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Cultivation of a Synergistetes Strain Representing a Previously Uncultivated Lineageemi 2135 916..928 Environmental Microbiology (2010) 12(4), 916–928 doi:10.1111/j.1462-2920.2009.02135.x Cultivation of a Synergistetes strain representing a previously uncultivated lineageemi_2135 916..928 S. R. Vartoukian, R. M. Palmer and W. G. Wade* culture-independent studies, but 40 strains from 11 King’s College London Dental Institute, Infection genera have been isolated (Hugenholtz et al., 2009). In Research Group, Guy’s Campus, London SE1 9RT, UK. humans, Synergistetes have frequently been detected at sites of disease: soft tissue wounds, cysts and abscesses; blood; peritoneal fluid; and the oral cavity in subjects with Summary dental disease (Munson et al., 2002; Horz et al., 2006; de Subgingival plaque samples obtained from human Lillo et al., 2006; Bernard et al., 2007; Jumas-Bilak et al., subjects with periodontitis, shown to include previ- 2007; Downes et al., 2009). ously uncultivable members of the phylum Syner- Oral Synergistetes taxa fall into two main Clusters, A gistetes, were used to inoculate Cooked Meat Medium and B (Vartoukian et al., 2007). The Synergistetes Cluster (CMM). The presence of Cluster A (uncultivable) Syn- A comprises 22 taxa, related to the TG5 group (Hugen- ergistetes was monitored by fluorescent in situ holtz et al., 2009) (Fig. 1). These taxa are exclusively oral hybridization (FISH) and quantitative PCR (Q-PCR). but none have ever been cultivated in the laboratory, Cluster A Synergistetes were found to grow in CMM in despite their widespread prevalence when molecular co-culture with other plaque bacteria and growth was methods are used for their detection (Vartoukian et al., stimulated by the addition of mucin and serum. 2009). It is estimated that half of the approximately 700 Plaque samples were also used to inoculate Blood taxa found in the human oral cavity have yet to be cultured Agar (BA) plates and growth of Cluster A Syner- (Paster et al., 2006). The study of such bacteria is clearly gistetes was revealed after anaerobic incubation, by important in order to improve our understanding of this colony hybridization with specific probes. Surface complex environment, its bacterial inhabitants, their inter- growth on the plates in regions identified by colony actions and their potential role in disease. Bacteria must hybridization was harvested and used to inoculate be cultivated in purity before they can be fully character- fresh plates, thus enriching for Cluster A Syner- ized not only with regard to their phenotypic and physi- gistetes. Cross-streaks of other plaque bacteria were ological properties, but also their virulence potential and also used to stimulate Synergistetes growth. In the susceptibility to antimicrobials. early passages, no discrete Synergistetes colonies Bacteria that are unable to grow in vitro may be depen- were seen, but after eight passages and the use of dent on specific factors or signals present only within the cross-streaks of other bacteria present in the source habitat (Lewis, 2007). The provision of such enriched community, colonies arose, which consisted factors, for example by artificially simulating the natural solely of Cluster A Synergistetes cells, as determined environment (Kaeberlein et al., 2002; Bollmann et al., by 16S rRNA gene PCR and cloning. This is the first 2007; Ferrari et al., 2008), may encourage growth. These report of the successful culture of a member of the essential factors may include specific nutrients not avail- uncultivable branch of this phylum. able in conventional culture media or chemical signals from other bacteria within the local community (Davey, Introduction 2008). Dental plaque is an example of a multi-species biofilm, within which a number of interspecies interactions Bacteria belonging to the recently described phylum Syn- and co-dependent relationships have been observed ergistetes (Jumas-Bilak et al., 2009) are widespread in (Marsh, 2005; Kuramitsu et al., 2007). An example of this the environment and in animals (Godon et al., 2005). As is the metabolic interaction between Tannerella forsythia yet, the vast majority of the 16S rRNA sequences that are and Fusobacterium nucleatum – production of N-acetyl available from the phylum have been obtained from muramic acid (NAM) by F. nucleatum supports the growth Received 4 September, 2009; accepted 18 November, 2009. *For of T. forsythia, which is unable to synthesize NAM, an correspondence. E-mail [email protected]; Tel. (+44) 20 essential factor in cell wall synthesis (Wyss, 1989). 71883872; Fax (+44) 20 71883871. Based on the hypothesis that Cluster A Synergistetes Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://www3.interscience.wiley.com/ have not been cultured because of their dependence on authorresources/onlineopen.html other dental plaque bacteria for growth, the aims of this © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd Cultivation of previously uncultivated Synergistetes 917 Fig. 1. Phylogenetic tree based on 16S rRNA gene sequence comparisons over 1221 aligned bases showing relationship between Synergistetes strain SGP1, Cluster A Synergistetes and representatives of other genera and groups within the phylum Synergistetes. Tree was constructed using the neighbour-joining method following distance analysis of aligned sequences. Numbers represent bootstrap values for each branch based on data for 500 trees. Accession numbers for 16S rRNA sequences are given for each strain. Scale bar shows number of nucleotide substitutions per site. work were: (i) to investigate whether as-yet-uncultivated highest linear regression coefficient (R2 value of 0.999), Cluster A Synergistetes are able to grow in co-culture in a large cycle threshold (Ct) value of 33 for the vitro with dental plaque bacteria in a complex broth no-template control and a distinct melting curve profile medium, and (ii) to attempt to isolate members of the confirming reaction specificity. Sequence analysis of Cluster A Synergistetes in pure culture on solid media, clone libraries prepared from the pooled products of two using colony hybridization as a guide to enrichment. replicate end-point PCR reactions with primers 763F/ 827R, mixed DNA template A6G and an annealing tem- Results perature of 57°C showed that all 42 clones sequenced were members of the Cluster A Synergistetes confirming Quantitative PCR assay development the validity of the primers. A dilution series of genomic A Quantitative PCR (Q-PCR) assay for Cluster A Syner- DNA was prepared from a mixed template sample taken gistetes was developed. Eight candidate primer sets from a Cooked Meat Medium (CMM) culture, and were found to give linear data over a range of at least Q-PCR assay was performed with primer sets 763F/ five of the nine calibration standards in dilution series. Of 827R and 1406F/1492R. Both primer sets showed good the primers targeting Synergistetes, 763F/827R demon- proportional amplification and linearity with R2 values strated the broadest range of linearity (10-1–10-8), the between 0.997 and 0.999. © 2010 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 12, 916–928 918 S. R. Vartoukian, R. M. Palmer and W. G. Wade ) Fig. 2. Cluster A Synergistetes 16S rRNA s 1000 gene copies in CMM broths inoculated with a mixed culture of sub-gingival plaque. 800 per ml (million s 600 rRNA gene copie 400 S 16 s tete 200 s ynergi S 0 0 5 10 15 20 25 30 35 ter A Days us Cl CMM Half-strength CMM CMM + 10 % sonicate CMM + 0.25 % mucin CMM + 5 µg / ml haemin CMM + 10 % serum Growth of cluster A Synergistetes in co-culture in Fig. 2. For all media except half-strength CMM, counts Cooked Meat Medium showed a marked increase until 14 days of growth, fol- lowed by a partial decrease at 33 days. In contrast, the Fluorescent in situ hybridization (FISH) analysis of the half-strength CMM showed only a modest increase, which plaque samples collected from subjects F5 and F6 con- continued until day 33. The corresponding 16S rRNA firmed the presence of Cluster A Synergistetes, including gene copy numbers for total bacteria (around 1010 gene Synergistetes OTU 3.3. Of the CMM broths and copies per ml of broth) increased up to day 14 and phosphate-buffered saline (PBS) controls inoculated with declined moderately thereafter. The doubling time for these samples, Cluster A Synergistetes were detected Cluster A Synergistetes was determined as 50.4 h for after 5 and 10 days anaerobic incubation in the CMM only. CMM/serum during log phase. The highest counts of In the original plaque samples, Synergistetes cells were Cluster A Synergistetes were found in CMM supple- curved bacilli 2–4 mm in length. At 5 days of culture, some mented with serum or mucin, while the half-strength CMM cells were elongated and 6–7 mm long. By 10 days, the had the lowest (Fig. 2). majority of the cells were filamentous, up to 13 mmin In all broths, the proportion of Cluster A Synergistetes to length. Subculture of the primary CMM cultures to fresh total bacterial count increased over time (Fig. 3). Cluster A CMM at 2, 5 and 10 days revealed, in each case, Cluster A Synergistetes were particularly enriched in CMM/serum Synergistetes by FISH after 5 days of incubation. No and CMM/mucin, making up 3.6% and 3.7%, respectively, Synergistetes were detected in subcultures to PBS at the of the total bacterial gene counts at 33 days (Fig. 3). FISH same time points. All positive CMM sub-cultures contained analysis confirmed the presence of Cluster A Syner- only small numbers of Synergistetes cells with a maximum gistetes in the CMM broths at all time points. Furthermore, of five fluorescent cells in each field of view equivalent to a the FISH images showed temporal changes in the abun- count of 4.5 ¥ 104 cells per ml of CMM broth.
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