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Journal of Bacteriology, June 2006, p. 4117-4124, Vol. 188, No. 11
0021-9193/06/$08.00+0     doi:10.1128/JB.01958-05

Rapid Succession within the Veillonella Population of a Developing Human Oral Biofilm In Situ

Robert J. Palmer Jr., Patricia I. Diaz,{dagger} and Paul E. Kolenbrander*

Oral Biofilm Communication Unit, Oral Infection and Immunity Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bldg. 30, Room 310, Bethesda, Maryland 20892

Received 21 December 2005/ Accepted 10 March 2006


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ABSTRACT
 
Streptococci are the primary component of the multispecies oral biofilm known as supragingival dental plaque; they grow by fermentation of sugars to organic acids, e.g., lactic acid. Veillonellae, a ubiquitous component of early plaque, are unable to use sugars; they ferment organic acids, such as lactate, to a mixture of shorter-chain-length acids, CO2, and hydrogen. Certain veillonellae bind to (coaggregate with) streptococci in vitro. We show that, between 4 and 8 hours into plaque development, the dominant strains of Veillonella change in their phenotypic characteristics (coaggregation and antibody reactivity) as well as in their genotypic characteristics (16S RNA gene sequences as well as strain level fingerprint patterns). This succession is coordinated with the development of mixed-species bacterial colonies. Changes in community structure can occur very rapidly in natural biofilm development, and we suggest that this process may influence evolution within this ecosystem.


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TEXT
 
In nature, bacterial biofilms are inherently multispecies communities (6). Human dental plaque is a paradigm biofilm, a complex community (20, 30) of intimately juxtaposed (19) microorganisms on which spatiotemporally resolved data are easily collected (26, 28). This community is highly resilient; under good dental hygiene practices, supragingival plaque is destroyed on a daily basis yet quickly and repeatedly reestablishes itself (22, 36). Two factors hypothesized as important for the establishment of these complex networks of interorganismal relationships are coaggregation and food webs.

Bacterial coaggregation, a type of cell-cell recognition, is studied almost exclusively in vitro and is typically identified by the formation of clumps (coaggregates) when a homogeneous suspension of one bacterium is mixed with that of a second bacterium (4). Coaggregation was first discovered in oral bacteria (12), and although similar interactions are being identified in bacteria from other microbial ecosystems (31), nowhere is coaggregation as clear and pervasive as in the bacterial flora of the human oral cavity (18). Recent experiments have provided direct evidence that coaggregation is a factor in establishing spatial relationships between certain streptococci and actinomyces in vivo (29).

Metabolic cooperation among bacteria may be important to the establishment of stable oral biofilm communities (3), and food webs can be set up through this cooperation. Streptococci make up 60 to 90% of the supragingival plaque biomass in the first 24 h of colonization (20, 27); they catabolize carbohydrates to short-chain organic acids, such as lactic acid and pyruvic acid (7). Veillonellae constitute as much as 5% of the initial plaque biomass (20, 27) but are unable to catabolize sugars. They rely on the fermentation of organic acids to propionic and acetic acids, carbon dioxide, and hydrogen (8, 32). Thus, a rudimentary food web whereby veillonellae depend upon organic acids produced by streptococci could exist. Proximity of producer to consumer could be important to such metabolite transfers, and a set of six oral streptococcal reference strains (4, 17) has been used to characterize coaggregation interactions of oral streptococci with other genera of oral bacteria. The majority of veillonellae isolated from saliva (90%) and from oral soft tissues (75%) either do not coaggregate with any of the six streptococcal reference strains or coaggregate solely with Streptococcus oralis J22 (15). In contrast, 83% of veillonellae from subgingival plaque coaggregate with multiple streptococcal reference strains (15). Also, veillonellae inoculated into the mouths of gnotobiotic rats are unable to establish themselves as monoinfections (single-species biofilms), yet when a strain of Veillonella is inoculated into rats already monoinfected with a strain of Streptococcus mutans that coaggregates with the Veillonella strain, then the number of veillonellae on the teeth of these coinfected animals is 1,000-fold higher than when a noncoaggregating Veillonella strain is used (23). Few data exist on proximity of veillonellae and streptococci in plaque. We hypothesized that cell-cell recognition between veillonellae and streptococci may be important in supragingival plaque formation, and we set out to examine this in a human model system.

Antibody reactivities and coaggregation properties of Veillonella reference strains. A polyclonal antibody, hereafter referred to as anti-1910, was used to characterize the antibody reactivities of twelve oral Veillonella strains (hereafter referred to as Veillonella reference strains), including all ATCC strains identified as human oral isolates. Antiserum was made (Covance Research Products, Denver, PA) by intramuscular injection into rabbits of a cell surface sonicate prepared from the promiscuously coaggregating Veillonella atypica strain PK1910 (14). Immunoglobulin G was purified using protein A columns (Pierce, Rockford, IL), absorbed overnight at 8°C against whole cells of Porphyromonas gingivalis W50 in the presence of protease inhibitors, and then repurified over a protein A column. Absorbed immunoglobulin G was fluorescently labeled using AlexaFluor (Invitrogen, Carlsbad, CA) dye according to the manufacturer's protocol. Veillonella reference strains beginning with PK and with N were isolated from the human oral cavity and identified to the species level (13). The remaining Veillonella strains were obtained from the American Type Culture Collection and are oral isolates except 10790, which is an intestinal isolate included in this study as the type strain of Veillonella parvula. Veillonella strains were grown on modified Schaedler's broth (Schaedler's broth without glucose and containing 21 ml/liter of 60% lactic acid syrup). Stationary-phase cells (500 µl) were washed twice in phosphate-buffered saline (PBS), allowed to react with 250 µl of fluorescently labeled primary antibody (5 µg protein/ml) at room temperature for 20 min, washed twice with PBS, and observed using a 100x, 1.3-numerical-aperture Plan Fluorotar lens mounted on a Leica DM LB2 microscope. Negative controls for antibody reactivity were Prevotella loescheii PK1295, Porphyromonas gingivalis W50, Haemophilus parainfluenzae 28, Streptococcus gordonii DL1, and Actinomyces naeslundii T14V. Coaggregation patterns occurring when reference veillonellae were partnered with the six previously described coaggregation reference strains of streptococci (4, 17) were determined using a visual assay (4). A score of "-" means no coaggregation (equivalent to 0 on the previously used scale), a score of "+" indicates a weak interaction with formation of small coaggregates (equivalent to 1 or 2 on the previous scale), and a score of "++" indicates a strong interaction with formation of large coaggregates that may settle to leave a clear supernatant (equivalent to 3 or 4 on the previous scale). The Veillonella reference strains were separated into two groups (Table 1); seven strains displayed promiscuous and strong coaggregation, whereas five strains had limited or no coaggregation. Anti-1910 recognized all of the strongly coaggregating veillonellae but only one of the weakly coaggregating strains.


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  		TABLE 1. Coaggregation and antibody reactivities of Veillonella reference strains

Localization of anti-1910-reactive veillonellae in undisturbed plaque. Confocal microscopy of intact supragingival plaque samples from four volunteers was performed to examine the locations of anti-1910-reactive veillonellae in situ. A retrievable-enamel-chip model (29) provided intact plaque after 4 and 8 hours of wear in the oral cavity. Enamel chips were cut from extracted human third molars, cleaned, sterilized, and inserted into bilateral mandibular stents. Nonsmoking, periodontally healthy volunteers wore the stents and were prohibited from eating or drinking (water excepted) while wearing the stents. If stent wear overlapped with a meal, the stents were removed and stored in a humid denture cup at 37°C until the volunteer's mouth was thoroughly rinsed with water after the meal. After the chips were removed from the stents, the adherent biofilms were stained (29) with fluorescently labeled primary antibodies together with nucleic acid stain and observed using a 63x, 0.9-numerical-aperture, water-immersible ("dipping") lens mounted on a Leica TCS SP2 laser confocal microscope. Anti-1910-reactive cells were present in the plaque of only one volunteer; reactive cells were undetectable in that individual's 4-h-old plaque (Fig. 1, top panels) but numerous in that volunteer's 8-h-old plaque (Fig. 1, bottom panels). They were frequently found intimately associated, i.e., they formed mixed-species colonies (Fig. 1, bottom panels), with other cells reactive with a second antibody (hereafter referred to as anti-RPS) against receptor polysaccharide (RPS), a streptococcal cell surface polysaccharide that is recognized by adhesins of other oral bacteria (5), including veillonellae (15), to mediate certain coaggregation interactions. Consistent with our previously published microscopy data (29), anti-RPS-reactive cells were found in all volunteers, the number of RPS-bearing cells in the biofilms increased from 4 to 8 h, and RPS-bearing cells formed mixed-species colonies with antibody-unreactive cells. Thus, even though anti-1910-reactive cells were found in only one volunteer's plaque, the plaque in all four volunteers developed in a consistent manner. Our present results demonstrate that the anti-1910-reactive cells become numerous in the biofilm only after mixed-species colonies containing anti-RPS-reactive cells become established and an intimate association between veillonellae and anti-RPS-reactive cells frequently occurs.


Figure 1
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  		FIG. 1. Anti-1910-reactive cells (green) appear between 4 and 8 h of plaque development and are associated with mixed-species colonies that contain RPS-bearing streptococci (red; anti-RPS immunostained). Four-hour-old (top panels) and 8-h-old (bottom panels) plaque are shown as maximum projections of confocal image stacks. The general nucleic acid stain (blue) was Syto 59 in the top panels and DAPI (4', 6'-diamidino-2-phenylindole) in the lower-left image. The right images are magnifications (roughly x3) of the center regions of the left images. The scale bar is 20 µm.

Isolation of veillonellae from plaque samples. Because veillonellae are a common albeit limited component of early plaque (9, 20, 28), we asked whether any veillonellae, regardless of antibody reactivity, were present in 4-h-old plaque from the volunteer who had anti-1910-reactive cells only at 8 h (volunteer 1) as well as whether any veillonellae were present in a volunteer who had no anti-1910-reactive cells at either time point (volunteer 2). Veillonella clinical isolates were obtained from retrievable enamel chips by mild sonication (Branson 150 sonic bath) of the chips in PBS (pH 7.4), followed by dilution plating on Veillonella enrichment agar (tryptone, 5 g/liter; yeast extract, 3 g/liter; sodium thioglycolate, 0.75 g/liter; 60% lactic acid syrup, 21 ml/liter; agar, 1.5 g/liter; vancomycin, 7.5 µg/ml). All isolates recovered on this enrichment medium were diplococci and formed gas when grown in modified Schaedler's broth. Veillonellae were isolated from chips at about the same frequency (roughly 2 x 103 CFU per chip after 4 hours of wear) for each volunteer, and counts were two- to threefold greater at 8 h than at 4 h. However, the anti-1910 reactivities of these veillonellae differed greatly between the individuals. Thirteen of the 39 isolates (2 of the 19 4-h-old isolates and 11 of the 20 8-h-old isolates) from volunteer 1 reacted with anti-1910, but none of the 40 isolates (20 at each time point) from volunteer 2 were anti-1910 reactive. Thus, despite the broad reactivity of anti-1910 with the panel of reference veillonellae, and despite the presence of veillonellae in both volunteers, anti-1910 reacted solely with isolates from one volunteer and then preferentially with those isolates obtained after 8 h.

Phylogenetic characterization of clinical isolates. We used 16S rRNA gene sequencing to phylogenetically characterize the 39 isolates from volunteer 1. Bacterial culture (3 µl) was mixed with 17 µl of Microlysis (The Gel Company, San Francisco, CA) and lysed by rapid heating and cooling according to the manufacturer's protocol. The Ex Taq-buffered (Takara Bio, Inc., Otsu, Shiga, Japan) 50-µl PCR mixtures contained 3 µl template (lysate), 400 µM of each deoxynucleoside triphosphate, 2.5 units Ex Taq Hot Start DNA polymerase (Takara Bio), and 0.6 µM of each primer. Universal primers D88 and E94 (30) were used to amplify approximately 1,500 bp of the 16S rRNA gene. PCRs were initially denatured at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 52°C for 30 seconds, and extension at 72°C for 2 min, with a final extension step at 72°C for 10 min. Primers D88 and F15 (30) were used to sequence (3730 DNA analyzer; Applied Biosystems, Foster City, CA) approximately 500 bp near the 5' region of the cleaned (PCR purification kit; QIAGEN, Santa Clarita, CA) PCR products. Sequences were assembled using Seqman II (Lasergene 6; DNASTAR, Inc., Madison, WI). BLAST (1) searches yielded initial identification. Phylogenetic trees were constructed via neighbor joining (33) in CLUSTAL_X version 1.64b (34). 16S rRNA gene sequences of Veillonella isolates are available in GenBank under accession numbers DQ123531 through DQ123569.

We also analyzed the relatedness of the 39 isolates at the strain level using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) fingerprinting, a technique that provides an assessment of diversity within a species (microdiversity) based on patterns produced by a set of PCR primers targeting highly conserved extragenic repetitive sequences from Escherichia coli and Salmonella enterica serovar Typhimurium (35). The 50-µl ERIC-PCR mixtures used primers ERIC1R and ERIC2 in the amplification cycle described previously (35) and contained 50 pmol of each primer, 100 ng template DNA, 3 mM MgCl2, 0.8 mM of each deoxynucleoside triphosphate, 5 µl 10x Taq buffer (QIAGEN), and 2 units Taq DNA polymerase (QIAGEN). Phoretix 1D Pro version 2003.02 (Nonlinear USA, Inc., Durham, NC) software was used for cluster analysis of fingerprints. Lane similarities were determined by applying the Dice coefficient to the peaks. Clustering was performed using the unweighted-pair group method with arithmetic means. Treeing of the ERIC sequences is not indicative of relative genetic relatedness between groups of strains; rather, it is an objective method for sorting the ERIC patterns into groups of similar strains. Similarity levels of ≥50% and ≥90% were chosen to separate, respectively, clusters of isolates and of siblings.

Congruent molecular taxonomic data were obtained by the two sequence-based methods (Fig. 2). Ribosomal gene sequencing showed that all isolates were indeed veillonellae and separated the isolates into three major groups (marked A1/A2, B, and C); ERIC fingerprinting gave seven clusters (numbered 1 to 7). Gene sequence group A1/A2 encompassed sequences that clustered with those of the reference strains of V. parvula and Veillonella dispar (species not separable by 16S analysis [21]) and contained 32 of the 39 clinical isolates. Subgroup A1 contained 16 of the 19 4-h-old isolates but only 5 of the 20 8-h-old isolates and corresponds almost exactly with ERIC clusters 1 and 2, the sole exception being the absence of R13 in the ERIC clusters. Ribosomal gene sequence subgroup A2 contained 10 of the 20 8-h-old isolates but only 1 4-h-old isolate and corresponds almost exactly to ERIC cluster 3, the sole exception now being the presence of isolate R13 in the ERIC cluster. Several ERIC cluster 3 isolates were "siblings" (here defined as ≥90% similar by ERIC analysis and, as discussed below, having identical antibody/coaggregation properties). Gene sequence group B contained five 8-h-old isolates and one 4-h-old isolate; the isolates in this group cluster with V. atypica rather than with V. parvula or V. dispar. Group B also corresponds exactly with ERIC clusters 4, 5, and 6. Gene sequence group C has only a single Veillonella reference sequence of human origin (Veillonella montpellierensis) and contains isolate R19, which is the sole member of ERIC cluster 7. ERIC results confirm that many of the 8-h-old isolates are unique strains, not simply 4-h-old isolates that display antigenic variation.


Figure 2
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  		FIG. 2. Phylogenetic and phenotypic characterization of veillonella isolates. Four-hour-old isolates are in red (isolate numbers R1 to R19); 8-h-old isolates are in blue (isolate numbers R40 to R60). The 16S rRNA gene sequence tree is on the left; ERIC fingerprinting (strain level relatedness using gel banding patterns) is on the right. Green lines demarcate groups (A1/A2, B, and C) in the gene sequence tree and clusters (numbered 1 to 7) in the ERIC dendrogram; horizontal dashed lines connect the related 16S groups and ERIC clusters. The red dot indicates the division point between group A1/A2 and group B; the green dot indicates the division point between subgroups A1 and A2; bootstrap values are indicated at branch points. Clusters (organisms with similar ERIC banding patterns) and siblings (isolates obtained more than once) were defined, respectively, as having ≥50% and ≥90% relatedness by clustering with the unweighted-pair group method with arithmetic means, and these levels of relatedness are indicated by the vertical dashed lines cutting the ERIC dendrogram. Coaggregation with S. oralis J22 and antibody reactivities (as shown in Table 1) of the isolates are indicated in columns to the left of the ERIC data. Note that certain isolates within ERIC cluster 3 that are molecularly defined as siblings have different phenotypes (e.g., R54 and R50; antibody reactivity is the same but coaggregation differs). ++, strong coaggregation or antibody reactivity; +, weak coaggregation or antibody reactivity.

Phenotypic characterization of clinical isolates. The isolates were characterized phenotypically through coaggregation interactions against the panel of reference streptococcal strains, and we made polyclonal antibody (hereafter referred to as anti-R1) against whole cells of the anti-1910-unreactive, 4-h-old R1 isolate and then tested the reactivities of the isolates against this antibody in an effort to obtain antibody useful for identifying anti-1910-unreactive veillonellae in intact plaque. Phylogenetic data were underscored by antibody reactivity patterns (Fig. 2). All of the ERIC cluster 1 and 2 isolates were anti-R1 reactive, and none reacted with anti-1910. All ERIC cluster 3 isolates, several of which were siblings by ERIC analysis, were anti-1910 reactive but also weakly anti-R1 reactive. All five antibody-unreactive isolates are found in congruent phylogenetic units, i.e., gene sequence group B and ERIC clusters 4, 5, and 6. Isolates that coaggregated did so only with streptococcal reference strain S. oralis J22 and were found at both isolation time points. Anti-R1 reacted with coaggregation-negative reference strains and clinical isolates as well as with coaggregation-positive reference strains and clinical isolates, including many that were anti-1910 reactive (Table 1 and Fig. 2). Likewise, no link between anti-1910 reactivity and coaggregation was observed in the clinical isolates (Fig. 2), even though this antibody is a good indicator of coaggregation within the reference strains (Table 1). Thus, in the clinical isolates (Fig. 2), coaggregation ability is not related to reactivity with either antibody. The broad anti-1910 reactivity in the reference strains thus has only a circumstantial relationship to coaggregation and likely relates to antigens borne in common by strains isolated from subgingival plaque. However, this same antibody identified isolates of supragingival origin (Fig. 1 to 3) and was useful in discerning differences in veillonellae between individuals. The coaggregation of the clinical supragingival plaque isolates with streptococcal partners is dramatically different from that of isolates obtained from subgingival plaque (including half of the Veillonella reference strains) and is similar to that displayed by isolates from saliva or oral soft tissues (15). Coaggregation promotes formation of mixed-species colonies between oral veillonellae and particular bacterial neighbors and thus positions veillonellae in specific habitats within the mouth.


Figure 3
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  		FIG. 3. Four-hour-old plaque (top panels) contains veillonellae reactive solely with anti-R1 (green). Eight-hour-old plaque (bottom panels) contains veillonellae reactive solely with anti-1910 (red) as well as veillonellae reactive with both antibodies (yellow). Antibody-reactive cells are typically associated with antibody-unreactive cells (blue). Staining: anti-R1, green; anti-1910, red; Syto 59 (nucleic acid stain), blue. The right images are magnifications (roughly x3) of the center regions of the left images. The scale bar is 20 µm.

Localization of anti-R1-reactive veillonellae in plaque. We next stained 4- and 8-h-old plaque samples on retrievable enamel chips with anti-R1 and anti-1910 simultaneously. Confocal microscopy confirmed that veillonellae present in 4-h-old plaque were reactive only with anti-R1 (Fig. 3, top panels), whereas veillonellae in 8-h-old plaque were either reactive with both antibodies (colocalization of anti-R1 with anti-1910) or reactive solely with anti-1910 (Fig. 3, bottom panels). Most antibody-reactive cells were in association with other bacteria.

Integration of veillonellae into the oral biofilm. Volunteer 1 showed a time-resolved pattern (Table 2): "4-h" cells dominated initially but were replaced by "8-h" cells within just 4 h, an extraordinarily rapid change early in oral biofilm development. Each population could fill a niche extant only in the presence of certain other bacteria, and coaggregation appears to set up some of these interactions. For example, we recently showed that, in an in vitro oral biofilm model, V. atypica PK1910 induces amylase in S. gordonii DL1 only when the two species are intimately juxtaposed (11). Our present results suggest that some veillonellae are adapted to the simple communities present in early plaque whereas others are adapted to the more complex communities present later, and the very specific coaggregation profile (coaggregation with S. oralis J22 only) underscores that veillonellae isolated from early supragingival plaque are quite different in their preference of community partners from veillonellae from other environments (e.g., the strains listed in Table 1). The reproducible appearance of specific niches in early, low-biomass plaque is likely to be based not just upon the presence of particular bacteria but also upon the juxtaposition of those bacteria at the micrometer scale as well as upon host-specific factors, such as composition and flux of saliva and gingival crevicular fluid.


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  		TABLE 2. Time-resolved Veillonella populations identified in this study

Diversity within veillonellae and impact on ecosystem stability. Classical bacterial taxonomy has established Veillonella species (V. parvula, V. dispar) based primarily on serologic and metabolic data, but these species are inseparable by 16S RNA gene sequence analysis. Microdiversity (differences within a species) is typically described by molecular analyses (16, 25), and although different molecular patterns are often linked to the different geographic or ecologic origins of the isolates, they are less often linked to phenotypic characteristics. Certain of our clinical isolates that had identical ERIC patterns displayed phenotypic differences. For example, strains R50 and R54 had identical ERIC patterns and identical antibody reactivities but different coaggregation properties. The combination of our molecular and phenotypic data (polyphasic taxonomy) suggests not only that oral veillonellae have significant microdiversity but also that microdiversity measured only at the molecular level, such as by strain fingerprinting, does not necessarily encompass the extent of (micro) diversity seen at a phenotypic level.

Within ecosystems, stability is correlated with diversity (2, 24), and resiliency in oral biofilms can be accentuated by high microdiversity coupled with high species diversity, each of which operates at multiple levels. Microdiversity within the veillonella population is evident in data for volunteer 1 (ERIC and 16S RNA gene sequence data), and the detection of anti-1910-reactive cells in the plaque of only that volunteer is suggestive of phenotypic diversity in veillonellae between individuals. Subject-dependent diversity is also observed in the total intestinal flora (10) as well as in the total 4-h-old oral biofilm microflora (9). We suggest that, in the oral cavity, these individualized flora arise from strong evolutionary pressure for the formation of multispecies biofilm communities that effectively utilize the limited, host-influenced nutritional sources in saliva, and human oral biofilms present a well-characterized microbial system in which polyphasic taxonomy can be used to test these links.


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ACKNOWLEDGMENTS
 
We thank J. Cisar for anti-RPS antibody.

This research was supported by the Intramural Research Program of the NIH, National Institute of Dental and Craniofacial Research.


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FOOTNOTES
 
* Corresponding author. Mailing address: National Institutes of Health, NIDCR, Building 30, Room 310, Bethesda, MD 20892-4350. Phone: (301) 496-1497. Fax: (301) 402-0396. E-mail: pkolenbrander{at}dir.nidcr.nih.gov. Back

{dagger} Present address: Department of Periodontology, School of Dentistry, University of North Carolina, Chapel Hill, NC 27599. Back


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Journal of Bacteriology, June 2006, p. 4117-4124, Vol. 188, No. 11
0021-9193/06/$08.00+0     doi:10.1128/JB.01958-05




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