R E S EA RCH AR T I C L E

Biofilm-growing intestinal anaerobic bacteria

Gianfranco Donelli1, Claudia Vuotto1, Rita Cardines2 & Paola Mastrantonio2

1Microbial Biofilm Laboratory, IRCCS Fondazione Santa Lucia, Rome, Italy; and 2Department of Infectious, Parasitic and Immune-mediated

Diseases, Istituto Superiore di Sanita, Rome, Italy

Correspondence: Gianfranco Donelli,

Microbial Biofilm Laboratory (LABIM), IRCCS

Fondazione Santa Lucia, Via Ardeatina 306,

00179 Rome, Italy. Tel.: +39 06 51501305/

+39 06 516501307; fax: +39 06 51501306;

e-mails: g.donelli@hsantalucia.it or

gianfranco.donelli@gmail.com

Received 14 October 2011; revised 1 March

2012; accepted 12 March 2012.

Final version published online 23 April 2012.

DOI: 10.1111/j.1574-695X.2012.00962.x

Editor: Thomas Bjarnsholt

Keywords

biofilm; adherence; anaerobes; intestine.

Abstract

Sessile growth of anaerobic bacteria from the human intestinal tract has been

poorly investigated, so far. We recently reported data on the close association

existing between biliary stent clogging and polymicrobial biofilm development

in its lumen. By exploiting the explanted stents as a rich source of anaerobic

bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacteri-

um, Finegoldia, Prevotella, and Veillonella, the present study focused on their

ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-

species biofilms. Experiments on dual-species biofilm formation were planned

on the basis of the anaerobic strains isolated from each clogged biliary stent,

by selecting those in which a couple of anaerobic strains belonging to different

species contributed to the polymicrobial biofilm development. Then, strains

were investigated by field emission scanning electron microscopy and confocal

laser scanning microscopy to reveal if they are able to grow as mono- and/or

dual-species biofilms. As far as we know, this is the first report on the ability

to adhere and form mono/dual-species biofilms exhibited by strains belonging

to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clos-

tridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium

necrophorum.

Introduction

Anaerobes contribute to form the largest and most diver-

sified microbial community of the human body, that is,

that of the gastrointestinal tract, exceeding by 2–4 log

units the aerobic flora. In the last years, metagenomic

experiments have shown that the vast majority of intesti-

nal bacteria belong to the phyla Firmicutes and Bacteroi-

detes (Fakhry et al., 2009). As the small intestine is

concerned, bacterial concentration ranges from 105 to 109

bacteria per gram of intestinal content and anaerobes are

mainly represented by Bacteroides, Bifidobacterium, Clos-

tridium, Finegoldia (formerly Peptostreptococcus), Fusobac-

terium, Prevotella, and Veillonella species (Berg, 1996).

The predominant anaerobes are represented by Bactero-

ides species that are bile-resistant, non-spore-forming,

Gram-negative rods (Wexler, 2007). It has been reported

that Bacteroides fragilis exhibits a high tolerance to bile

salts as well as a great ability to utilize a broad spectrum

of polysaccharides and to vary surface antigens to evade

host immune responses; all these features presumably

explain its persistence in high numbers in the intestine

(Pumbwe et al., 2007). Bifidobacteria are non-spore-form-

ing, filamentous Gram-positive anaerobic rods that inha-

bit the gastrointestinal tract and the vaginal mucosa.

Recent studies have suggested that their interaction with

intestinal epithelial cells is effective on the integrity of

mucosa that is protected from inflammation presumably

by metabolites produced by these anaerobes (Amit-

Romach et al., 2010). Clostridia are spore-forming, micro-

aerophilic Gram-positive rods, and the species Clostridium

baratii, Clostridium bifermentans, and Clostridium perfrin-

gens are frequently isolated at the intestinal level while the

sporadically occurring microorganism Clostridium difficile

is known to overgrow in the intestine of patients treated

with broad-spectrum antibiotics (Gorbach, 1996). A

recent characterization of multidrug-resistant C. difficile

clinical isolates showed that antibiotic resistance provides

this pathogen with potential advantages over the co-resi-

dent gut flora (Spigaglia et al., 2011). Thus, C. difficile,

ª 2012 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 65 (2012) 318–325Published by Blackwell Publishing Ltd. All rights reserved

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also for the recent emergence of new hypervirulent epi-

demic strains, is considered an increasingly alarming

nosocomial enteric pathogen (Bartlett, 2010). Finegoldia,

formerly known as Peptostreptococcus, is a genus consist-

ing of Gram-positive anaerobic cocci, occurring in short

chains, in pairs or as single cells. Species belonging to the

genus Finegoldia, slow-growing commensals in the mouth

and in the intestinal tract, can cause septicemia and

abscesses in the brain, lungs, and liver of immunosup-

pressed patients (Brook, 2008). The genus Fusobacterium

includes 13 species of Gram-negative, strictly anaerobic,

non-spore forming, and spear-shaped bacilli. The most

frequent isolates in clinical specimens are Fusobacterium

necrophorum and Fusobacterium nucleatum, both belong-

ing to the normal flora of the oral cavity, intestinal tract,

and vagina. Particularly, F. necrophorum accounts for

25% of the isolates from liver abscesses (Huggan & Mur-

doch, 2008).

Prevotella genus, constituted by nonmotile, Gram-nega-

tive anaerobic rods, includes 20 different species known

to contribute in causing periodontitis, abscesses, bactere-

mia, wound, and urogenital tract infections (Alauzet

et al., 2010). Veillonella genus consists of small, strictly

anaerobic Gram-negative cocci lacking of flagella, spores,

and capsule. In the human intestine, Veillonella spp. con-

tribute to dehydroxylation of bile acids and have been

suggested as causative agents of opportunistic infections

(Verma et al., 2010).

The ability of the above-mentioned anaerobic species

to form biofilm and/or co-aggregate has been rarely

reported in the intestinal tract if compared with the num-

ber of studies on the development of anaerobes as multi-

species biofilms in the oral cavity (Kolenbrander, 2011;

Marsh et al., 2011) and in several chronic infections

including chronic wounds, cystic fibrosis, and otitis media

(James et al., 2008; Burmølle et al., 2010; Thornton et al.,

2011).

Sandra MacFarlane group reported (Ahmed et al.,

2007) on the intestinal occurrence of Bacteroides and Bifi-

dobacteria as microcolonies identified by fluorescence in

situ hybridization and on their distribution throughout

the mucus layer observed by confocal laser scanning

microscopy (CLSM). Then, we reported results on the

development of a multi-species biofilm in the lumen of

clogged biliary stents as consequence of the ascending

colonization from duodenum of aerobic and anaerobic

bacteria (Guaglianone et al., 2008). More recently, How-

ard Ceri group proposed a model for culturing mucosal

anaerobic bacteria recovered from colonic biopsies to

develop multi-species biofilms (Sproule-Willoughby et al.,

2010). The identification by conventional and molecular

techniques of both culturable and nonculturable sessile-

growing bacterial and fungal species in the biliary sludge

has revealed the occurrence of anaerobes in the 57% of

the examined biliary stents and has confirmed the poly-

microbial nature of the biofilm developing in their lumen

(Guaglianone et al., 2010).

Using the clogged biliary stent as a model of multi-

species biofilm development and exploiting the large

number of explanted stents as a generous source of anaer-

obic strains belonging to the genera Bacteroides, Clostrid-

ium, Fusobacterium, Finegoldia, Prevotella, and Veillonella,

the present study investigated on their ability to adhere,

to grow in sessile mode, and to form in vitro mono- or

dual-species biofilms.

Materials and methods

Bacterial strains

The anaerobic strains, here identified by the codes

assigned when isolated from the explanted biliary stents,

are the following: C. baratii strain CbaBs33, C. bifermen-

tans strain CbiBs1, C. difficile strain CdiBs21, Clostridium

fallax strain CfaBs3, C. perfringens strain CpeBs31, Fine-

goldia magna strain FmBs21, F. magna strain FmBs12,

B. fragilis strain BfBs12, Bacteroides oralis strain BoBs32,

F. necrophorum strain FnBs4, Parabacteroides distasonis

strain PdBs7, Prevotella intermedia strain PiBs18, Veillo-

nella spp. strain VBs4.

Culture conditions

All anaerobic strains, maintained on Brucella agar supple-

mented with vitamin K (0.5 mg L�1), haemin (5 mg L�1),

and 5% defibrinated sheep red blood cells, were routinely

cultured in brain heart infusion broth (BHI) containing

the above-mentioned supplements. All bacterial cultures

were anaerobically grown at 37 °C in an anaerobic

chamber.

Quantitative biofilm production assay

Bacterial strains were grown anaerobically at 37 °C in pre-

reduced BHI broth for a time ranging from 24 to 72 h,

depending on the strain. Each well of a 96-well flat-

bottomed plastic tissue culture plate (three wells for each

strain) was filled with 20 lL of the broth culture (adjusted

to 0.5 McF) and 180 lL of fresh BHI supplemented with

1% glucose. As a control, a well with fresh BHI supple-

mented with 1% glucose without bacteria has been used.

The plate was covered with a lid and incubated anaerobi-

cally for 48 h at 37 °C. Then, the content of each well was

removed, and the wells were carefully washed three times

with 200 lL of PBS. The plate was dried for 1 h at

60 °C and stained for 5 min with 150 lL of 2% Hucker’s

FEMS Immunol Med Microbiol 65 (2012) 318–325 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Biofilm-growing intestinal anaerobic bacteria 319

crystal violet. Excess stain was rinsed off by rinsing the

plate under tap water, and the plate was dried for 10 min

at 60 °C. Each assay was performed in triplicate and

repeated three times. The dye bound to the adherent cells

was solubilized with 150 lL of 33% (v/v) glacial acetic

acid per well. The optical density (OD) of each well was

measured at 570 nm using a microplate photometer (Mul-

tiscan FC; Thermo Scientific). The cut-off OD (ODc) is

defined as three standard deviations above the mean OD

of the negative control. According to the defined ODc,

all the strains were classified on the basis of their adher-

ence ability into the following categories: nonadherent

(OD � ODc), weakly adherent (ODc < OD � 29ODc),

moderately adherent (2ODc < OD � 49ODc), and

strongly adherent (49ODc < OD) (Stepanovic et al., 2000).

To further investigate by field emission scanning elec-

tron microscopy (FESEM) and CLSM the ability of single

bacterial strains to form biofilm, each well of a 24-well

plastic tissue culture plate, with a 13-mm diameter glass

coverslip placed on the bottom, was filled with 200 lL of

a broth culture (adjusted to 0.5 McF) of each strain and

1.8 mL of prereduced BHI broth supplemented with 1%

glucose and incubated for 48 h at 37 °C.As the ability to grow in a mixed biofilm of the couples

of anaerobic strains isolated from biliary stents (Veillonel-

la spp. strain VBs4 + F. necrophorum strain FnBs4; B. fra-

gilis strain BfBs12 + F. magna strain FmBs12; C. difficile

strain CdiBs21 + F. magna strain FmBs21), the following

protocol was applied: 200 lL from each broth culture of

the two strains, at the same OD (adjusted to 0.5 McF),

were mixed in a well with 1.6 mL of prereduced BHI

broth supplemented with 1% glucose and incubated

anaerobically for 48 h at 37 °C. After incubation, the

content of each well was removed and the wells were

washed carefully three times with PBS.

Field emission scanning electron microscopy

(FESEM)

Bacterial biofilms, obtained as described earlier, were fixed

with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH

7.4) at room temperature for 30 min, postfixed with 1%

OsO4 in 0.1 M phosphate buffer for 20 min and dehy-

drated through graded ethanol (30°, 50°, 70°, 85°, 95°,100°). After critical point drying in hexamethyldisilazane

and gold coating by sputtering, biofilm samples were

examined by a field emission scanning electron micro-

scope (Sigma-Zeiss) at an accelerating voltage of 5 kV.

Confocal laser scanning microscopy (CLSM)

Biofilms grown on coverslips were fixed with 3.7% para-

formaldehyde at room temperature for 30 min and

stained with the LIVE/DEAD® BacLightTM Bacterial Via-

bility Kit (Invitrogen, Molecular Probes®) by adding, in

each well of a 24-well plate, 3 lL of the dye mixture in

1 mL of distilled water for 15 min at room temperature

in the dark. The stain was aspirated, and the coverslips

was gently washed twice with distillate water. This kit

employs two nucleic acid stains differing in their ability to

penetrate healthy bacterial cells: green-fluorescent SYTO®

9 stain and red-fluorescent propidium iodide stain. When

used alone, SYTO® 9 stain labels both live and dead

bacteria. In contrast, propidium iodide penetrates only

bacteria with damaged membranes, reducing SYTO® 9

fluorescence when both dyes are present. Thus, live bacte-

ria with intact membranes fluoresce green, while dead

bacteria with damaged membranes fluoresce red. The exci-

tation/emission maxima for these dyes are about 480/

500 nm for the SYTO® 9 and 490/635 nm for propidium

iodide. Fluorescence from stained biofilms was viewed

using a CLSM (Nikon C1si), the mounted specimens were

observed using a 109 lens and the acquired images of the

biofilms were at a resolution of 512 9 512 pixels.

Results

Anaerobic strains were investigated for their ability to

adhere in vitro, and the relative results are shown in

Table 1 and Fig. 1. Among the Gram-negative anaerobic

strains tested for their quantitative biofilm production,

those belonging to the species B. fragilis, F. necrophorum,

P. intermedia, and Veillonella spp. were strongly adherent;

B. oralis was moderately adherent; and P. distasonis was

weakly adherent. As the Gram-positive anaerobic strains

are concerned, those belonging to the species C. baratii,

C. fallax, C. perfringens, C. bifermentans, and F. magna

were strongly adherent and only the C. difficile strain was

moderately adherent.

Particularly, the ability of different species to coexist in

a unique microbial community or to develop as a dual-

species biofilm was investigated by focusing our experi-

ments on the couples of anaerobic strains belonging to

different species occurring within the same biliary stent.

Then, the skill of these strains to grow together and/or

to co-aggregate in a mutualistic mode was explored by

FESEM (Figs 2–4).As the F. necrophorum strain FnBs4 + Veillonella spp.

strain VBs4-mixed biofilm is concerned, the scanning

electron microscopy analysis reveals (Fig. 2c) a thick bio-

film characterized, after 48 h of incubation, by an EPS

matrix denser than that observed in the respective mono-

species biofilms (Fig. 2a and b).

Scanning electron micrographs of the mono-species

biofilms of F. magna strain FmBs12 (Fig. 3a) and

B. fragilis strain BfBs12 (Fig. 3b) revealed, after 48-h

ª 2012 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 65 (2012) 318–325Published by Blackwell Publishing Ltd. All rights reserved

320 G. Donelli et al.

incubation, a rich EPS matrix in the former, while in the

latter, bacteria grow in sessile mode and appear tightly

aggregated but not immersed in a dense matrix. In the

biofilm formed by both strains, B. fragilis strain BfBs12

seems to grow immersed in the matrix produced by the

F. magna strain FmBs12.

A similar condition was observed when we tested

F. magna strain FmBs21 + C. difficile strain CdiBs21. In

fact, as evidenced in the insert of Fig. 4c, a closer interac-

tion between the F. magna EPS matrix and the C. difficile

cell surface was detected with respect to that described ear-

lier between F. magna FmBs12 and B. fragilis strain BfBs12.

This co-aggregation phenomenon between F. magna

strain FmBs21 and C. difficile strain CdiBs21 was also

documented by CLSM, the images (Fig. 5) showing that

the two strains cooperate with each other to form a

homogeneous biofilm (Fig. 5c) while they show a dense

and thick appearance (Fig. 5a) or a thin and sparse one

(Fig. 5b) when grown as mono-species biofilms.

Discussion

The molecular bases of microbial interactions and the

biofilm development have been largely investigated in a

number of species inhabiting the oral cavity, including

microaerophilic, tolerant, and strictly anaerobic bacteria.

Also microbial co-aggregation was firstly reported to

occur among different oral bacteria, in which a highly

specific phenomenon of recognition and adhesion, that is

currently believed to facilitate the integration of microor-

ganisms into biofilm-growing polymicrobial communities,

has been described (Gibbons & Nygaard, 1970). In recent

years, mutualistic multi-species biofilms built in vitro

by oral strains belonging to the species Aggregatibacter

actinomycetemcomitans, F. nucleatum, and Veillonella spp.

were also described (Periasamy & Kolenbrander, 2009).

On the other hand, heating and sugar-addition experi-

ments revealed that interactions between couples of oral

and intestinal strains – B. adolescentis and F. nucleatum;

Actinomyces naeslundii and C. perfringens; F. nucleatum

and Lactobacillus paracasei – and within intestinal strains

are mediated by lectin–carbohydrate interactions (Ledder

et al., 2008).

Taking into consideration this background, our results

allowed to reveal for different intestinal anaerobic isolates,

belonging to the genera Bacteroides, Clostridium, Fusobac-

terium, Finegoldia, Prevotella, and Veillonella, the ability

to adhere on abiotic surfaces, to develop as mono-species

biofilms, and to interact with each other giving rise to

dual-species biofilms.

As the adhesion properties are concerned, the quanti-

tative biofilm production assay ranked the isolates

(Table 1) as strongly adherent (77%), moderately adher-

ent (15.4%) and weakly adherent (7.6%) strains.

The here-described ability to adhere and to grow as bio-

film exhibited to a different extent by the investigated

strains is in agreement with data previously reported by

MEAN ODs0 0.5 1 1.5 2 2.5 3 3.5 4 4.5

CbaBs33

CbiBs1

CdiBs21

CfaBs3

CpeBs31

FmBs12

FmBs21

BfBs12

BoBs32

FnBs4

PdBs7

PiBs18

VBs4

Fig. 1. Mean ODs of the stained bacterial biofilm obtained from the

analyzed anaerobic strains.

Table 1. Mean OD values of anaerobic strains measured by the quantitative biofilm production assay

FEMS Immunol Med Microbiol 65 (2012) 318–325 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Biofilm-growing intestinal anaerobic bacteria 321

others on the species B. fragilis, C. perfringens, F. magna,

P. bivia, and Veillonella spp. In fact, B. fragilis was reported

to contain putative luxR orthologues, which could respond

to exogenous homoserine lactones and modulate biofilm

formation, bmeB efflux pump expression, and susceptibil-

ity to antibiotics (Pumbwe et al., 2008). Further, Varga

et al. (2008) firstly described that a type IV pilus (TFP)-

related gliding motility is necessary for an optimal biofilm

formation together with the functional CcpA protein in

C. perfringens. On the contrary, there is no evidence so far

on F. magna ability to form biofilm even if the presence

on their surface of hair-like projections able to mediate

interactions between neighboring cells has been recently

reported. In fact, after their removal following treatment

with proteases, bacteria no longer form aggregates, suggest-

ing a significant role of these surface proteins in bacterial

clumping (Frick et al., 2008).

Thus, as far as we know, our data are currently the

only available on the adhesiveness and/or biofilm-forming

ability of anaerobic strains belonging to the species

B. oralis, C. difficile, C. baratii, C. fallax, C. bifermentans,

F. magna, F. necrophorum.

In light of the current knowledge on the mutualistic

co-aggregation phenomena and on the basis of the in vivo

finding of each couple of anaerobic strains tested in vitro

—F. necrophorum strain FnBs4 + Veillonella spp. strain

(a)

(b)

(c)

Fig. 2. FESEM micrographs of mono-species biofilms of Fusobacterium

necrophorum strain FnBs4 (a) and Veillonella spp. strain VBs4 (b) and

of a biofilm formed by both strains (c), obtained at an accelerating

voltage of 5 kV (50009 magnification).

(a)

(b)

(c)

Fig. 3. FESEM micrographs of mono-species biofilms of Finegoldia

magna strain FmBs12 (a) and Bacteroides fragilis strain BfBs12 (b) and

of a biofilm formed by both strains (c), obtained at an accelerating

voltage of 5 kV (50009 magnification).

ª 2012 Federation of European Microbiological Societies FEMS Immunol Med Microbiol 65 (2012) 318–325Published by Blackwell Publishing Ltd. All rights reserved

322 G. Donelli et al.

VBs4, B. fragilis strain BfBs12 + F. magna strain FmBs12,

C. difficile strain CdiBs21 + F. magna strain FmBs21 – we

can assume a synergistic interaction of the involved

species in forming biofilm.

In fact, through the analysis of the high-resolution elec-

tron micrographs obtained by FESEM, we can distinguish

between the two biofilm-forming bacterial species accord-

(a)

(b)

(c)

Fig. 4. FESEM micrographs of mono-species biofilms of Finegoldia

magna strain FmBs21 (a) and Clostridium difficile strain CdiBs21 (b)

and of a biofilm formed by both strains (c), obtained at an

accelerating voltage of 5 kV (50009 magnification). In the insert,

the matrix-mediated close interaction between the two strains

growing in the biofilm is shown at higher magnification (10 0009

magnification).

(a)

(b)

(c)

Fig. 5. CLSM micrographs of mono-species biofilms of Finegoldia

magna strain FmBs21 (a) and Clostridium difficile strain CdiBs21 (b)

and of a biofilm formed by both strains (c), observed using a 109

lens and acquired at a resolution of 512 9 512 pixels.

FEMS Immunol Med Microbiol 65 (2012) 318–325 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Biofilm-growing intestinal anaerobic bacteria 323

ing to their highly different features (rod- or spear-shaped

bacilli vs. cocci). This morphological approach allows

evaluating the more or less balanced presence of each

species in the mixed biofilm and their skill to interact

with each other.

Particularly, a mature biofilm, exhibiting a ‘common’

EPS matrix denser than that produced by Veillonella spp

strain VBs4 alone, was developed within 48 h by F. necro-

phorum strain FnBs4 + Veillonella spp. strain VBs4.

On the other hand, our data on F. necrophorum strain

FnBs4 biofilm seem to confirm that the production of an

extra-cellular polysaccharide matrix is not an intrinsic

feature of the species belonging to the genus Fusobacteri-

um, as already known for F. nucleatum, nevertheless

reported to be able to co-adhere and form biofilm (Zilm

& Rogers, 2007).

Anyway, a synergy between Fusobacterium and Veillo-

nella in developing a dual-species biofilm is presumably

present. In fact, as it is already known, Fusobacterium has

strong adhesive properties because of the presence of lec-

tins that mediate not only the adhesion to epithelia but

also the co-agglutination with other bacteria (Roberts,

2000) by playing its pivotal role of ‘bridging microorgan-

ism’ in inter-species adherence and multi-species oral

biofilm (Kaplan et al., 2009). On the other hand, Veillo-

nella is not able to catabolize sugars, so its growth

depends on acetic, propionic, butyric, and lactic acids

provided by Fusobacterium.

As the interaction between the FESEM investigated

strains of B. fragilis and F. magna is concerned, a syner-

gistic co-aggregation can be hypothesized on the basis of

the largely increased production of EPS matrix in the

mixed biofilm.

On the basis of data obtained by FESEM and CLSM

investigations, the sticky biofilm constituted by C. difficile

strain CdiBs21 + F. magna strain FmBs12 appears quite

different from the thin and sparse biofilm developed by

C. difficile alone or the dense and thick one exhibited by

F. magna. Thus, according to our data, two different

hypotheses can be taken into consideration: (1) C. difficile

strain CdiBs21 coexists in biofilm growing together with

the strong biofilm-producer F. magna strain FmBs12; or

(2) C. difficile strain CdiBs21, classified by the quantita-

tive biofilm production assay as a weakly adherent strain,

became able to grow as biofilm as consequence of a close

interaction with the F. magna, direct or mediated by

a biofilm-promoting substance released by the latter. It

should be considered that both the coexistence and the

possibly induced growth as biofilm of C. difficile could

protect the microorganism from the action of antimicro-

bial drugs thus causing the failure of the targeted anti-

biotic therapies. In fact, our findings could well explain

why C. difficile relapses occur in 15–20% of CDI patients.

As a whole, our data suggest the possibility that non- or

weak biofilm-producing bacteria could benefit from living

in biofilms developed by other strong biofilm-forming

species.

According to our knowledge, this is the first report on

adherence and/or biofilm formation displayed by strains

belonging to the anaerobic species B. oralis, C. difficile,

C. baratii, C. fallax, C. bifermentans, F. magna, F. necro-

phorum.

Our intention is to continue in studying the mecha-

nisms of mono- and mixed-biofilms formation of anaero-

bic bacteria to elucidate their scarcely investigated role in

a number of acute or chronic severe infections in

humans.

Acknowledgements

The authors are indebted to Dr Fabrizio Barbanti and

Dr Emilio Guaglianone for their continuous advice and

skilled assistance in performing experiments in anaerobic

chamber and in scanning electron microscopy investiga-

tions, respectively. The authors report no conflicts of

interest. The authors alone are responsible for the content

and writing of the paper.

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