Metatranscriptomic analysis unveils functional alterations in subgingival biofilm of young smokers with periodontitis

The objective was to assess the influence of smoking on the subgingival metatranscriptomic profile in young patients affected by stage III/IV and generalized periodontal disease. Six young patients affected by periodontitis, including both smokers and non-smokers (n=3/group), were selected, following STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for case-control reporting. Periodontal clinical measurements and subgingival biofilm samples were collected. RNA was extracted from biofilm and sequenced through Illumina HiSeq. Differential expression analysis utilized Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and differentially expressed genes were identified using the Sleuth package in R with a statistical cutoff of ≤0.05. A total of 3351 KEGGs were identified in the subgingival biofilm of both groups, and smoking habits altered the functional behavior of the subgingival biofilm, resulting in 304 differentially expressed KEGGs between groups. Moreover, seven pathways were modulated: glycan degradation, galactose metabolism, glycosaminoglycan degradation, oxidative phosphorylation, peptidoglycan biosynthesis, butanoate metabolism, and glycosphingolipid biosynthesis. Besides, smoking also altered the Antibiotic Resistance Genes (ARGs) levels in subgingival biofilm through the significant over-expression of genes related to beta-lactamase, permeability, antibiotic efflux pumps, and antibiotic-resistant synthetases. Given the limitations of a small sample size, our data suggests that smoking may influence the functional behavior of the subgingival biofilm, modifying pathways that negatively impact the behavior of the subgingival biofilm, potentially leading to a more virulent community.

本研究旨在评估吸烟对患有III/IV期广泛性牙周病的年轻患者龈下宏转录组谱（subgingival metatranscriptomic profile）的影响。研究选取6名牙周炎年轻患者，其中吸烟组与非吸烟组各3例，遵循STROBE（流行病学观察性研究报告规范，Strengthening the Reporting of Observational Studies in Epidemiology）的病例对照研究报告指南。收集牙周临床指标及龈下生物膜样本后，从生物膜中提取RNA并通过Illumina HiSeq进行测序。差异表达分析采用京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）富集法，差异表达基因通过R语言的Sleuth包鉴定，统计阈值设定为≤0.05。结果显示，两组龈下生物膜中共鉴定出3351个KEGG条目；吸烟习惯改变了龈下生物膜的功能特征，导致组间出现304个差异表达KEGG条目。此外，7条通路受到调控，包括聚糖降解、半乳糖代谢、糖胺聚糖降解、氧化磷酸化、肽聚糖生物合成、丁酸代谢及糖鞘脂生物合成。同时，吸烟还通过显著上调β-内酰胺酶、通透性、抗生素外排泵及抗生素耐药合成酶相关基因的表达，改变了龈下生物膜中抗生素耐药基因（Antibiotic Resistance Genes，ARGs）的水平。尽管存在样本量较小的局限性，本研究数据表明，吸烟可能影响龈下生物膜的功能特征，通过调控相关通路对其行为产生负面影响，进而可能形成更具毒性的群落。