Application of MLST and pilus gene sequence comparisons to investigate the population structures of Actinomyces naeslundii and Actinomyces oris

Abstract

Actinomyces naeslundii and Actinomyces oris are members of the oral biofilm. Their identification using 16S rRNA sequencing is problematic and better achieved by comparison of metG partial sequences. A. oris is more abundant and more frequently isolated than A. naeslundii. We used a multi-locus sequence typing approach to investigate the genotypic diversity of these species and assigned A. naeslundii (n = 37) and A. oris (n = 68) isolates to 32 and 68 sequence types (ST), respectively. Neighbor-joining and ClonalFrame dendrograms derived from the concatenated partial sequences of 7 house-keeping genes identified at least 4 significant subclusters within A. oris and 3 within A. naeslundii. The strain collection we had investigated was an under-representation of the total population since at least 3 STs composed of single strains may represent discrete clusters of strains not well represented in the collection. The integrity of these sub-clusters was supported by the sequence analysis of fimP and fimA, genes coding for the type 1 and 2 fimbriae, respectively. An A. naeslundii subcluster was identified with both fimA and fimP genes and these strains were able to bind to MUC7 and statherin while all other A. naeslundii strains possessed only fimA and did not bind to statherin. An A. oris subcluster harboured a fimA gene similar to that of Actinomyces odontolyticus but no detectable fimP failed to bind significantly to either MUC7 or statherin. These data are evidence of extensive genotypic and phenotypic diversity within the species A. oris and A. naeslundii but the status of the subclusters identified here will require genome comparisons before their phylogenic position can be unequivocally established.

Figure 1. Neighbor-Joining tree of concatenated sequences of the 7 house-keeping genes of A. naeslundii and A. oris.

Bar is 0.005 substitutions per site.

Figure 2. 50% majority-rule consensus tree derived using ClonalFrame.

Six ClonalFrame runs were conducted using the default settings, the initial 50,000 iterations were discarded and the next 50,000 iterations were sampled at every 100^th generation was sampled. Therefore, 501 trees per run were calculated and the data of the six runs were combined and the consensus tree was drawn in MEGA4 . Scale is coalescent units.

Figure 3. Neighbor-Joining tree of partial 16S rRNA gene sequences.

Approximately 1400 bp sequences of 16S rRNA genes selected A. naeslundii and A. oris strains indicated by strain name and [sequence type] were determined. Sequences of A. naeslundii (X81062.1; ST77), A. viscosus (X82453), A. johnsonii (AB545933.1) and A. oris (GQ421308; ST16) type strains were included for comparative purposes. STs 1–68 are A. oris and 69–100 are A. naeslundii. Only bootstrap values of >60 are shown for clarity. Bar is 0.001 substitutions per site.

Figure 4. 16S rRNA signature differentiating A. naeslundii and A. oris 16S rRNA sequences.

16S rRNA sequences were aligned using BioEdit and visually compared to detect sequence signatures that differentiated bewteen the species. Figure lists strain names and [ST]. The first base is base 1010 in 16S rRNA sequence of A. naeslundii NCTC 10301 (X81062). Signature bases are marked with arrowheads.

Figure 5. Neighbor-joining tree showing relationships between fimA sequences.

Partial fimA sequences of A. oris, A. naeslundii, A. johnsonii and A. viscosus strains were determined, aligned using BioEdit and a neighbour-joining tree was calculated and visualised using MEGA4. Strain 117 is an additional A. johnsonii strain, see Table S1.

Figure 6. Neighbor-joining tree showing relationships between fimP sequences.

Partial fimA sequences of A. oris, A. naeslundii, A. johnsonii and A. viscosus strains were determined, aligned using BioEdit and a neighbour-joining tree was calculated and visualised using MEGA4. fimP sequence 120 is from A. viscosus ATCC19246 [AF106034]. Strain 117 is an additional A. johnsonii, see Table S1.

Figure 7. Relative attachment of actinomyces strains to salivary proteins.

Bacteria were bound to salivary proteins and saliva preparations and labelled using the fluorescent stain SYTO 13. Bacterial cell binding was normalised such that maximum was 100 and equivalent to all added bacteria binding to substrate. The substrates were (A), a preparation of proline-rich proteins in the presence of lactose (B) MUC7 and (C) statherin. An is the A. naeslundii type strain [ST 77], Ao is the A. oris type strain [ST 16], Aj is the A. johnsonii type strain and Av is the A. viscosus type strain.