Biofilm-Forming Capacity on Clinically Isolated Streptococcus constellatus from an Odontogenic Subperiosteal Abscess Lesion

S. Treptococcus constellatus is a part of normal commensal flora in the mouth, oropharynx and gastrointestinal tract, and composes the S. Treptococcus anginosus Group (SAG), together with S. Treptococcus anginosus and S. Treptococcus intermedius [1,2]. S. Treptococcus constellatus has been cultured from dental caries and periodontitis lesions, but has also been found to cause purulent infections with abscess formation in various regions of the body, especially in thorax and head and neck regions, despite this organism appears to be innocuous at its natural habitat [3-5]. In contrast, S. anginosus and S. intermedius in SAG show tropism for abdomen and genitourinary tract, and for liver and Central Nerve System (CNS), respectively. There apparently appears to be species bias to some infections, but mechanisms behind such phenomena are not fully understood [6,7].


Introduction
S. Treptococcus constellatus is a part of normal commensal flora in the mouth, oropharynx and gastrointestinal tract, and composes the S. Treptococcus anginosus Group (SAG), together with S. Treptococcus anginosus and S. Treptococcus intermedius [1,2]. S. Treptococcus constellatus has been cultured from dental caries and periodontitis lesions, but has also been found to cause purulent infections with abscess formation in various regions of the body, especially in thorax and head and neck regions, despite this organism appears to be innocuous at its natural habitat [3][4][5]. In contrast, S. anginosus and S. intermedius in SAG show tropism for abdomen and genitourinary tract, and for liver and Central Nerve System (CNS), respectively. There apparently appears to be species bias to some infections, but mechanisms behind such phenomena are not fully understood [6,7].
In this study, we found that S. Treptococcus constellatus strain H39 isolated from a subperiosteal abscess lesion due to the infection of root canal system of a tooth, has a capacity to produce viscous materials in spent culture media and form dense meshwork structures, a typical phenotype for biofilm-forming bacteria [8], around cells on agar plates. This phenotype was also observed on type strains for S. Treptococcus constellatus. As far as we retrieved, only a small number of literatures are available regarding biofilm-phenotypic characterization of SAG [4,9,10]. Therefore, we also examined the capacity of this isolate and type strains of SAG, to form biofilm on abiotic materials.
S. Treptococcus constellatus ATCC 27823, S. intermedius ATCC 27335 and S. anginosus ATCC 33397 were used as the type strains for SAG, and the cultures were maintained as described above.

Abstract
S. Treptococcus constellatus, a member of the S. Treptococcus anginosus Group (SAG), and known as part of indigenous oral microbiota, has been described to cause abscesses in various regions of the body, despite this organism appears to be innocuous, in general at its habitat. In this communication, we report a biofilm-forming capacity of a facultative anaerobic gram-positive coccus isolated as a dominant bacterium in an odontogenic subperiosteal abscess lesiony. The clinical isolate designated as strain H39 formed dense meshwork structures around the cells that are typical for biofilm forming bacteria, and produced viscous materials in its spent culture media. The 16S rRNA gene sequence of strain H39 was 99% homologous to that of S. Treptococcus constellatus ATCC 27823, a type strain for S. Treptococcus constellatus. Phylogenetic analysis using data sets of recN, groEL, tuf and 16S rRNA genes showed a sister relationship between strain H39 and S. Treptococcus constellatus ATCC 27823. Dense meshwork-like structures found on strain H39 were observed on S. Treptococcus constellatus ATCC 27823, but not on S. intermedius ATCC 27335 and S. anginosus ATCC 33397. Biofilm assay using 96-wells polystyrene microtiter plates revealed that S. Treptococcus constellatus strains H39 and ATCC 27823 can form dense biofilm on abiotic materials consistently. S. intermedius ATCC 27335 was able to form biofilm on microtiter plates at a lesser extent to those of S. Treptococcus constellatus strains. S. anginosus ATCC 33397 did not form biofilm on an abitotic material. As conclusions, dense meshwork structures around the cells of S. Treptococcus constellatus, and the capacity of S. Treptococcus constellatus and S. intermedius to form biofilm on abiotic materials as observed in this study might be related to the pathogenicity of these two organism and the tropism of organisms in SAG. As recently suggested, phylogenetic analysis could be a powerful tool for differentiating and identifying clinical isolates belong to SAG.

Identification by phylogenetic analysis
Identification of organisms in SAG had long been problematic due to the considerable genetic similarity within the group and heterogeneous phenotypes on each species in SAG, but phylogenetic analysis using several housekeeping gene sequences is recently acknowledged as a powerful tool to divide SAG in three species [11][12][13][14]. Therefore, one of clinical isolates designated as strain H39 was subjected to phylogenetic analysis, using partial sequences of 16S rRNA, recN, groEL and tuf genes.

Measurement of viscosity
The viscous material-producing ability of clinical isolates was evaluated by measuring the viscosity of spent culture media, as described elsewhere [16]. Arbitrarily selected eight clinical strains and type strains of SAG were grown in trypticase soy broth (BBL Microbiology Systems, Cockeysville, MD, USA), supplemented with 0.5% yeast extract (BBL Microbiology Systems) (TSB-Y) for 48 h. The viscosity was measured as shearing stress between a rotor and a rotor shaft at 50 rpm, 20°C, using a rotary viscometer (Toki-sangyo, Tokyo, Japan), in accordance with the manual provided by the manufacturer. The measurement was performed five times on each sample, and the results of four independent cultures were averaged to give the final value. Statistical differences between the bacterial cultures and control medium were determined using an unpaired t-test, with the level of significance set at P<0.05.

Preparation for Scanning Electron Microscopy (SEM)
For SEM studies, cells scraped from 1-day-old cultures were collected on a piece of filter paper (Glass fiber GA55, Toyo Roshi, Tochigi, Japan), fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (Mitsubishi Chemical Medience, Tokyo, Japan) for 2 hours, and with 1% OsO 4 in 0.1M phosphate buffer for 1 h at 4°C and dehydrated through an ethanol series and 2-methyl-2-propanol, followed by platinum ion coating (E-1030, HITACHI, Tokyo, Japan). Specimens were examined with a scanning electron microscope (S-4800, HITACHI), at an accelerating voltage of 3 kV.

Biofilm formation assay
The ability to form biofilm was determined, as described previously [17]. Briefly, the seed cultures of S. Treptococcus constellatus strains H39 and ATCC 27823, S. intermedius ATCC 27335 and S. anginosus ATCC 33397 were prepared as described above, and diluted to an O.D. of 0.1 at 620 nm in the same medium. Next, 150 μl diluted culture was transferred to three sterile polystyrene microtiter plate wells (IWAKI, Tokyo, Japan) per strain. Sterile TSB-Y was used as a control. The plates were prepared in duplicate and incubated at 37°C for 24 h. Unbound cells were removed by inversion of the microplate and tapping on absorbent paper towel. Adhered cells were then stained with 0.1% crystal violet for 15 min. Excess stain was removed by washing with PBS. The stained wells were recorded photographically using a camera (PC1089, Canon, Tokyo, Japan) set. The crystal violet was then solublized by the addition of 95% ethanol, and absorbance at 570 nm was measured by spectrophotometer U-2000 (Hitachi). Each strain was assayed in three wells on each plate and the whole experiment was repeated three times.

Identification of strain H39
The nucleotide sequence of 16S rRNA gene of strain H39 (accession number: AB749301) was identical and highly homologous to that of S. Treptococcus constellatus ATCC 27823 (accession number: AF104676).

Viscous material productivity
Strain H39 and type strains of SAG were inoculated into TSB-Y and grown for 48 h. The viscosities of the spent culture media were measured using a rotary viscometer. Strain H39 produced materials in vitro that were highly viscous, as compared with the control medium ( Figure 2). The viscosity of spent culture medium of S. Treptococcus constellatus ATCC 27823 was as high as those of the clinical isolates. The viscosities of S. intermedius ATCC 27335 and S. anginosus ATCC 33397 were similar to that of control medium, correlated to its cell surface structures (Figure 2).

Biofilm phenotype on strain H39 and type strains of SAG
SEM observation revealed that strain H39 showed surfaceassociated dense meshwork-like structures around the cells (Figure 3). Similar surface structure was observed on S. Treptococcus constellatus ATCC 27823, but not on S. intermedius ATCC 27335 and S. anginosus ATCC 33397 (Figure 3).

Biofilm formation assay
The ability to form biofilm was investigated for S. Treptococcus constellatus strains H39 and ATCC 27823, S. intermedius ATCC 27335 and S. anginosus ATCC 33397, using crystal violet microtiter plate assay. Strain H39 was consistently able to form biofilm on flatbottomed polystyrene microtiter plates (Figure 4). Type strains of S. Treptococcus constellatus and S. intermedius were able to form biofilm on abiotic materials. The capacity to form biofilm on S. Treptococcus S. Treptococcus sanguinis), which caused greater chemotaxis than did S. aureus. Interestingly, SAG and viridans streptococci were equally engulfed by human neutrophils than S. aureus, but killed more slowly and less completely. Our previous studies showed that the resistance of biofilm-forming Prevotella intermedia strains to phagocytosis by human neutrophils were more significant than those of strains lacking biofilm phenotype [16,17,26]. We hypothesize that biofilm bacteria, being held together by exopolysaccharides as biofilm matrices, might present a huge physical challenge for phagocytosing neutrophils. As a consequence of these neutrophils being frustrated by their inability to phagocytose this bacterial mass, this might trigger the unregulated release of bactericidal compounds that could cause tissue injury [27], as shown in the inflammatory pathway associated with lung injury or chronic wounds [28,29]. Further investigation is needed to clarify whether inconsistency of biofilm phenotype in SAG gives influence of their interaction with human neutrophils and propensity to cause abscesses.
As described above, only a few studies have done regarding biofilm phenotype on S. Treptococcus constellatus. Landrygan-Bakri et al.
constellatus ATCC 27823 seemed to be comparable to that of strain H39, but S. intermedius ATCC 27335 was weaker than that of strain H39. S. anginosus ATCC 33397 could not form biofilms on microtiter plates (Figure 4).

Discussion
Clinical studies [3,18] have shown that S. Treptococcus constellatus has the capacity to cause invasive purulent infection relatively in superficial areas through the body, while S. intermedius and S. anginosus in SAG exhibit a marked association with the infection in the central nervous system and gastrointestinal tracts, respectively. This organism is not frequently isolated from abscess lesions deep inside of the body, but several clinical reports showed the association of S. Treptococcus constellatus with brain abscesses [19,20], endocarditis [21][22][23], and sepsis after dental treatment [24]. The remarkable propensity to cause abscess or purulent infection observed on three species in SAG remind us an idea that there might be a unique interaction between the organisms in SAG and human neutrophils. In this regards, Wanahita et al. [25] reported that SAG organisms stimulated less chemotaxis than did Staphylococcus aureus, in contrast to viridans Streptococci (S.   plates, but lacked dense meshwork structures. Presumably there might be different systems to induce biofilm growth between S. Treptococcus constellatus and S. intermedius.
In this study, we examined one clinical isolate and three type strains for SAG, and we found diversity of cell surface structures between the organisms in SAG. In addition to the previously known various properties of SAG which cause ambiguity in determining pathogenic potential of organisms in SAG, the phenotypic difference found in this study might influence their pathogenicity and tropism in the human body. It is known that S. Treptococcus constellatus is sometimes coisolated with Eikenella species [30], or Prevotella species [31,32], so the capacity to form single/multi species biofilm on S. Treptococcus constellatus should be further investigated.