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Food-borne Enterococci Integrate Into Oral Biofilm: An In Vivo Study Ali Al-Ahmad, Dr. rer. nat.,* Julia Maier, cand. Dr. med. dent.,* Marie Follo, Dr. rer. nat.,† ¨ ller, Technician* Annette Wittmer, Prof. Dr. med.,‡ Elmar Hellwig, Prof. Dr. med. Bettina Spitzmu ¨ bner, Prof. Dr. med.,§ and Daniel Jonas, Prof. Dr. med.,k Dent,* Johannes Hu Abstract Introduction: Enterococci, particulary Enterococcus faecalis, are still a primary concern in endodontic infections. To date, enterococci have been considered to be only transiently present in the oral cavity. The aim of this study was to examine whether different enterococci from food are able to reside in oral biofilm. Methods: Six healthy volunteers wore dental splints loaded with enamel slabs. After 3 days, the volunteers consumed cheese containing enterococci. The fate of the enterococci was analyzed by culture technique and 16S rRNA gene sequencing. All isolates were characterized genotypically by macrorestriction analysis (SmaI) and pulsed-field gel electrophoresis. E. faecalis was also analyzed by using fluorescent in situ hybridization (FISH). Results: E. faecalis, E. faecium, E. avium, and E. durans were detected in the initial biofilm after 2 hours, as well as in the 5-day-old oral biofilm. E. faecalis, E. faecium, and E. avium isolated from the initial biofilm and from the 5-day-old biofilm, as well as those isolated from cheese, showed genetic homogeneity. E. faecium and E. avium had integrated into a pre-existing 3-day-old biofilm. No genetic similarity between E. durans strains isolated from cheese and those from the initial and 5-day-old oral biofilm was detected. E. faecalis was also detected in the oral biofilm by using FISH. Conclusions: Food-borne enterococci, particularly E. faecalis, might not only be transient but could also survive in the oral biofilm and become a source for endodontic infections. Moreover, genotypic analysis is required to study the source of oral enterococci. (J Endod 2010;36:1812–1819)
Key Words Duplex FISH, endodontic infection, enterococci, Enterococcus faecalis, food, oral biofilm
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nterococci are ubiquitous and can be isolated from a wide range of habitats, such as water, plants, fermented food, and the gastrointestinal tract of humans and animals (1). Members of these bacteria are associated with periradicular lesions in root-filled teeth in which therapy has failed (2–7). In several studies summarized by Schirrmeister et al (6) and Siqueira and Roˆc¸as (7), Enterococcus faecalis was revealed to be the predominant infectious agent associated with secondary endodontic infections. To date, enterococci, especially E. faecalis, are not considered to be part of the normal oral microbiota (8, 9). Even in the saliva of patients with endodontic therapy, E. faecalis has rarely been isolated (6, 10). The origin of enterococci in treated root canals is therefore still unclear (11, 12). Razavi et al (12) detected enterococci in rinse samples of volunteers 100 minutes after consumption of this fermented food. The authors suggested that fermented food could be an origin for endodontic infections with enterococci. However, the authors did not investigate the possible incorporation of enterococci into oral biofilms. Because the association of enterococci with subgingival periodontitis has been previously reported (13, 14), the question arises as to whether enterococci are able to integrate into supragingival oral biofilm and constitute a persistent source for endodontic infections. In a first step to answer this question, we recently demonstrated that endodontic and salivary isolates of E. faecalis were able to integrate into a biofilm cultivated from human salivary bacteria in a biofilm reactor in vitro (15). These results led to the hypothesis that E. faecalis coaggregates with other oral bacteria in vivo as a member of the normal supragingival plaque. However, biofilm formation in the oral cavity cannot be simulated in vitro because of the complicated composition of oral microorganisms and of the salivary pellicle that has formed on enamel (16, 17). Fluorescence in situ hybridization (FISH) was shown previously to be adequate to detect E. faecalis in clinical samples (18). The combination of FISH and confocal laser scanning microscopy (CLSM) has already been used to analyze initial and mature oral biofilms without the destruction of their natural structures (16, 19). To determine the origin of oral bacteria, genotypic analysis and comparison to strains derived from other environments such as fermented food are necessary. For similar purposes, macrorestriction analysis and pulsed-field electrophoresis (PFGE) were shown to be useful for enterococci (20, 21). The capability of E. faecalis to incorporate in supragingival plaque has not been investigated in situ until now, even though this oral site is the origin of diseases such as caries and periodontitis and could also be the origin of endodontic infections. Thus, the purpose of the present in vivo study was to analyze the origin of different enterococci in
From the *Department of Operative Dentistry and Periodontology, †Department of Hematology and Oncology, Core Facility, and ‡Institute for Medical Microbiology and Hygiene, Albert-Ludwigs-University, Freiburg; and §Division of Infectious Diseases and kDepartment of Environmental Health Sciences, University Hospital Freiburg, Freiburg, Germany. Address requests for reprints to Dr Ali Al-Ahmad, Department of Operative Dentistry and Periodontology, University School and Dental Hospital, Hugstetter Straße 55, D-79106 Freiburg, Germany. E-mail address:
[email protected]. 0099-2399/$ - see front matter Copyright ª 2010 American Association of Endodontists. doi:10.1016/j.joen.2010.08.011
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Basic Research—Biology the oral cavity of 6 subjects after they consumed cheese containing enterococci. Saliva, initial adhesion, and the oral biofilm were analyzed by culture technique. In addition, E. faecalis was analyzed by using FISH and CLSM. All enterococcal isolates from the different subjects were genetically compared with the cheese isolates by using macrorestriction analysis in combination with PFGE.
Material and Methods Study Design: Subjects and Specimens Six healthy subjects with an age range of 25–26 years participated in the study. The volunteers were not enrolled into the study if any of the following criteria were present: severe systemic diseases, pregnancy or lactation, use of antibiotics within the past 30 days, positive for enterococci isolation from saliva or plaque probes of the subjects before the start of the study, or participation in another clinical study during the course of the previous 3 months. All subjects were instructed not to consume fermented food that could contain enterococci such as sausages and cheeses. Dental oral examinations were carried out by an experienced dentist. No signs of gingivitis or caries were found. Informed written consent was given by the subjects before participation in the study, and the study design was reviewed and approved by the Ethics Committee of the University of Freiburg (Proposal 180/09), Germany. Specimens were prepared as described in an earlier study in detail (22). In brief, cylindrical enamel slabs with a surface area of 19.63 mm2 were prepared from the labial surfaces of bovine incisors from 2-year-old cattle. All cattle were tested to be negative for bovine spongiform encephalopathy (BSE). The enamel surfaces were polished by grinding with abrasive paper (400–4000 grit). The surface of the enamel specimen was monitored by using impinging light microscopy (Leica Wild M3Z, Wetzlar, Germany). Disinfection of the bovine enamel slabs (BES) was conducted by using ultrasonication with NaOCl for 3 minutes, followed by 3-minute treatment in ethanol. The BES were then washed with distilled water and stored for 24 hours in distilled water before use. The subjects wore dental splints loaded with 8 BES in an approximal position as shown in Fig. 1. During meals and the daily oral hygiene procedure, the splint was removed and stored in 0.9% sodium chloride solution. After 3 days, the absence of enterococci in the formed oral biofilm on 4 BES and in saliva was confirmed by culture technique and by FISH. Four new BES were added to the splint, and the remaining 4 BES were left in place until the end of the cultivation period of 8 days. At this point the volunteers consumed 10 g of the French cheese Brie de Meaux, which was previously characterized concerning its enterococcal content. Table 1 shows the number of colony-forming units (CFUs) of the different enterococcal species isolated from this cheese. After 120 minutes the initial adhesion on 4 BES and the saliva from each subject were microbiologically tested for the presence of enterococci
Figure 1. Dental cast of upper jaw exhibiting the acrylic appliance with BES. Exposed surfaces are fixed toward the tooth enamel in the approximal positions. (This figure is available in color online at www.aae.org/joe/.)
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by using the culture technique as well as FISH. Four new BES were added, and the subjects again consumed 10 g of the same batch of cheese. Five days later the formed biofilm was analyzed by culture technique and FISH-CLSM. Enterococci that were isolated from cheese as well as from saliva, the initial biofilm, new 5-day-old oral biofilm, and 8-day-old biofilm that had already existed before cheese consumption were all genetically analyzed by using macrorestriction analysis and PFGE.
Isolation and Identification of Enterococci One gram of cheese was placed in 10 mL of sodium citrate buffer (2%, w:w) and homogenized for 1 minute by using an Ultraturax homogenizer (Janke & Kunkel KG, Staufen, Germany) and mixed vigorously for 30 seconds (Vortex genie 2; Scientific Industries, Inc, Bohemia, NY). Subsequently, different dilutions (10–1–10–5) were prepared in the same buffer solution. Bile esculin agar plates (selective for enterococci) were used to plate 100 mL of each dilution. The agar plates were incubated at 37 C for 3 days under aerobic conditions and 5% CO2. The plating was conducted in triplicate, and CFUs were counted by using the Gel Doc EQ Universal Hood (Bio-Rad Life Science Group, Hercules, CA). To determine the level of enterococci present in the oral biofilm, BES overgrown with biofilm were washed with 0.9% sodium chloride. The dentin side and the margins were brushed off with sterile small sponge pieces (Voco GmbH, Cuxhaven, Germany), with sterile tweezers to remove microorganisms not adherent to the enamel side. The enamel slabs overgrown with biofilm were then placed into sterile tubes with 1 mL 0.9% sodium chloride, vortexed, and treated for 30 seconds in an ultrasonic bath on ice. A dilution series was prepared in 0.9% sodium chloride. To determine the number of enterococci present in saliva, undiluted 1 mL unstimulated saliva and dilution series (10–1–10–3) were prepared. The plating, incubation, and counting of CFU were carried out as described above. Enterococcal isolates were Gram-stained, and bacterial cell morphologies were determined by using light microscopy (Axioscope; Zeiss, Jena, Germany; 1000 magnification). A number of biochemical tests were used to differentiate the enterococci, including fermentation of arabinose (ARA), sorbit (SOR), methyl-a-d-glucopyranoside (MDG), and growth on hydrogen sulfide, indole, motility (SIM) agar. In addition, the commercially available API 20 Strep (Bio Merieux, Marcy-1’Etoile, France) was used to confirm species identity. All enterococcal isolates were also identified by 16S rRNA gene sequencing to confirm or revise the biochemical identification. DNA was extracted by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. 16S rRNA gene amplification was performed in a total volume of 50 mL, containing 5 mL 10 polymerase chain reaction (PCR) buffer (Qiagen), MgCl2 (2.5 mmol/L), 200 mmol/L of each deoxyribonucleoside triphosphate (dNTP), 2 U Taq polymerase (Qiagen), and 300 nmol/L of reverse and forward primer. For amplification of the 16S rRNA gene, a set of primers (forward primer TP16U1: 5’-AGAGTTTGATC[C/A]TGGCTCAG-3’ and reverse primer RT16U6: 5’-ATTGTAGCACGTGTGT[A/C]GCCC-3’) were used as published previously (6). The 1018 base pair–long PCR products were extracted and purified by using the GFX PCR DNA and gel band purification kit (Amersham Biosciences Europe GmbH, Freiburg, Germany). Purified PCR products were sequenced by using the BigDye terminator kit v1.1 cycle sequencing kit (Applied Biosystem, Darmstadt, Germany) and the ABI 310 Genetic Analyzer (GMI, Inc, Ramsey, MN). The sequencing reaction was performed with TP16U1 as the sequencing primer. Sequences were analyzed by using the BLAST program from NCBI (http://www.ncbi.nih.gov/BLAST). Sequence comparison to GenBank data entries served as a confirmation or revision of the detected and identified bacterial strains. Food-borne Enterococci Integrate Into Oral Biofilm
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E. faecalis E. faecium E. avium E. durans
Cheese (CFU/g)
Saliva (CFU/mL)
Initial adhesion (CFU/cm2)
New 5-day-old biofilm* (CFU/cm2)
Previously grown biofilm† (CFU/cm2)
4.2 108 1 106 1 105 1 105
1.6–4.8 105 (5) 1 104 (2) nd 1 103 (4)
4.7 103 to 3.2 104 (6) 5 103 to 1.4 104 (5) 4.7 103 (1) 5 103 (1)
4.7 103 to 4.2 104 (5) 5 103 to 1 104 (2) 5 103 (1) 5 103 (2)
5 103 to 3.3 104 (5) 5 103 (1) 5 103 (1) 4.7 103 (1)
Values in parentheses represent the number of subjects from whom the strain was found in the particular samples. *New biofilm represents enterococci integrated in a new biofilm formed in situ 5 days after cheese was consumed. † Previously grown represents enterococci integrated in a biofilm that was previously formed for 3 days before cheese consumption, and that was allowed to mature for additional 5 days after cheese consumption.
Duplex FISH FISH was conducted according to Amann (23), with modifications as described previously (15). In brief, biofilms grown on enamel samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (1.7 mmol/L KH2PO4, 5 mmol/L Na2HPO4 with 0.15 mol/L sodium chloride, pH 7.2) for 12 hours at 4 C. After fixation all specimens were washed with PBS and fixed again in an ethanol containing solution (50% in PBS, v/v) for 12 hours. The probes were washed twice with PBS and then incubated in a solution containing 7 mg of lysozyme per mL of 0.1 mol/L Tris-HCl-5 mmol/L ethylenediaminetetraacetic acid (EDTA), pH 7.2, for 10 minutes at 37 C to permeabilize cells within the plaque biofilm. At this point the biofilms were dehydrated through a series of ethanol washes containing 50%, 80%,
and 100% ethanol for 3 minutes each. The specimens were then incubated with the oligonucleotide probes at a concentration of 50 ng each per 20 mL of hybridization buffer (0.9 mol/L NaCl, 20 mmol/L Tris-HCl [pH 7.2], 25% formamide [v/v], and 0.01% sodium dodecylsulfate [w/ v]). Hybridization was conducted in 96-well plates (Greiner bio-one, Frickenhausen, Germany) at 46 C for 2 hours. After probe hybridization, specimens were incubated for 15 minutes at 48 C in wash buffer containing 20 mmol/L Tris-HCl (pH 7.5), 5 mmol/L EDTA, 159 mmol/L NaCl, and 0.01% sodium dodecylsulfate (w/vol). All high-power liquid chromatography purified oligonucleotide probes used in this study were synthesized commercially and labelled at their 5’ ends with different fluorochromes (Thermo Electron GmbH, Ulm, Germany). The oligonucleotide probe Efs 129
Figure 2. CLSM images from different sections of in situ oral biofilm. Red spots shown by arrows represent E. faecalis.
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Figure 3. PFGE patterns and dendrogram of E. faecalis strains isolated from cheese and oral cavities of the different subjects. Lower parentheses show the strains that were isolated from volunteers and genetically identical to the cheese strain. Upper 2 parentheses show genetically identical strains that are isolated from different volunteers. R above the PFGE indicates the reference strain S. aureus NCTC 8325. Scale at top of dendrogram depicts percentage of similarity.
(5’-CCCTCTGATGGGTAGGTT-3’) described by Behr et al (24) was 5’labelled with Cy3 and used to visualise E. faecalis as well as with the gene probe EUB 338 (5’-GCTGCCTCCGTAGGAGT-3’) described by Amann et al (25), which was 5’-modified with fluorescein and used to visualize the entire bacterial population within the biofilm specimen. The specificity of the E. faecalis probe was confirmed by using different microorganisms. These included several species that are taxonomically close to E. faecalis such as Streptococcus spp. (16).
CLSM and Image Analysis The biofilms were analyzed in a chambered coverglass (m Slide 8 well; ibidi GmbH, Munich, Germany) by CLSM (Leica TCS SP2 AOBS, Mannheim, Germany) by using a 63 water immersion objective (HCX PL APO/bd.BL 63.0x1.2W; Leica). Excitation of the FISH probes was carried out at 488 nm for fluorescein and at 546 nm for CY3. Fluorescence emission was measured at 493–538 nm for fluorescein and at 568–621 nm for CY3. To minimize spectral overlap between the probes, confocal scanning was carried out sequentially for each image. A total of 6 biofilm points, consisting of 3 points for each of the 2 BES, were analyzed for every experimental period. Image analysis was conducted as described previously (16). The biofilm was examined at several locations. Within each area the thickest point was measured by determination of the upper and lower boundaries of the biofilm. This procedure was repeated twice so that an average could be determined from the 3 measurements. Biofilms were scanned from these 3 starting points, generating sections of thickness of approximately 0.5 mm each JOE — Volume 36, Number 11, November 2010
at 2-mm intervals throughout the biofilm layers (to avoid overlaps). Standard images were made with a zoom setting of 1.7, corresponding to physical dimensions of 140 140 mm for each image. The area of each section was transformed into a digital image containing 1024 1024 pixels. The areas measured were from 3 separate and representative locations on the BES that were covered with biofilm.
DNA Fingerprinting To compare the food-borne enterococcal isolates with those from the subjects’ oral cavities, PFGE was performed as described by Matushek et al (26) and Klare et al (21), with the modifications as described below. All cultures were grown overnight in brain heart infusion broth (Merck, Darmstadt, Germany), and 1 mL of each overnight culture was transferred to a microcentrifuge tube and harvested by centrifugation (4000g, 5 minutes, Eppendorf model 5415C). The harvested cells were washed twice in 1 mL cold sterile TEN buffer (0.1 mol/L Tris Cl, 0.15 mol/L NaCl, 0.1 mol/L EDTA). The centrifuged and washed cells were resuspended in 140 mL cell suspension buffer (20 mmol/L NaCl, 10 mmol/L Tris Cl, [pH 7.2], 50 mmol/L EDTA) and warmed to 55 C. Twenty microliters of 25 mg/mL lysozyme solution (Sigma, St Louis, MO) and 140 mL 1.2% InCert-agarose (Lonza, Basel, Switzerland) solution made up in TE buffer and warmed to 55 C were added. This mixture was then pipetted into a plug mold (BioRad Laboratories) and allowed to solidify for 1 hour in the refrigerator. Each plug was placed into 1 mL of EC-lysis buffer (6 mmol/L Tris HCl [pH 7.4], 1 mol/L NaCl, 0.1 mol/L EDTA, 0.5% Brij 58, 0.2% Food-borne Enterococci Integrate Into Oral Biofilm
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Figure 4. PFGE patterns and dendrogram of E. faecium strains isolated from cheese and oral cavities of the different subjects. Parentheses show the strains isolated from volunteers and genetically identical to cheese strain.
deoxycholate, 0.5% Sarkosyl) to which lysozyme (Sigma) at 1.5 mg/mL was added. The plugs were incubated overnight at 37 C with gentle shaking (Thermomixer, Eppendorf, Germany). Plugs were then washed with 1 mL EC buffer each and transferred to 1 mL ESP buffer (10 mmol/L Tris HCl [pH 7.4], 1 mmol/L EDTA) to which proteinase K (Sigma) at a concentration of 100 mg/mL and 1% sodium dodecylsulfate were added. The overnight incubation was conducted again at 55 C with gentle shaking (Thermomixer). Plugs were washed with 14 mL TE buffer (10 mmol/L Tris Cl [pH 7.4], 0.1 mmol/L EDTA) twice for 60 minutes each and stored in TE until restriction digestion. For electrophoresis, the plug was cut into small slices and placed in a total volume of 100 mL enzyme mixture containing 98 mL restriction buffer and 2 mL 10 U/mL SmaI (New England BioLabs, Beverly, MA). After
incubation overnight at 25 C, PFGE of the trimmed plug slices was subsequently performed by using the CHEF-DR II apparatus (BioRad Laboratories) with the following ramped pulse times: 1–11 seconds for 13 hours and 11–30 seconds for 13 hours in 1% agarose gel (LE GP agarose; Biozym, Hess, Oldendorf, Germany) at 6 V/cm and 14 C. The running gel was prepared in 0.5% TBE buffer. DNA bands were visualized by ethidium bromide staining. All fingerprints were analyzed by use of the BioNumerics software (Applied Maths, SintMartens-Latem, Belgium), with the control strain Staphylococcus aureus NCTC 8325 as a molecular size standard for normalization. After assignation of bands, percentages of similarities between the fingerprints were calculated with 1% tolerance and 0.5% optimization by using the Dice coefficient. Cluster analysis was performed with the
Figure 5. PFGE patterns and dendrogram of E. avium strains isolated from cheese and oral cavities of the different subjects. Parentheses show the strains isolated from volunteers and genetically identical to the cheese strain.
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Figure 6. PFGE patterns and dendrogram of E. durans strains isolated from cheese and oral cavities of the different subjects. Parentheses show genetically identical strains that were isolated from different volunteers.
unweighted pair group method by using arithmetic averages (UPGMA) and presented as dendrograms. Banding patterns with up to 3 fragment differences were considered as possibly related.
Results Four different enterococcal species, E. faecalis, E. faecium, E. avium, and E. durans, could be isolated from cheese samples (Table 1). CFUs of E. faecalis were highest (4.2 108/g), followed by E. faecium (1 106/g) and E. avium and E. durans (1 105/ g). Two hours after consumption of 10 g of cheese, E. faecalis could be isolated from 5 subjects at the highest level at a range of 105 CFU/ mL from saliva, whereas E. faecium was found in the saliva of only 2 subjects at a level of 104 CFU/mL. E. durans was detected in the saliva of 4 subjects in amounts of 103 CFU/mL. E. avium was not detectable in the saliva of any volunteer 2 hours after cheese consumption. Considering the initial adhesion 2 hours after cheese consumption, E. faecalis was detected in all 6 volunteers, followed by E. faecium, which was found in the initial adhesion of 5 subjects. E. avium and E. durans were only detectable in the initial biofilm of 1 volunteer and at a lower amount of 103 CFU/cm2. E. faecalis (up to 104 CFU/cm2) could be isolated in 5 subjects from the secondary biofilm formed in vivo 5 days after the cheese was consumed. E. faecium and E. durans were detected in the secondary oral biofilm from only 2 volunteers at an amount of up to 104 and 103 CFU/cm2, respectively. E. avium was revealed in this second oral biofilm in only 1 subject. From the oral biofilm previously grown for 3 days and which was allowed to mature for additional 5 days after cheese consumption, E. faecalis was isolated from 5 subjects at up to 104 CFU/cm2, whereas all other enterococci were detected in the biofilm from only 1 volunteer each and at a lower amount of 103 CFU/cm2. Fig. 2 shows different images from the CLSM of a biofilm formed in vivo after cheese consumption. Red spots representing E. faecalis could be found in different sections of the biofilm, suggesting that this species had integrated into the oral biofilm. PFGE of SmaI restriction patterns with corresponding dendrograms of all enterococci strains isolated from cheese and from all subjects are shown in Figs. 3–6. Genotyping resulted in different clusters of the isolated strains for each species. E. faecalis strains isolated from saliva, initial biofilm, and from secondary biofilm formed after cheese consumption for 5 days could be identified as being similar to the cheese isolate (Fig. 3, cluster in parentheses). Some strains from the secondary biofilm as well as from previously grown (3 days before cheese consumption) biofilm and from saliva showed 100% homogeneity (Fig. 3, cluster in parentheses). Genetic JOE — Volume 36, Number 11, November 2010
similarity was revealed for E. faecium from the cheese strain as well as of strains from the initial biofilm, the 5-day-old secondary biofilm, and the 8-day-old oral biofilm that had already existed before cheese consumption took place (Fig. 4, parentheses). As shown in Fig. 5 (cluster in parentheses), E. avium and the cheese isolate of this species were genetically identical, whereas no genetic similarity could be shown between E. durans strains isolated from the initial biofilm and 5-day-old biofilm and cheese strain (Fig. 6). However, strains of E. durans isolated from different compartments of the oral cavity from different volunteers showed 100% genetic homogeneity (Fig. 6, parentheses).
Discussion Enteroccocci have only rarely been recovered from the healthy human oral cavity (3–5, 9, 12, 27), although members of these bacteria were frequently found to be associated with periradicular lesions in root-filled teeth in which therapy had failed (2–4, 6, 7, 28). However, E. faecalis was repeatedly shown to be the predominant species in root-filled teeth associated with apical periodontitis, as recently summarized by Schirrmeister et al (5, 6) and Siqueira and Roˆc¸as (7). E. faecalis was also detected with a high prevalence in primary endodontic infections (29). Antibiotic resistance in E. faecalis and E. faecium and a possible contribution to horizontal gene transfer as a result of this resistance underline the growing attention being paid to enterococci in the oral cavity (30, 31). Moreover, E. faecalis is one of the most discussed microorganisms in endodontic infections and has been shown to possess different putative virulence factors (27, 32), which were also found in endodontic strains (15). Because enterococci are still not considered as belonging to the normal oral microbiota, the question arose about the origin of these bacteria in infected root canals. In a recent review, Zehnder and Guggenheim (11) considered the appearance of enterococci and particularly E. faecalis in infected root canals as a mysterious phenomenon that could be due to the transient character of these bacteria in the oral cavity. This transient presence was suggested by Razavi et al (12), who showed that E. faecalis could be isolated from the oral cavity 100 minutes after consumption of enterococci-positive cheese harboring this species. On the basis of these findings, fermented food has been believed to be the source of enterococci in the oral cavity. However, the enterococcal content of oral dental biofilm after consumption of such food was not investigated by the authors. The presence of enterococci in these biofilms would indicate that enterococci not only can be transiently present in the oral cavity but might also survive in the dental biofilm, allowing them to serve as a source for endodontic infections.
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Basic Research—Biology A recently published study by our group showed that endodontic and salivary isolates of E. faecalis integrated into biofilm from human salivary bacteria cultivated in vitro and became subsequently resident in the biofilm cultivated in a biofilm reactor (15). However, as was suggested in this earlier study, the oral biofilm cannot be simulated in vitro because of the complex interactions of different parameters in the oral cavity, such as saliva and salivary pellicle, as well as the coaggregation processes among the different bacterial species involved in oral biofilm formation in vivo (16, 17). The present in vivo study has therefore focused on the hypothesis that enterococci from fermented food will not only be transiently present in the oral cavity but will also survive in the dental oral biofilm. The splint system used for the BES was shown to be useful for in vivo biofilm studies, because no disturbance of the oral biofilm can be caused by the tongue or the cheeks (16, 33). BES were used as a substratum for biofilm formation because the chemical properties of human and bovine enamel are quite similar (34). Furthermore, it is more feasible to harvest standardized samples of a homogenous quality and large surface area from bovine rather than from human teeth. To allow genotyping comparisons with the strains isolated from cheese, CFUs of enterococci were determined in each of the oral samples. In addition, the occurrence of enterococci within the oral biofilm was confirmed by a specific oligonucleotide probe for E. faecalis in a duplex FISH assay as described earlier (15). The combination of FISH with CLSM was also used by Zapata et al (35) to visualize infected dentin by E. faecalis in situ. The proof of bacterial growth within the biofilm structure enables the survival of bacteria in endodontic infections. This was reported even for species that are not frequently isolated from infected root canals, such as Bacillus subtilis (36). The fact that E. faecalis was shown to reside within different layers of the oral biofilm indicates the ability of enterococci not only to aggregate with different oral bacteria in the dental oral supragingival plaque but also to be part of the persistent biofilm in infected root canals, leading to failure of endodontic therapy. The SmaI macrorestriction followed by PFGE was shown to be an appropriate and discriminative method for the genotyping of enterococci (21). In regard of E. faecalis and E. faecium, good correlations between sequence typing and PFGE data have been reported (37–40). In the present study, the genotyping results revealed that E. faecalis, E. faecium, and E. avium originating from cheese are not only transiently present in the oral cavity as reported by other authors (11, 12), but they can also be resident in the oral biofilm, serving as a possible source for root canal infections and endodontic disease. However, because the capability of enterococci to reside for a period of time longer than 5 days was not tested in this study, it should be taken into consideration that enterococci could disappear from the oral cavity if they do not proliferate as well as other typical oral microbiota. The absence of genotypic homology between the cheese isolates and some of the strains recovered from the different volunteers could be caused by high diversity in the Enterococcus strains present in this fermented food. However, strains from other environmental sources cannot be excluded, because enterococci, particularly E. faecalis, are ubiquitous. Another limitation is the difficulty in cultivating all enterococci established in the oral cavity after cheese consumption, especially because of the semi-planktonic character of salivary bacteria, as well as bacteria desorbed from biofilm after ultrasonication (15). Only one recent comprehensive study from Sun et al (41) has looked at the genotypic structure of E. faecalis collected from 3.7% of 2839 marginal periodontitis patients and from 9.5% of 21 apical periodontitis patients from primary and secondary infections (Sun, personal communication, 2009) by using multilocus sequence typing (MLST). The authors found that although a high amount of genetic diversity was 1818
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shown, the strains could be grouped with the known isolates from the international MLST database, which led the authors to the conclusion that enterococci should not be considered as a genus that is not part of the normal oral microbiota. Overall, within the limitations of the small sample size of 6 volunteers, the results presented in the current study support this suggestion. The occurrence of enterococci in infected root canals could originate from the dental oral biofilm. In this context, the relatedness of enterococci and streptococci should be kept in mind; both groups belong to the same family of lactobacilli (Streptococcaceae). However, the hospital-adapted genetic complexes revealed by MLST showed a high rate of recombination in E. faecalis (39). This underlines the possibility of acquiring antibiotic resistance genes (41) and gives enterococci, particularly E. faecalis, a special status when they are detected in endodontic infections. Future studies should try to detect any possible connections between E. faecalis strains isolated from endodontic infections and those from other hospital infections. In particular, the aspect of antibiotic resistance should remain of primary concern. Endodontists studying root canal infections need to include endodontic clinical isolates of E. faecalis to analyze properties of these strains, ie, putative virulence factors or antibiotic resistance genes. Possible effects on the proliferation and differentiation of eukaryotic cells like osteoblast-like cells should be studied to determine the role of this species in the development of periradicular disease. This point was recently urged by Siqueira and Roˆc¸as (7), who summarized the state of the art of microbial diversity in endodontic infections and emphasized the need to investigate the role of bacterial diversity revealed in endodontic infections. A genotyping comparison of a large number of such isolates would add knowledge about the degree of relatedness of the endodontic E. faecalis pool to other clonal groups of this species deposited in international databases. In conclusion, our results indicate that food-borne enterococci can integrate into dental oral biofilm in vivo, allowing them to become a potential source for endodontic infections. However, studying a higher sample size of volunteers, in addition to genotypic comparison of food and oral isolates of enterococci, should be conducted to clarify the possible transmission of these bacteria by fermented food because enterococci could survive in the oral cavity in predilection sites, especially where oral hygiene measures do not reach. In future studies the prevalence of enterococci should be investigated in a wide range of patients with different levels of oral hygiene. Furthermore, genotypic analysis is critical to better understand the different possible sources of these microorganisms in endodontic infections. The role of probiotics based on enterococci in multispecies root canal infection has to be taken into consideration in future studies.
Acknowledgments The authors thank Gabriele Braun for her excellent technical help. The authors deny any conflicts of interest related to this study.
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