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.

本研究的目的是评估吸烟对受III/IV期及广泛性牙周病影响的年轻患者龈下代谢转录组谱的影响。选取了包括吸烟者与非吸烟者（每组n=3）在内的6名患有牙周炎的年轻患者，并遵循STROBE（加强流行病学观察性研究报告）指南进行病例对照报告。收集了牙周临床测量指标和龈下生物膜样本。从生物膜中提取RNA并通过Illumina HiSeq进行测序。差异表达分析采用了京都基因与基因组百科全书（KEGG）富集分析，并使用R中的Sleuth包识别差异表达基因，统计阈值设定为≤0.05。两组龈下生物膜中共鉴定出3351个KEGG，吸烟习惯改变了龈下生物膜的功能行为，导致两组间有304个差异表达的KEGG。此外，七条通路受到调节：糖类降解、半乳糖代谢、糖胺聚糖降解、氧化磷酸化、肽聚糖生物合成、丁酸代谢和糖鞘脂生物合成。此外，吸烟还通过β-内酰胺酶、通透性、抗生素外排泵和抗生素耐药合成酶相关基因的显著过表达，改变了龈下生物膜中抗生素耐药基因（ARGs）的水平。鉴于样本量较小的局限性，我们的数据表明，吸烟可能影响龈下生物膜的功能行为，调节对龈下生物膜行为产生负面影响的通路，可能导致更具致病性的菌群。