Department of Ophthalmology, University Hospitals Leuven, Leuven, Belgium.
KU Leuven, Biomedical Sciences Group, Department of Neurosciences, Research Group Ophthalmology, Leuven, Belgium.
Transl Vis Sci Technol. 2022 Mar 2;11(3):2. doi: 10.1167/tvst.11.3.2.
To clarify the short-term effect of topical anesthetics on 16S ribosomal ribonucleic acid amplicon sequencing results in ocular surface microbiome research.
Both eyes of 24 eligible volunteers undergoing general anesthesia were sampled. Before sampling, a drop of artificial tears or a drop of topical anesthetic was applied in a randomized way. By using artificial tears as a control, we assured blinding of the executer and took a potential diluting effect into account. Bacterial DNA was extracted using the QIAGEN RNeasy PowerMicrobiome Kit with specific adaptations. Amplified DNA was sequenced with the Illumina MiSeq sequencing platform.
Four sample pairs were excluded due to low yield of bacterial DNA. In the remaining 20 sample pairs, no differences were observed with topical anesthetics at the levels of amplicon sequence variants (ASVs), phylum, genera, or alpha and beta diversity. Weighted UniFrac distance confirmed that the intraindividual distance between the right and left eye was smaller than the effect of the topical anesthetic. Interestingly, however, we identified Cutibacterium as a potential discriminative biomarker for topical anesthetic use. Overall, a significantly higher number of observed reads were assigned to genera with Gram-positive characteristics.
Based on our targeted, double-blinded, within-subject study, topical anesthetics do not affect the overall sequencing results but display a specific effect on Cutibacterium. When comparing research results, the impact of topical anesthetics on prevalence and abundance of Cutibacterium should be considered.
Understanding and standardization of sampling techniques are indispensable to properly execute clinical microbiome research.
阐明局部麻醉剂对眼表微生物组研究中 16S 核糖体核糖核酸扩增子测序结果的短期影响。
对 24 名接受全身麻醉的合格志愿者的双眼进行采样。在采样前，以随机方式滴加人工泪液或局部麻醉剂。通过将人工泪液作为对照，我们确保了执行者的盲法，并考虑了潜在的稀释效应。使用 QIAGEN RNeasy PowerMicrobiome 试剂盒进行细菌 DNA 提取，并进行了特定的调整。使用 Illumina MiSeq 测序平台对扩增 DNA 进行测序。
由于细菌 DNA 产量低，有 4 对样本被排除。在剩余的 20 对样本中，使用局部麻醉剂在扩增子序列变异体（ASV）、门、属或α和β多样性水平上均未观察到差异。加权 UniFrac 距离证实，个体右眼和左眼之间的个体内距离小于局部麻醉剂的影响。然而，有趣的是，我们发现表皮葡萄球菌是局部麻醉剂使用的潜在鉴别生物标志物。总体而言，观察到的读频数与革兰氏阳性特征的属的分配显著增加。
基于我们的靶向、双盲、个体内研究，局部麻醉剂不会影响整体测序结果，但对表皮葡萄球菌有特定影响。在比较研究结果时，应考虑局部麻醉剂对表皮葡萄球菌的流行和丰度的影响。
马晓莉
