背景:大多数关于圆盘中细菌菌群的文献在利用传统培养方法和靶向单一细菌方面处于不利地位,粉刺杆菌。
目的:我们的目的是使用下一代基因组工具记录正常和退化椎间盘之间细菌菌群的多样性,以入围潜在病原体。
方法:实验病例对照研究方法:采用16S宏基因组测序来分析来自脑死亡器官自愿供体(n=20)和40个退化椎间盘的MRI正常健康椎间盘中的细菌多样性。手术(Modic(MC)=20和Non-Modic(NMC)=20)。使用通用细菌引物341F和806R扩增V3-V4区,并且使用ILUMINANOVOSEQ6000平台对文库进行测序。在具有最少100OTU(操作分类单位)的细菌处设置统计显著性,并且存在于至少70%的样品中。使用QIIME-2流水线处理QC过滤的读段。使用Greengenes/SILVA参考数据库对合并的读段进行OTU聚类和分类学分类。通过使用LC-MS/MS方法鉴定样品中的细菌代谢物来进行验证。
结果:丰富的细菌在多样性上差异很大,正如Alpha和Beta多样性分析所证明的那样,存在于所有对照和变性样品中。对照组细菌属数量为27个(14-革兰氏阳性:13-革兰氏阴性),Modic组中的23(10-革兰氏阳性:11-革兰氏阴性),和16(11-革兰氏阳性:5-革兰氏阴性)在非Modic组中。在Modic组中,发现革兰氏阴性菌OTU占优势(占鉴定的总细菌的50%以上),而在对照组和非Modic组中,革兰氏阳性菌的OTU占优势。物种水平分析显示了大量的机会性革兰阴性病原体,如铜绿假单胞菌,Sphingomonospaucibacillus,光盘中的五重杆菌和Modic变化,超过非Modic光盘。细菌代谢物和群体感应分子如N-癸基-L-高丝氨酸内酯的存在,6-羟基烟酸,2-氨基苯乙酮,4-羟基-3-聚异戊烯基苯甲酸酯,PE(16:1(9Z)/18:0)和邻苯二甲酸验证了椎间盘中细菌的定植和细胞间通讯,排除了污染理论。痤疮杆菌并不是三组圆盘中的任何一组中的主要细菌,实际上在对照圆盘中的丰度顺序位于第16位(0.72%),在Modic光盘中排名第七(1.41%),在非Modic光盘中排名第12位(0.53%)。
结论:这项研究发现,在退化的椎间盘中,革兰氏阴性菌占优势,并强调痤疮梭菌可能不是唯一的引起退化的细菌。这可能归因于环境,饮食,和样本人群的生活习惯。虽然这项研究没有揭示确切的病原体,这可能为未来的研究铺平道路。
结论:这些发现要求进一步研究细菌谱与椎间盘退变表型的因果关系以及表型驱动的临床治疗方案。
The majority of literature on bacterial flora in the disc stands disadvantaged in utilizing traditional culture methods and targeting a single bacterium, Cutibacterium acnes.
Our objective was to document the diversity in the bacterial flora between normal and degenerated discs for shortlisting potential pathogens using next-generation genomic tools.
Experimental case-control
study.
Researchers employed 16S metagenome sequencing to profile bacterial diversity in magnetic resonance imaging normal healthy discs from brain-dead organ voluntary donors (n=20) and 40 degenerated disc samples harvested during surgery (Modic [MC]=20 and non-Modic [NMC]=20). The V3-V4 region was amplified using universal bacterial primers 341F and 806R, and the libraries were sequenced using Illumina NovoSeq 6000 platform. Statistical significance was set at bacteria with a minimum of 100 operational taxonomic unit (OTU) and present in at least 70% of the samples. The quality check-filtered reads were processed using the QIIME-2 pipeline. The OTU clustering and taxonomic classification were carried out for the merged reads using the Greengenes/SILVA reference database. Validation was done by identification of bacterial metabolites in samples using the liquid chromatography-mass spectrometry approach.
Abundant bacteria differing widely in diversity, as evidenced by Alfa and Beta diversity analysis, were present in all control and degenerative samples. The number of bacterial genera was 27 (14-gram-positive: 13-gram-negative) in the control group, 23 (10-gram-positive: 11-gram-negative) in the Modic group, and 16 (11-gram-positive: 5-gram-negative) in the non-Modic group. In the Modic group, gram-negative bacteria OTUs were found to be predominant (more than 50% of the total bacteria identified), whereas in control and non-Modic groups the OTUs of gram-positive bacteria were predominant. Species-level analysis revealed an abundance of opportunistic gram-negative pathogens like Pseudomonas aeruginosa, Sphingomonos paucibacillus, and Ochrobactrum quorumnocens in the discs with Modic changes, more than in non-Modic discs. The presence of bacterial metabolites and quorum-sensing molecules like N-decanoyl-L-homoserine lactone, 6-hydroxynicotinic acid, 2-aminoacetophenone, 4-hydroxy-3-polyprenylbenzoate, PE (16:1(9Z)/18:0) and phthalic acid validated the colonization and cell-cell communication of bacteria in disc ruling out contamination theory. Cutibacterium acnes was not the predominant bacteria in any of the three groups of discs and in fact was in the 16th position in the order of abundance in the control discs (0.72%), seventh position in the Modic discs (1.41%), and 12th position (0.53%) in the non-Modic discs.
This
study identified a predominance of gram-negative bacteria in degenerated discs and highlights that Cutibacterium acnes may not be the only degeneration-causing bacteria. This may be attributed to the environment, diet, and lifestyle habits of the sample population. Though the
study does not reveal the exact pathogen, it may pave the way for future studies on the subject.
These findings invite further investigation into causal relationships of bacterial profile with disc degeneration phenotypes as well as phenotype-driven clinical treatment protocols.