RNA-oligonucleotide quantification technique (ROQT) for the enumeration of uncultivated bacterial species in subgingival biofilms

Abstract

Approximately 35% of the species present in subgingival biofilms are as yet uncultivated, so their role in periodontal pathogenesis is unknown. The aim of the present study was to develop a high throughput method to quantify a wide range of cultivated and uncultivated taxa in subgingival biofilm samples associated with periodontal disease or health. Oligonucleotides targeting the 16S ribosomal DNA gene were designed, synthesized and labeled with digoxigenin. These probes were hybridized with the total nucleic acids of pure cultures or subgingival biofilm samples. Target species included cultivated taxa associated with periodontal health and disease, as well as uncultivated species, such as TM7 sp. OT 346, Mitsuokella sp. OT 131 and Desulfobulbus sp. OT 041. Sensitivity and specificity of the probes were determined. A Universal probe was used to assess total bacterial load. Sequences complementary to the probes were used as standards for quantification. Chemiluminescent signals were visualized after film exposure or using a CCD camera. In a pilot clinical study, 266 subgingival plaque samples from eight periodontally healthy people and 11 patients with periodontitis were examined. Probes were specific and sensitivity reached 10(4) cells. Fusobacterium nucleatum ss. polymorphum and Actinomyces gerencseriae were the most abundant cultivated taxa in clinical samples. Among uncultivated/unrecognized species, Mitsuokella sp. OT 131 and Prevotella sp. OT 306 were the most numerous. Porphyromonas gingivalis and Desulfobulbus sp. OT 041 were only detected in patients with periodontitis. Direct hybridization of total nucleic acids using oligonucleotide probes permitted the quantification of multiple cultivated and uncultivated taxa in mixed species biofilm samples.

Figure 1

RNA oligonucleotide quantification technique (ROQT) membrane used to assess probe sensitivity. Total nucleic acids extracted from selected bacterial species were used as targets in the horizontal lanes. Oligonucleotide probes in the vertical lanes were hybridized against these targets. (A) Targets at 10⁶ to 10³ bacterial cells. because of higher than ideal concentration of the Campylobacter rectus probe, signals became saturated for both cell concentrations. (B) Targets at 10⁵ to 10⁴ bacterial cells.

Figure 2

Assessment of probe specificity. Total nucleic acids (TNA) from selected bacterial species were used as targets in the horizontal lanes. Probes to the same species were hybridized against these targets in the vertical lanes. This figure shows nine of the 23 target taxa tested.

Figure 3

Effects of different levels of species in mixtures on signal intensity. Aa, Aggregatibacter actinomycetemcomitans; Ao, Actinomyces odontolyticus; Cr, Campylobacter rectus; Cs, Capnocytophaga sputigena; Pe, Porphyromonas endodontalis; Pm, Parvimonas micra; Sp, Streptococcus parasanguinis.

Figure 4

A checkerboard membrane showing hybridization of clinical samples with oligonucleotide probes. Probes for cultivated species (black) and as yet uncultivated species (green) are listed across the top. Each horizontal lane represents the total nucleic acids (TNA) from a sample from the indicated numbered tooth. Standards comprised a mixture of ‘complementary’ sequences from all the test taxa at 0.004 and 0.04 pM, respectively. Teeth marked with an asterisk (*) were absent.

Figure 5

(A) Mean estimated ‘counts’ ( ×10⁵ ± SEM) of 20 bacterial taxa in 266 subgingival plaque samples obtained from eight periodontally healthy people and 11 patients with periodontitis. The species were ordered according to mean counts. Taxa in black represent cultivated species, whereas those in orange represent uncultivated taxa. (B) Mean estimated ‘counts’ (× 10⁵ ± SEM) of 20 bacterial taxa in 266 samples obtained from eight periodontally healthy people (green) and 11 patients with periodontitis (red). The species were ordered according to mean counts in health. Taxa in black represent cultivated species, while those in orange represent uncultivated taxa.

Figure 5

(A) Mean estimated ‘counts’ ( ×10⁵ ± SEM) of 20 bacterial taxa in 266 subgingival plaque samples obtained from eight periodontally healthy people and 11 patients with periodontitis. The species were ordered according to mean counts. Taxa in black represent cultivated species, whereas those in orange represent uncultivated taxa. (B) Mean estimated ‘counts’ (× 10⁵ ± SEM) of 20 bacterial taxa in 266 samples obtained from eight periodontally healthy people (green) and 11 patients with periodontitis (red). The species were ordered according to mean counts in health. Taxa in black represent cultivated species, while those in orange represent uncultivated taxa.