背景:在这项研究中,我们首次使用metaranscriptomics来研究微生物组成,功能签名,以及牙髓感染中抗菌素耐药性(AR)基因的表达。
方法:从十颗牙齿收集根管样本,包括5例原发性和5例持续性/继发性牙髓感染。从牙髓样品中提取RNA,并且在NovaSeq6000系统(Illumina)上进行RNA测序。使用Kraken2细菌数据库进行分类分析。然后,具有分类学分类的序列针对通用蛋白质知识库进行了注释,以进行功能注释,并针对综合抗生素抗性数据库进行了AR样基因鉴定。
结果:变形杆菌,拟杆菌,Firmicutes,放线菌代表优势门,而镰刀菌,螺旋藻,而Synergistetes是非优势门。前十种主要以专性(或准专性)厌氧菌为代表,包括革兰氏阴性(例如,Capnocytophagasp.口服分类单元323,具核梭杆菌,中间介体普雷沃特拉,Prevotellaoris,连翘制革菌和连翘制革菌。口服分类单元HOT-286)和革兰氏阳性物种(例如,Olsenellauli和Parvimonasmicra)。编码月光蛋白的转录本(例如,糖酵解蛋白,平移伸长因子,伴娘,和热休克蛋白)高表达,可能影响细菌粘附,生物膜的形成,主机防御逃避,和炎症诱导。牙髓细菌表达的基因赋予对牙科常用抗生素类的抗性,四环素和lincosamide耐药基因的高流行和高表达。抗生素外排和抗生素靶标改变/保护是主要的耐药机制。
结论:转移转录组学揭示了潜在的牙髓病原体的活性,它们表达了推定的毒力因子和可能与抗微生物药物耐药性有关的广泛基因。
BACKGROUND: In this study, we used metatranscriptomics for the first time to investigate microbial composition, functional signatures, and antimicrobial resistance gene expression in endodontic infections.
METHODS: Root canal samples were collected from ten teeth, including five primary and five persistent/secondary endodontic infections. RNA from endodontic samples was extracted, and RNA sequencing was performed on a NovaSeq6000 system (Illumina). Taxonomic analysis was performed using the Kraken2 bacterial database. Then, sequences with a taxonomic classification were annotated against the Universal Protein Knowledgebase for functional annotation and the Comprehensive Antibiotic Resistance Database for AR-like gene identification.
RESULTS: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria represented the dominant phyla, whereas Fusobacteria, Spirochetes, and Synergistetes were among the nondominant phyla. The top ten species were mainly represented by obligate (or quasiobligate) anaerobes, including Gram-negative (eg, Capnocytophaga sp. oral taxon 323, Fusobacterium nucleatum, Prevotella intermedia, Prevotella oris, Tannerella forsythia, and Tannerella sp. oral taxon HOT-286) and Gram-positive species (eg, Olsenella uli and Parvimonas micra). Transcripts encoding moonlighting proteins (eg, glycolytic proteins, translational elongation factors, chaperonin, and heat shock proteins) were highly expressed, potentially affecting bacterial adhesion, biofilm formation, host defense evasion, and inflammation induction. Endodontic bacteria expressed genes conferring resistance to antibiotic classes commonly used in dentistry, with a high prevalence and expression of tetracycline and lincosamide resistance genes. Antibiotic efflux and antibiotic target alteration/protection were the main resistance mechanisms.
CONCLUSIONS: Metatranscriptomics revealed the activity of potential endodontic pathogens, which expressed putative virulence factors and a wide diversity of genes potentially involved in AR.