Isolation, identification, and significance of salivary Veillonella spp., Prevotella spp., and Prevotella salivae in patients with inflammatory bowel disease

Abstract

The global prevalence of inflammatory bowel disease (IBD) is on the rise, prompting significant attention from researchers worldwide. IBD entails chronic inflammatory disorders of the intestinal tract, characterized by alternating flares and remissions. Through high-throughput sequencing, numerous studies have unveiled a potential microbial signature for IBD patients showing intestinal enrichment of oral-associated bacteria. Simultaneously, the oral microbiome can be perturbed by intestinal inflammation. Our prior investigation, based on 16S rRNA amplicon sequencing, underscored elevated abundance of Veillonella spp. and Prevotella spp. in the salivary microbiomes of IBD patients. Noteworthy, Prevotella salivae emerged as a distinct species significantly associated with IBD. P. salivae is an under-recognized pathogen that was found to play a role in both oral and systemic diseases. In this study, we delve deeper into the salivary microbiomes of both IBD patients and healthy controls. Employing diverse cultivation techniques and real-time quantitative polymerase chain reactions (RT-qPCR), we gauged the prevalence and abundance of Veillonella spp., Prevotella spp., and P. salivae. Our isolation efforts yielded 407 and 168 strains of Veillonella spp., as well as 173 and 90 strains of Prevotella spp., from the saliva samples of IBD patients and healthy controls, respectively. Veillonella-vancomycin agar emerged as the discerning choice for optimal Veillonella spp. cultivation, while Schaedler kanamycin-vancomycin agar proved to be the most suitable medium for cultivating Prevotella spp. strains. Comparing our RT-qPCR findings to the previous 16S rRNA amplicon sequencing data, the results corroborated the higher abundance of Veillonella spp., Prevotella spp., and P. salivae in the saliva of IBD patients compared to healthy controls. However, it's worth noting that in contrast to RT-qPCR, the 16S rRNA amplicon sequencing data revealed greater absolute abundance of all three bacterial groups in both IBD patients and controls.

Keywords: 16S rRNA amplicon sequencing; Prevotella salivae; Prevotella spp.; Veillonella spp.; inflammatory bowel disease; real-time quantitative PCR.

Figure 1

Diverse morphological and growth patterns of Prevotella salivae. (A) Illustrates a single strain of P. salivae (OMI1400), showcasing distinct sizes, shapes, and colors on the same agar medium. (B) Additional morphologies of P. salivae (OMI1399). (C) P. salivae (OMI1399) after one week of incubation. (D) P. salivae (OMI1399) after two weeks of incubation, with a noticeable central bull-eyed pigmentation. (E) 1. Satellite colonies of P. salivae (small peripheral) (OMI1400) orbiting a central colony of P. melaninogenica (OMI1328). 2. Self-satellitism of P. salivae (OMI1400), depicting small peripheral colonies encompassing a larger colony of itself or the identical species. (F) 1. Magnified section of Figure E (1) distinctly showcasing the satellitism interaction between P. salivae and P. melaninogenica. 2. An enlarged section of Figure E (2), explicitly illustrating the self-satellitism phenomenon of P. salivae.

Figure 2

Growth and inhibition zones from P. salivae and P. melaninogenica cross-streaks. (A) Schaedler KV agar plate showing different P. melaninogenica strains (vertical) cross-streaked with different P. salivae strains (horizontal). (B) Enlarged intersection between P. salivae and P. melaninogenica streaks clearly showing the inhibition of P. melaninogenica in the contact zone with P. salivae.

Figure 3

Unrooted phylogenetic trees based on 16S rRNA sequences, comparing MALDI-TOF and 16S rRNA sequencing identifications. The first name indicates species identification by MALDI-TOF, while the second name represents the identification based on 16S rRNA sequence analysis. (A) Phylogenetic tree of 41 Veillonella spp. isolates (B) Phylogenetic tree of the 28 investigated Prevotella spp. isolates.

Figure 4

Comparative analysis of absolute abundance: RT-qPCR vs. 16S rRNA amplicon sequencing in both IBD patients and healthy controls for (A) Veillonella genus, (B) Prevotella genus, and (C) P. salivae. Significance levels are denoted as (*), (**), and (***) for P < 0.05, P < 0.01, and P < 0.001, respectively.