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The composition and relative abundance of bacterial species change throughout the development of dental caries; however, how these changes relate to clinical symptoms remains elusive. In this study, we explored the relationship between clinical symptoms and specific microorganisms in advanced dentinal caries.A total of 111 permanent premolars and molars were used to simulate the progression from caries to pulpitis indirectly. Clinical symptoms were evaluated, and teeth were diagnosed according to the diagnostic criteria of the American Association of Endodontics. Samples were collected for 16S ribosomal DNA sequencing. Associations between the microbiota and clinical symptoms/diagnosis and the relationship between alpha diversity and clinical symptoms/diagnosis were evaluated independently by the linear discriminant analysis effect size and Spearman rank correlation analyses.The 16S ribosomal DNA sequences were assigned to 13,852 operational taxonomic units. The linear discriminant analysis effect size and Spearman correlations unveiled negative associations between the relative abundance of Bacteroidia and Gammaproteobacteria and referred pain, Gammaproteobacteria and the electric pulp test response, and Actinomyces and Propionibacterium and diagnosis (r < 0.0, P < .05). Conversely, the relative abundance of Lactobacillus was positively correlated to referred pain, the cold test, the percussion response, and diagnosis (r > 0, P < .05). Lactobillus reuteri, a probiotic bacterium, was more abundant in teeth with referred pain and teeth diagnosed with symptomatic irreversible pulpitis. The Spearman correlation between alpha diversity and clinical symptoms/diagnosis was not significant (P > .05).Clinical symptoms and diagnosis were significantly associated with specific microorganisms in the most advanced layers of dentinal caries.