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,Fusobacterium,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, Clostridium fallax,Clostridium bifermentans,Finegoldia magna, and Fusobacterium necrophorum.
Anaerobes contribute to form the largest and most diversified 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 intestinal bacteria belong to the phyla Firmicutes and Bacteroidetes (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, Clostridium, Finegoldia (formerly Peptostreptococcus), Fusobacterium, Prevotella, and Veillonella species (Berg, 1996).
The predominant anaerobes are represented by Bacteroides 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-forming, filamentous Gram-positive anaerobic rods that inhabit 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, microaerophilic Gram-positive rods, and the species Clostridium baratii, Clostridium bifermentans, and Clostridium perfringens 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-resident gut flora (Spigaglia et al., 2011). Thus, C. difficile, also for the recent emergence of new hypervirulent epidemic strains, is considered an increasingly alarming nosocomial enteric pathogen (Bartlett, 2010). Finegoldia, formerly known as Peptostreptococcus, is a genus consisting 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 immunosuppressed 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 belonging 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 & Murdoch, 2008).
Prevotella genus, constituted by nonmotile, Gram-negative anaerobic rods, includes 20 different species known to contribute in causing periodontitis, abscesses, bacteremia, 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. contribute 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 number 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 Bifidobacteria 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, Howard 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 polymicrobial 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 anaerobic strains belonging to the genera Bacteroides, Clostridium, 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
The anaerobic strains, here identified by the codes assigned when isolated from the explanted biliary stents, are the following: C. baratii strain CbaBs33, C. bifermentans strain CbiBs1, C. difficile strain CdiBs21, Clostridium fallax strain CfaBs3, C. perfringens strain CpeBs31, Finegoldia 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, Veillonella spp. strain VBs4.
All anaerobic strains, maintained on Brucella agar supplemented 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 prereduced 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 μL of the broth culture (adjusted to 0.5 McF) and 180 μL of fresh BHI supplemented with 1% glucose. As a control, a well with fresh BHI supplemented with 1% glucose without bacteria has been used. The plate was covered with a lid and incubated anaerobically for 48 h at 37 °C. Then, the content of each well was removed, and the wells were carefully washed three times with 200 μL of PBS. The plate was dried for 1 h at 60 °C and stained for 5 min with 150 μL of 2% Hucker's 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 μL 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 (Multiscan 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 adherence ability into the following categories: nonadherent (OD ≤ ODc), weakly adherent (ODc < OD ≤ 2×ODc), moderately adherent (2ODc < OD ≤ 4×ODc), and strongly adherent (4×ODc < OD) (Stepanovic et al., 2000).
To further investigate by field emission scanning electron 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 μL 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 (Veillonella spp. strain VBs4 + F. necrophorum strain FnBs4; B. fragilis strain BfBs12 + F. magna strain FmBs12; C. difficile strain CdiBs21 + F. magna strain FmBs21), the following protocol was applied: 200 μL 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 dehydrated 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 microscope (Sigma-Zeiss) at an accelerating voltage of 5 kV.
Confocal laser scanning microscopy (CLSM)
Biofilms grown on coverslips were fixed with 3.7% paraformaldehyde at room temperature for 30 min and stained with the LIVE/DEAD® BacLight™ Bacterial Viability Kit (Invitrogen, Molecular Probes®) by adding, in each well of a 24-well plate, 3 μL 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 bacteria with intact membranes fluoresce green, while dead bacteria with damaged membranes fluoresce red. The excitation/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 10× lens and the acquired images of the biofilms were at a resolution of 512 × 512 pixels.
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.
Table 1. Mean OD values of anaerobic strains measured by the quantitative biofilm production assay
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 experiments 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 biofilm 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 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 interaction between the F. magna EPS matrix and the C. difficile cell surface was detected with respect to that described earlier 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.
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 microorganisms 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 experiments 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, Fusobacterium, 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 quantitative biofilm production assay ranked the isolates (Table 1) as strongly adherent (77%), moderately adherent (15.4%) and weakly adherent (7.6%) strains.
The here-described ability to adhere and to grow as biofilm exhibited to a different extent by the investigated strains is in agreement with data previously reported by 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 susceptibility 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, suggesting 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 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 electron micrographs obtained by FESEM, we can distinguish between the two biofilm-forming bacterial species according 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. necrophorum 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 Fusobacterium, 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 Veillonella 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 lectins 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 microorganism’ in inter-species adherence and multi-species oral biofilm (Kaplan et al., 2009). On the other hand, Veillonella 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 synergistic 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 quantitative 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 antimicrobial drugs thus causing the failure of the targeted antibiotic 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. necrophorum.
Our intention is to continue in studying the mechanisms of mono- and mixed-biofilms formation of anaerobic bacteria to elucidate their scarcely investigated role in a number of acute or chronic severe infections in humans.
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 investigations, respectively. The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.