Culture-independent characterisation of human faecal flora using rRNA-targeted hybridisation probes

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Culture-independent characterisation of human faecal

flora using rRNA-targeted hybridisation probes

Joël Doré, Geneviève Gramet, Isabelle Goderel, Philippe Pochart

To cite this version:

Original

article

Culture-independent

characterisation

of

human

faecal flora

_using

rRNA-targeted hybridisation

probes

Joël Doré*

Geneviève Gramet Isabelle

_Goderel,

Philippe

Pochart

Institut national de la recherche

_agronomique,

Domaine de

Vilvert,

78352

_{Jouy-en-Josas cedex,}

France

Abstract -

_Comparison

of cultural and

microscopic

counts indicates that a

_significant

fraction of human faecal flora is nonculturable. To characterise human faecal flora

using quantitative

dot-blot

hybridisation,

a set of ten ribosomal ribonucleic acid

(rRNA)-targeted

oligonucleotide probes

were used to

_quantify

rRNA abundance of different microbial groups within total RNA from faeces of ten individuals. The three domains and

_major

groups of the domain bacteria were

_{investigated.}

Cultural counts

were

_compared

with rRNA relative abundance for total

_{Bacteroides, Bifidobacterium}

and Escherichia coli. Bacterial rRNA

_represented

78.2 f 4.3

%

_{(range 59-94)}

of total rRNA. The archaeal and

eucaryal

rRNAs

only

showed a minor contribution to total rRNA

_(0.28

and 2.02

_{%, respectively),}

the added contribution of all three domains

accounting

for 80.5 ! 4.5 %

_{(range 61-100)}

of total rRNA. Additive

_{probes targeting}

high

G

+

C

Gram-positives

(HGC)

and other bacteria

_(NHGC)

hybridised

with 1.4 and 80.6

%

of bacterial

_{rRNA, respectively.}

Total Bacteroides and

_{enterics-group}

rRNAs accounted for 35.5 ±4.5 % and 2.7 ±0.7 % of bacterial

_{rRNA, respectively.}

These results

_suggested

a _very

_significant

contribution of low G + C

_{Gram-positives}

(LGC)

to total bacteria.

Assuming

that Bacteroides and enterics were the

_major

Gram-negatives

present, LGC could be calculated to account for 47 to 67

%

of bacterial rRNA. Partial

_probing

of LGC accounted for 24 ! 5.5 % of bacterial rRNA. Cultural counts were more variable between individual faecal

samples

than were rRNA relative abundances for all three groups enumerated.

Average

counts of Bacteroides and

_{Bifidobacterium, expressed}

as _percent of total

_anaerobes,

were two-fold

_higher

than the

_{corresponding}

rRNA

_{indices, indicating}

a

_systematic

overestimation of

these groups. This could be due to an underestimation of total anaerobes. Our

results indicate that culture-based

techniques

introduce a bias in the

_appreciation

of the

_biodiversity

of the human

_gut

flora. LGC are

_apparently

more

_susceptible

to cultural underestimation. Their

diversity

as well as their functional contribution to the

_{gut ecosystem}

_may therefore need reconsideration. A

_complete

molecular

inventory

of human faecal

_{flora, currently underway}

in our

_laboratory,

and

probe-based

_technologies

will

_{certainly help}

to this end.

_©

_{Inra/Elsevier,}

Paris

molecular ecology

/

microbial diversity

/

rRNA

_/

hybridisation

/

faecal flora

*

Résumé - Caractérisation moléculaire de la microflore _{digestive humaine par}

hybridation quantitative a l’aide de sondes ciblant les ARNr. La

comparaison

des dénombrements au

_microscope

ou _par culture anaérobie de la flore fécale

hu-maine

_indique

l’existence d’une fraction non-cultivable

importante.

Nous avons utilisé

l’hybridation quantitative

en dot-blot _pour caractériser la structure et la diversité de la flore fécale humaine par une méthode

indépendante

de la culture. Un

ensem-ble de dix sondes ciblant les ARNr a

permis

la

quantification

relative des ARNr

de divers _{groupes microbiens} dans les selles de dix volontaires sains. Les trois do-maines

_primordiaux

et les groupes

majeurs

du domaine Bacteria ont été étudiés. Le dénombrement par culture de

Bacteroides,

Bifidobacterium

et Escherichia coli a

été

_comparé

avec la

_{quantification}

relative des ARNr

_{correspondants.}

L’ARNr du

domaine Bacteria a

_représenté

_{78,2 f 4,3 %}

[extrêmes

59-94]

de l’ARNr total. Les ARNr des domaines Archaea et

_Eucarya

n’ont

présenté qu’une

contribution minime à l’ARNr total

_(0,28

et

2,02

%,

respectivement),

portant

la contribution des trois domaines à

80,5 f 4,5 %

[61-100]

de l’ARNr total. Deux sondes additives ciblant les Gram

positifs

à haut

_(G

+

_C)

%

_(HGC)

et les autres bactéries

_(NHGC)

ont reconnu

1,4

et

80,6

% de l’ARNr

_bactérien,

respectivement.

Les ARNr des Bacteroides et du groupe des

entériques

ont

représenté

35,5:t 4,5

% et

2,7 f 0,7 %

de l’ARNr

bactérien,

respectivement.

Ces résultats

_suggéraient

une contribution

_importante

des bactéries Gram

_positives

à bas

_(G

+

_C)

%

(LGC)

dans la flore totale. Si Bacteroides et les

entériques

sont bien les Gram

_négatifs

dominants de la

_flore,

la contribution attendue des LGC est de 47 à 67

%

de l’ARNr bactérien. Une sonde reconnaissant

_{partiellement}

le groupe LGC a donné une contribution s’élevant à 24 iL

_5,5

% de l’ARNr bactérien.

Les dénombrements par culture anaérobie se sont montré

plus

variables entre indi-vidus que ne l’étaient les

quantifications

relatives d’ARNr pour les trois groupes micro-biens concernés. Les moyennes de dénombrements de Bacteroides et

_{Bifidobacterium,}

exprimées

en _pourcent de la flore

_totale,

étaient

_supérieures

d’un facteur 2 aux

quan-tités relatives d’ARNr

_{correspondantes, suggérant}

une surestimation

_{systématique}

de

ces _{groupes par} la culture. Cela

_pourrait

être dû à une sous-estimation de la flore

totale. Nos résultats

indiquent

que la culture anaérobie biaise la reconnaissance de la biodiversité réelle de

l’écosystème digestif

humain. Les LGC sont

plus

sensibles à

une sous-estimation _par la culture. Leur diversité et leur contribution fonctionnelle à

l’écosystème digestif

devront être réévaluées. L’inventaire moléculaire

complet

d’une flore fécale

humaine,

en cours dans notre

_laboratoire,

va _permettre d’avancer dans ce sens et de

compléter

le

panel

actuel de sondes

d’hybridation.

©

Inra/Elsevier,

Paris

écologie moléculaire

_/

diversité microbienne

/

ARNr

/

hybridation

/

flore fécale humaine

1. INTRODUCTION

Human colonic microflora is

_composed

of more than

1011

microbial cells

per gram of intestinal

contents,

belonging

to as _many as 400 different

_species

(Conway, 1995).

Their

_recognition

has been

possible

so far due to culture-based

enumeration and identification under strict anaerobic conditions.

_{Nevertheless,}

culture-based

techniques

introduce a bias illustrated

by

the

recognition

of a

fraction of not

_yet

cultured

components

within this

ecosystem,

representing

10

to 90

%

of the total anaerobic

_flora,

_according

to various studies

_(Moore

and

Holdeman, 1974;

Finegold

et

_al.,

_1983;

_Langendijk

et

_al.,

_1995;

Wilson and

Blitchington,

1996).

Modern molecular

_techniques

based

on _sequence

_comparisons

of nucleic

ribonucleic acid

_(rRNA)-based

methods have proven most

_appropriate

_(Pace

et

_al.,

_1985;

_Ward,

_1989;

_Sayler

and

_Layton,

_1990;

Stahl and

_Amann,

_1991;

Ward et

_{al., 1992;}

Olsen and

_{Woese, 1993; Stahl,}

_1993a,

_b;

Mackie and

_Doré,

1995).

The

_highly

conserved

_regions

of

rRNA

molecules can serve as

_target

sequences for universal or domain

_probes

(discriminating

between the domains

Archaea,

Bacteria and

_Eucarya,

sensu Woese et

_al.,

_1990).

The intermediate and more variable

_regions

are

_appropriate

for the

design

of _genus-,

species- and,

sometimes, strain-specific hybridisation probes.

In the

present

study,

we used

rRNA-based

quantitative

dot-blot

hybridi-sation for the characterisation of the

_community

structure of human faecal microflora. The three domains and

major

groups of the domain bacteria were

investigated.

Whenever

_possible,

cultural counts were

compared

with

rRNA

relative abundance.

2. MATERIALS AND METHODS

2.1. Total faecal RNA extractions

Human faecal

_samples

_(0.2

_{g in 2.2} mL _screw-cap

_tube)

were

_initially

sub-jected

to direct

_phenol

extraction with mechanical

_disruption

_{(Mini-beadbeater,}

Biospec Products, Bartlesville, OK,

USA)

using

0.2 g zirconium beads as

pre-viously

described

_(Stahl

et

_{al., 1988;}

Doré et

_al.,

_1998).

Nucleic acid extracts

were

quantified spectrophotometrically.

2.2. Probes and

_labelling

Synthetic high-performance

liquid

chromatography

(HPLC)

purified

oligonu-cleotide

probes

(Appligene,

Illkirch,

France)

were

5’-end

labelled with

32p

by

using polynucleotide

kinase

_(Gibco-BRL,

_Eragny,

_France)

and

_b-

_32p

_]ATP

(specific

activity

of

>5 000

Ci/mmol;

Amersham,

Les

_Ulis,

_France).

The

effi-ciency

of

_labelling,

assessed

_by

_{thin-layer chromatography}

_using

polyethylene-imine cellulose

_plates

and 0.5 M ammonium carbonate as mobile

phase,

was

always

above 75

%.

2.3. Total RNA dot-blot

_{hybridisation}

Samples

of 0.5 to 500 _ng total

RNA

from control _pure cultures and 0.1 to 1 vg total RNA from frozen faecal

_samples

were vacuum-blotted on

_Nytran

N

membranes

_using

the Minifold I

_(Schleicher

and

_Schuell).

_{Hybridisations}

were

performed

as

_previously

described

(Raskin

et

_{al., 1994;}

Doré et

_al.,

_1998).

The

probes

used are shown in table I.

Quantification

of

hybridisation signals

on dot-blots involved

_{radioimaging using}

the Instant

_Imager

_{(Packard Instrument).}

Figure

1 illustrates the results of

_{parallel hybridisation}

on similar total faecal

RNA

dilutions.

2.4. Units

All

_quantitative

rRNA measurements were standardised

_using

rRNA

Bacteroides

_vulgatus

_{(ATCC 8482),}

E. coli

_(Boehringer

Mannheim rRNA

stan-dards),

Saccharomyces

cerevisiae

_(authors’

_collection)

and Methanobrevibacter smithii

_{(ATCC 35061).}

Results are

expressed

as

_percent

of total or bacterial

rRNA,

as measured

_using

a universal or Bacteria domain

_probe

and

_given

as

mean f standard error of the mean of

_triplicate

RNA dilutions for each faecal

extract.

2.5. Microbial enumerations

For the enumeration of total

Bacteroides,

dilutions of human faecal

samples

were

_{plated using}

brain-heart infusion and

exposed

to air for 1 h to

_preselect

for

_{non-extremely oxygen-sensitive microorganisms, prior}

to incubation under strict anaerobic conditions at

37 °C

for 48 h

_(Corthier

et

_al.,

_1996).

Colonies

were transferred onto

_Nytran-Plus

membranes

_(Schleicher

and

_Schuell,

Ec-quevilly,

France)

following

the manufacturer’s recommendations.

_Colony

hy-bridisation was used for the enumeration of colonies

_belonging

to the

_target

group

using

the

Bacteroides-specific

oligonucleotide probe

(Doré

et

al.,

1998).

Bifidobacterium

spp. and E. coli were

classically

enumerated

_using

selective

media

_(Beerens

and

_Drigalski

media under anaerobic and aerobic

_conditions,

respectively).

Total anaerobes were enumerated

_using

the medium of Wilkins

Chalgren

in

_deep

_agar tubes. Microbial

populations

are

given

as

_percent

of total

culturable anaerobes.

2.6. Human volunteers

Human adults received unrestricted

_western-type

diets and none received

antibiotics or laxatives within 3 weeks

prior

to

_sampling.

All volunteers _gave informed consent to the

_research,

which was

approved by

the local ethics

committee.

3.

RESULTS

AND DISCUSSION

The

present

work demonstrates the use of

_quantitative

dot-blot

hybridisation

using rRNA-targeted probes

for the

analysis

of the structure of the human faecal microflora. It can be

_applied

to frozen faecal

_samples.

3.1. Structure of human faecal flora at the level of the domains

Ribosomal RNA indices indicated that the Bacteria domain was

_always

highly

dominant. Bacterial

rRNA

_represented

78.2 ! 4.3 %

_{(range 59-94)}

of total

rRNA.

It was the dominant

hybridisation signal

in all

samples

analysed.

The archaeal and

_eucaryal

rRNAs

_only

showed a minor contribution to total

RNA

_(0.28

and 2.02

_%,

_{respectively).}

The archaeal

_signal

was considered

representative

of

_{methanogenic populations,}

the

_only

_gut

archaea described

to date.

_Only

six of ten

_samples

showed a

_signal

_greater

than 0.1

%

archaeal

rRNA. For these

samples,

a

Methanobacteriales-specific

probe

was

further

used

to evaluate the

_proportion

of this _group, to which all current isolates of human

the

_currently

known

_diversity

within

_methanogenic

_archaea,

as

_performed

for

other

ecosystems

(Raskin

et

_al.,

_1994).

The

_{eucaryal hybridisation signal}

is

suspected

to come from residual RNA from

_sloughed

_enterocytes.

The added contribution of all three domains accounted for 80.5 ± 4.5

%

_(range

_61-100)

of total

rRNA.

An

_{aspecific signal}

for the universal

_probe

would

_explain

such observations.

_Furthermore,

we have indications that the Bacteria and

_Eucarya

domain

_probes,

_designed

_{years ago} with a limited _sequence database derived

from cultured

_{microorganisms}

_(Amann

et

_al.,

_1990),

are more

_specific

than

expected.

This would also lead to an underestimation of the added contribution of all three domain

_probes

to the universal

_{signal. Besides,}

the two lowest results

(N 61

%)

corresponded

to

_samples

for which the RNA

_yields

were the lowest

(<

300 _vg total

_RNA/g

_faeces).

3.2. Structure of the human faecal flora at the level of bacterial

groups

Two additive

_{probes targeting}

the same

_region

of 23S rRNA

(Roller

et

_al.,

1994),

the one

specific

for

_high

G + C

_{Gram-positives}

_(HGC)

and the other for all other bacteria

_(NHGC),

_gave

_{hybridisation}

intensities

_representing

1.4 and 80.6

%

of bacterial

_rRNA,

_{respectively.}

The _average sum of the

_{corresponding}

signals

was

82.0 ! 8.4 %

(range 51-140)

of total bacterial rRNA. This _very

marked variation was due to measurements with the NHGC

_{targeted probe.}

Besides,

the

_signals

obtained with a

Bifidobacterium genus-specific probe

(see

later)

were

_always

_greater

than those obtained with the HGC

_{probe, suggesting}

a

_systematic

underestimation of HGC

_by

the latter

_probe.

Total Bacteroides and

_{enterics-group}

rRNAs

accounted for

35.5 ±4.5%

(range

22-61)

and 2.7 ±0.7

%

_{(range 1.2-9.3)}

of bacterial

_{rRNA, respectively.}

Low

G

+

C

Gram-positives

(LGC),

including

the _genera

_{Streptococcus,}

Lac-tococcus,

Enterococcus, Lactobacillus, Clostridiv,m, Eubacterium,

Ruminococ-cus and

_others,

could

_only

be accounted for

by

a broad _group

probe

whose

rRNA

_target

has mismatches for some

_{representatives}

of all concerned _genera.

It could therefore

_{only give}

a

_partial

measure of LGC. Their

rRNA

_represented

24.2 ! 5.5

%

_(range

_22-61)

of bacterial

rRNA

based on this

probe. Hence,

us-ing

the

_currently

available

_{probe panel covering}

the _genera Bacteroides and

Bifidobacterium,

and the enterics and LGC groups

(latter,

partial),

two thirds of the bacterial rRNA could be attributed to identified

_phylogenetic

_groups

(figure

2).

Assuming

that Bacteroides and enterics were the

_major

Gram-negatives

present,

as

_expected

from all microbial enumerations done so

_far,

bacterial rRNA not accounted for

_by

the

_probes

used in this

_study

_may be

es-sentially

from LGCS. LGC would then account for 47 to 67

%

of bacterial rRNA.

Alternatively,

the fraction of the human faecal flora that has so far eluded cul-tivation could account for a

significant

_part

of

the current

_discrepancy

between the domain

_{probe targeting}

a conserved

_region

and _group

probes

solely

based

on _sequences derived from cultured

_organisms.

This

_hypothesis

could be

investi-gated using

several

_{probes targeting}

other known

_{Gram-negative}

_gut

anaerobes such as Fibrobacteriaceae for which a

_{family-specific}

_probe

is

_available,

or

cov-ering

the

proteobacteria

for which former cultural characterisations indicated the _presence of

_only

_gamma

_(enterics)

and delta

_{(sulphate-reducers)}

_groups at

3.3. Cultural counts versus relative rRNA abundance

Cultural counts were more variable between individual faecal

samples

than were

rRNA

relative abundances for all three groups enumerated.

Average

counts of Bacteroides and

_{Bifidobacterium,}

expressed

as

_percent

of total

anaer-obes,

were two-fold

_higher

than the

_{corresponding}

rRNA indices

(figure 3).

This

suggested

a

_systematic

overestimation of these _genera

_using

cultural

_techniques.

This could be due to an underestimation of total

anaerobes,

a

hypothesis

sup-ported by

numerous

_examples

of

_comparisons

of total

_microscopic

and cultural

counts which indicate that

_only

10 to 90

%

of total observable

microorgan-isms in situ are culturable

(Moore

and

Holdeman, 1974;

Finegold

et

_{al., 1983;}

Langendijk

et

al., 1995;

Wilson and

_{Blitchington,}

_1996).

We are

_currently

performing

a

complete

molecular

_inventory

of human faecal

flora based on

_sequencing

of cloned 16S

_rDNA,

which will resolve certain

ambiguities

revealed

_by

the

_{hybridisation}

approach.

ACKNOWLEDGEMENTS

This work was funded in _part

_by

_grants

from the Bureau des Ressources

Génétiques

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