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Growth stimulation of ‘difficult-to-culture’ oral bacterial strains by siderophores: Lawn cultures of Prevotella sp. HOT-376 (KCL-E7_34) were prepared by inoculating plates of Fastidious Anaerobe Agar (FAA, Lab M, UK) with 50 μl suspensions (McFarland standard 1 turbidity), prepared from a four-day plate culture. Plates of blood Agar Base No. 2 (Lab M, UK) + 5% horse blood (BA) were inoculated with 50 μl suspensions (McFarland standard 4) of Fretibacterium fastidiosum DSM 25557T from a seven-day plate culture. Test agents were placed in a central well in each plate in 1.5, 15 or 150 μg amounts. The agents tested included the siderophores desferricoprogen (EMC Microcollections, Germany), pyoverdines-Fe-complex (Sigma-Aldrich, UK) and ferrichrome-Fe-free (Santa Cruz, USA); siderophore-like molecules 2,3-dihydroxybenzoic acid (Acros Organics, Belgium) and salicylic acid (ChemCruz Biochemicals, USA); and the iron chelator ferric citrate (Sigma-Aldrich, UK). The positive control was filtered supernatant of a 72-hr broth culture of F. nucleatum and negative controls were the agent diluents. Plates were incubated in an anaerobic workstation (Don Whitley Scientific Ltd.) with an atmosphere of 80% nitrogen, 10% hydrogen and 10% carbon dioxide at 37°C for up to 35 days. The diameter and relative density of any zone of growth were recorded, and used to arbitrarily grade growth stimulation relative to positive (++++) and negative controls (-). The experiment was performed in triplicate.
Development of an in-vitro culture model for the cultivation and isolation of previously-uncultivated oral bacteria: Four subjects with chronic periodontitis, none of whom had received periodontal or antimicrobial therapy within the previous three months, were recruited for the study with their informed written consent. Ethical approval was granted by the South West London REC 3 Research Ethics Committee (REF: 10/H0803/161). Subgingival plaque samples were collected with a sterile curette from two deep (7–9 mm) periodontal pockets from each subject, pooled and suspended in Reduced Transport Medium (RTM) [35].
Samples were transported to an anaerobic workstation within 1 hr of collection. After vortexing for 1 min, a 10-fold dilution series of each sample was prepared in RTM. Pre-reduced BA plates were inoculated with 50 μl of each 102-, 105- and 106-diluted plaque suspension. 150 μl neat plaque suspension (matched to the plaque that was diluted and used for culture), 15 μg siderophore (pyoverdines-Fe or desferricoprogen), or 150 μl sterile water (negative control) were added to a central well in the plates, and plates were incubated anaerobically.
Sub-culture and identification of colonies of interest in mixed cultures: After 8–28 days of incubation, culture plates were inspected under a dissecting microscope and any colonies of interest (namely microcolonies or colonies showing satellitism: growing either around or on larger colonies) were passaged onto: (a) fresh BA plates, cross-streaked with helper strains (Propionibacterium acnes ATCC 6919, Fusobacterium nucleatum subspecies polymorphum NCTC 10562, or colonies in proximity to the colonies of interest) and where relevant, supplemented with either pyoverdines-Fe or desferricoprogen; or (b) cellulose acetate membranes overlying lawn cultures of the helper strains. Once pure, sub-cultured colonies were identified by partial 16S rRNA gene sequencing with primer 519R (as described previously [36]) after PCR of extracted DNA or ‘touch’-PCR of single colonies with ‘universal’ primers 27FYM and 1492R [37]. For any taxa: (a) found to have less than 98.5% similarity to 16S rRNA gene sequences in the Human Oral Microbiome (HOMD) and GenBank nucleotide databases, or (b) with more than 98.5% 16S rRNA gene sequence similarity to previously-uncultivated phylotypes, clone libraries were prepared (as described previously [36]) from amplicons representing 16S rRNA gene ‘touch’-PCR of single colonies with ‘universal’ primers; and 20 clone inserts were sequenced per library to confirm purity. The full length of the 16S rRNA gene for any such novel taxon was sequenced with multiple primers for triple coverage [36].
Pyrosequencing analysis of subgingival plaque samples before and after culture: 500 μl of neat plaque suspension from subjects SP9 and SP10 were subjected to propidium monoazide (PMA) treatment prior to DNA extraction. PMA dye is cell membrane-impermeable and selectively binds to/modifies DNA from cells with damaged cell membranes; consequently, only DNA from intact cells was amenable to PCR amplification later [38]. Briefly, 1.25 μl of 20 mmol/L PMA (Cambridge Biosciences, UK) was added to the plaque suspensions, which were incubated in the dark with occasional agitation (5 min) and then placed on ice under a 500 W halogen lamp for 3 min, with occasional agitation. DNA was extracted from the samples using the GenElute Bacterial Genomic DNA kit (Sigma-Aldrich, UK), following the protocol for Gram positive bacteria. At 14 d of growth, colonies were harvested from half of the surface of culture plates derived from 102- and 105-dilutions of samples SP9 and SP10, and suspended in 2 ml PBS. 500 μl of each suspension was PMA-treated and DNA was extracted as described above. An approximately 500 bp region of the 16S rRNA gene (covering V1–V3) was PCR-amplified from extracted DNA samples using composite fusion primers comprising universal 16 S primers (27FYM and 519R) along with Roche GS-FLX Titanium Series adapter sequences (A & B) for 454 pyrosequencing using the Lib-L emPCR method. Previously described unique 12 base error-correcting Golay barcode sequences [39] were incorporated into the forward primers (5′-CCATCTCATCCCTGCGTGTCTCCGACTCAG-NNNNN NNNNNNN-AGAGTTTGATYMTGGCTCAG-3′). The appropriate barcoded A-27FYM and the B-519R (5′-CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-GWATT ACCGCGGCKGCTG-3′) primers were used in PCRs with Extensor Hi-Fidelity PCR Mastermix (Thermo Scientific). There was an initial denaturation step of 5 mins at 95°C followed by 25 cycles of 95°C for 45 s, 53°C for 45 s, 72°C for 1 m 30 s and a final extension of 72°C for 15 mins. PCR amplicons were subsequently purified using the QIAquick PCR purification kit (Qiagen) following the manufacturer's instructions. The size and purity of the amplicons was checked using the Agilent DNA 1000 kit and the Agilent 2100 Bioanalyzer. The amplicons were quantitated by means of a fluorometric assay using the Quant-iT Picogreen fluorescent nucleic acid stain (Invitrogen) and then pooled at equimolar concentrations (1×109 molecules/μl). emPCR and undirectional sequencing of the libraries was performed using the Lib-L kit and Roche 454 GS-FLX Plus Titanium chemistry by the the Department of Biochemistry, University of Cambridge, UK. The mothur pipeline [40] was used for denoising, trimming, alignment to the Silva reference alignment and removal of chimerae. Sequences were identified using the classify.seqs command.
Partial 16S rRNA gene sequences for the novel taxa detected in this study have been submitted to the DDBJ/EMBL/GenBank databases under the following accession numbers: Bacteroidetes bacterium HOT-365 SP18_29, KT861603; Chloroflexi taxon Anaerolineae bacterium HOT-439 SP9_5, KT861598; Chloroflexi taxon Anaerolineae bacterium HOT-439 SP10_2, KT861597; Chloroflexi taxon Anaerolineae bacterium HOT-439 SP19_9, KT861599.
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