPE reaction mixture. The mixture undergoes a thermal program

involving 20-25 cycles of denaturation (96 oC), annealing (60-64

oC) and single-

base extension (72 oC). The products were purified and removed of the excess

ddNTP residual by shrimp alkaline phosphatase (SAP) digestion. An

appropriate amount of the product mixture was mixed with synthetic

oligonucleotide standards, and then subjected to capillary electrophoresis. From

the electropherograms, the extended primers were recognized by means of

consistent fragment sizes and the colors of the extended ddNTP.

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Fig. 3. A graphical representation of PCR amplicon mass obtained at different number

of thermal cycles. Exponential amplification was achieved from cycle 10 to 20,

and plateau after 20 cycles. To minimize PCR bias that can affect the

determination of relative abundance of bacterial targets, total PCR-amplified

16S rRNA genes were obtained after 20 cycles of PCR amplification, purified

and then used for HOPE.

Fig. 4. The actual electropherograms of HOPE products obtained in the presence of

total PCR-amplified 16S rRNA genes from (a) Bacteroides thetaiotaomicron

BCRC10624 clone and (b) human feces H3. Black, red and green peaks

represented primers that were extended with dTAMRA-ddCTP, dROX-ddTTP,

dR6G-ddATP respectively.

Fig. 5. A statistical interpretation to the HOPE results. (a) The relative abundances of B.

fragilis cluster, B. fragilis subcluster, and the P. distasonis cluster against the

total amplified 16S rRNA genes were averaged out within the individual sample

type. (b) B. massiliensis, B. vulgatus, B. eggerthii, B. uniformis, B. intestinalis,

B. caccae and B. fragilis were detected and their average relative abundances

within the B. fragilis cluster were illustrated. A predominant portion within the

B. fragilis cluster remains unidentified. (c) A multidimensional scaling (MDS)

plot obtained by first performing a Euclidean distance square-root

transformation on the raw data to generate a similarity matrix. The matrix then

undergoes a 100-iterations MDS analysis.

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Figure 1.

Bacteroides tectus JCM 10003 cloneBacteroides tectus JCM 10003 (AB200228)

Bacteroides pyogenes JCM 6294 clone Bacteroides pyogenes JCM 6294 (AB200229)

Bacteroides salyersiae JCM 12988 clone

Bacteroides salyersiae WAL 10018 (AY608696)

Bacteroides nordii JCM 12987 cloneBacteroides nordii WAL 11050 (AY608697)

Bacteroides thetaiotaomicron BCRC 10624 cloneBacteroides thetaiotaomicron ATCC 29148 (L16489)

Bacteroides fragilis BCRC 10619 cloneBacteroides fragilis (M11656)

Bacteroides caccae JCM 9498 cloneBacteroides caccae ATCC 43185T (X83951)

Bacteroides acidifaciens JCM 10556 clone

Bacteroides acidifaciens A32 (AB021162) Bacteroides intestinalis JCM 13266 cloneBacteroides intestinalis JCM 13266 (AB214329)

Bacteroides uniformis JCM 5828 cloneBacteroides uniformis 23-18 (AB247142)

Bacteroides eggerthii JCM 12986 cloneBacteroides eggerthii (AB050107)

Bacteroides stercoris JCM 9496 cloneBacteroides stercoris ATCC 43183T (X83953)

Bacteroides helcogenes JCM 6297 clone

Bacteroides helcogenes JCM 6927 (AB200227) Bacteroides vulgatus BCRC 12903 cloneBacteroides vulgatus (M58762)

Bacteroides massiliensis JCM 12982 cloneBacteroides massiliensis B84634 (AY126616)

Parabacteroides goldsteinii JCM 13446 cloneParabacteroides goldsteinii (AY974070)Parabacteroides distasonis JCM 5825 clone

Parabacteroides distasonis JCM 5825 (AB238922)

Parabacteroides merdae JCM 9497 clone

Parabacteroides merdae JCM 13405 (AB238929)Bacteroides coprocola JCM 12979 cloneBacteroides coprocola M16 (AB200224)

Bovine-specific uncultivated Bacteroidales



Swine-specific uncultivated Bacteroidales

Canines/Felines-specific uncultivated Bacteroidales0.02

Ä Pg_dtc732

Ä Bsls1016

Ä Bnd136

Ä Bth584

ﾐ Bfrg1026

O Bcc1066

O Badf1009

O Bitt141

ズ Bufm1018

ズ Begt999

ズ Bhcg171

± Bvg1016

± Bmsl1000

± Bcpc1015

Ä, #

Bth

274

[T]

ﾐ, # Bth274 [C]

# Bth274 [C]

ﾐ,O

, ズ, ±

, #

Bfrg

602

#

Bac303

#

U1390

# B

dts

_g

p980

Bfrg

602

Ä Reaction one:

Bth274 [T]_15dA

Pg_dtc732_20dA

Bsls1016_10dA

Bnd136

Bth584_16dA

ﾐ Reaction

two:

Bfrg602_19dA

Bfrg1026_20dA

Bth274_15dA

O Reaction three:

Bfrg602_19dA

Bcc1066_15dA

Badf1009

Bitt141

ズ Reaction four:

Bfrg602_19dA

Bufm1018_20dA

Begt999

Bhcg171_8dA

±Reaction five:

Bfrg602_19dA

Bvg1016

Bmsl1000_19dA

Bcpc1015_24dA

# Reaction six:

U1390_17dA

Bac303_5dA

Bfrg602_19dA

Bdts_ gp980

Bth274 [C]_15dA

Bth274 [T]_15dA

Bacteroides tectus JCM 10003 cloneBacteroides tectus JCM 10003 (AB200228)

Bacteroides pyogenes JCM 6294 clone Bacteroides pyogenes JCM 6294 (AB200229)

Bacteroides salyersiae JCM 12988 clone

Bacteroides salyersiae WAL 10018 (AY608696)

Bacteroides nordii JCM 12987 cloneBacteroides nordii WAL 11050 (AY608697)

Bacteroides thetaiotaomicron BCRC 10624 cloneBacteroides thetaiotaomicron ATCC 29148 (L16489)

Bacteroides fragilis BCRC 10619 cloneBacteroides fragilis (M11656)

Bacteroides caccae JCM 9498 cloneBacteroides caccae ATCC 43185T (X83951)

Bacteroides acidifaciens JCM 10556 clone

Bacteroides acidifaciens A32 (AB021162) Bacteroides intestinalis JCM 13266 cloneBacteroides intestinalis JCM 13266 (AB214329)

Bacteroides uniformis JCM 5828 cloneBacteroides uniformis 23-18 (AB247142)

Bacteroides eggerthii JCM 12986 cloneBacteroides eggerthii (AB050107)

Bacteroides stercoris JCM 9496 cloneBacteroides stercoris ATCC 43183T (X83953)

Bacteroides helcogenes JCM 6297 clone

Bacteroides helcogenes JCM 6927 (AB200227) Bacteroides vulgatus BCRC 12903 cloneBacteroides vulgatus (M58762)

Bacteroides massiliensis JCM 12982 cloneBacteroides massiliensis B84634 (AY126616)

Parabacteroides goldsteinii JCM 13446 cloneParabacteroides goldsteinii (AY974070)Parabacteroides distasonis JCM 5825 clone

Parabacteroides distasonis JCM 5825 (AB238922)

Parabacteroides merdae JCM 9497 clone

Parabacteroides merdae JCM 13405 (AB238929)Bacteroides coprocola JCM 12979 cloneBacteroides coprocola M16 (AB200224)




Swine-specific uncultivated Bacteroidales

Canines/Felines-specific uncultivated Bacteroidales0.02

Ä Pg_dtc732

Ä Bsls1016

Ä Bnd136

Ä Bth584

ﾐ Bfrg1026

O Bcc1066

O Badf1009

O Bitt141

ズ Bufm1018

ズ Begt999

ズ Bhcg171

± Bvg1016

± Bmsl1000

± Bcpc1015

Ä, #

Bth

274

[T]

ﾐ, # Bth274 [C]

# Bth274 [C]

ﾐ,O

, ズ, ±

, #

Bfrg

602

#

Bac303

#

U1390

# B

dts

_g

p980

Bfrg

602

Ä Reaction one:

Bth274 [T]_15dA

Pg_dtc732_20dA

Bsls1016_10dA

Bnd136

Bth584_16dA

ﾐ Reaction

two:

Bfrg602_19dA

Bfrg1026_20dA

Bth274_15dA

O Reaction three:

Bfrg602_19dA

Bcc1066_15dA

Badf1009

Bitt141

ズ Reaction four:

Bfrg602_19dA

Bufm1018_20dA

Begt999

Bhcg171_8dA

±Reaction five:

Bfrg602_19dA

Bvg1016

Bmsl1000_19dA

Bcpc1015_24dA

# Reaction six:

U1390_17dA

Bac303_5dA

Bfrg602_19dA

Bdts_ gp980

Bth274 [C]_15dA

Bth274 [T]_15dA

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Figure 2.

Size

3’ 5’

5’ 3’

3’ 5’

Capillary electrophoresis

Internal standards

PA

5’ 3’

3’ 5’

ddCTP

ddATP

ddTTP

ddGTP

SAP enzymatic digestion

C

Purified PCR

amplicons

Fluorophore-

Labeled ddNTPs

DNA polymerase Primer mixture

Target A

5’ 3’

3’ 5’Target B

PA

PB

PC

PD

5’ 3’

3’ 5’

HOPE reactions

(20 – 25 cycles)

AG

3’ 5’AGA

Target A

Target B

60 – 64 o C

Primers anneal

3’ 5’C AG

3’ 5’AGA

Target A

Target B

72 o C

Primers extend

96 o C

Amplicons denature

PB

PC

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0

10

20

30

40

50

60

10 15 20 25 30 35 40

No. of cycles

Am

plic

on

mas

s (

10

3n

g)

Municipal Influent

Swine Influent

0

10

20

30

40

50

60

10 15 20 25 30 35 40

No. of cycles

Am

plic

on

mas

s (

10

3n

g)

Municipal Influent

Swine Influent

Figure 3.

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Multip

lex T

ube 2

Bth274 [T]_15dA

Bth274 [C]_15dA

Bfrg602_19dA

Dye intensity (rfu)

Bfrg1026_20dA

Inte

rna

l sta

nd

ard

Dye intensity (rfu)

Multip

lex T

ube 1

Inte

rna

l sta

nd

ard

Bth274 [C]_15dA

Bth274 [T]_15dABth584_16dA

Bfrg602_19dABcc1066_15dA

Multip

lex T

ube 3

Inte

rna

l sta

nd

ard

Multip

lex T

ube 4

Bfrg602_19dABufm1017_20dA

Inte

rna

l sta

nd

ard

Multip

lex T

ube 5

Bfrg602_19dA

Bvg1016

Inte

rna

l sta

nd

ard

Bfrg602_19dAU1390_17dA

Bac303_5dA

Multip

lex T

ube 6

Dye intensity (rfu)

Dye intensity (rfu)Dye intensity (rfu)Dye intensity (rfu)

Multip

lex T

ube 2

Bth274 [T]_15dA

Bth274 [C]_15dA

Bfrg602_19dA

Dye intensity (rfu)

Bfrg1026_20dA

Inte

rna

l sta

nd

ard

Dye intensity (rfu)

Multip

lex T

ube 1

Inte

rna

l sta

nd

ard

Bth274 [C]_15dA

Bth274 [T]_15dABth584_16dA

Bfrg602_19dABcc1066_15dA

Multip

lex T

ube 3

Inte

rna

l sta

nd

ard

Multip

lex T

ube 4

Bfrg602_19dABufm1017_20dA

Inte

rna

l sta

nd

ard

Multip

lex T

ube 5

Bfrg602_19dA

Bvg1016

Inte

rna

l sta

nd

ard

U1390_17dA

Inte

rna

l sta

nd

ard

Multip

lex T

ube 6

Dye intensity (rfu)

Dye intensity (rfu)Dye intensity (rfu)Dye intensity (rfu)

Bdts_gp980_12dA

Fig

ure

4(A

).

Fig

ure

4(B

).

Dye intensity (rflu)

Btn274 [T]_15dA

Dye intensity (rflu)

Bth584_16dA

dTAMRA-standard

dROX-standard

Bac303_5dA

Bth274 [T]_15dA

U1390_17dABfrg602_19dA

dTAMRA-standard

dROX-standard

dR6G-standard

Mu

ltiple

x T

ub

e 1

in p

rese

nce

of B

. the

taio

tao

mic

ron

clo

ne

Mu

ltiple

x T

ub

e 6

in p

rese

nce

of B

. the

taio

tao

mic

ron

clo

ne

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0 10 20 30 40 50 60

Others

B. fragilis

B. caccae

B. acidifaciens

B. intestinalis

B. uniformis

B. eggerthii

B. helcogenes

B. vulgatus

B. massiliensis

B. coprocola

Relative abundance (%)-10

0

10

20

30

40

50

Munic

ipal

WW

Sw

ine

WW

Pig

Cow

Hu

man

Others

B. fragilis cluster

P. distasonis cluster

B. fragilis subcluster [extends with ddCTP]

B. fragilis subcluster [extends wtih ddTTP]

Re

lative

abundance (

%)

(A) (C) (B)

Human Pig Cow

H1

H2

H3P1, P2, P3Swine WW

C1, C2, C3

Municipal

WW

Stress: 0

Legend:

Figure 5

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Documents

Relative Abundance of Bacteroides spp. in Stools and Wastewaters as Determined by Hierarchical