The Effect of Topical Anesthetics on 16S Ribosomal Ribonucleic Acid Amplicon Sequencing Results in Ocular Surface Microbiome Research

Abstract

Purpose To clarify the short-term effect of topical anesthetics on 16S ribosomal ribonucleic acid amplicon sequencing results in ocular surface microbiome research. Methods 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. Results 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. Conclusions 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. Translational Relevance Understanding and standardization of sampling techniques are indispensable to properly execute clinical microbiome research.

DNA extraction protocol impacts ocular surface microbiome profile

“…RNeasy scores consistently good with a low standard deviation and low interquartile range, meaning there is high consistency between the different volunteers. Of note, the relative abundances of the samples extracted with RNeasy are very similar to our previously described results where volunteers were sampled under general anaesthesia (Delbeke et al, 2022;Table 5). This underscores the repeatability of this extraction protocol and also highlights that the difference in sampling depth (soft versus firm swabbing), as earlier described, might only be a minor contributor to the retrieved sequencing differences in different publications (Dong et al, 2011).…”

“…However, in line with data from gut microbiota research, the biggest impact is expected to result from differences in DNA extraction protocols ( Costea et al, 2017 ). After reporting on the immediate effect of topical anaesthetics on ocular microbiome results ( Delbeke et al, 2022 ), we now compare different protocols for community DNA extraction. It is well-known that DNA extraction methods for complex microbial communities affect the observed species diversity downstream ( Costea et al, 2017 ; Ahannach et al, 2021 ), as Gram-negative and Gram-positive bacteria have different cell wall structures.…”

“…Comparison of relative abundances (≥1%) of the core genera as described in our systematic review [combining different extraction protocols (n = 359)](Delbeke et al, 2021) (1), the healthy controls (n = 20) from our previous publication analysed with RNeasy(Delbeke et al, 2022) (2) and the sequencing results of our current project per used extraction protocol with and without decontamination steps.…”

Validation of 16S rRNA Gene Sequencing of the Periocular Microbiome and Lack of Alteration by Topical Eyedrops

“… 26 Second, topical anesthetic was used prior to sampling to ensure patient comfort, although it has not been found to alter results of 16S rRNA analysis. 27 Finally, the swab used for 16S rRNA analysis was performed prior to swabs for conventional culture in all participants, and the impact (or lack thereof) of this approach is unknown.…”

Gut microbiota is a potential factor in shaping phenotypic variation in larvae and adults of female bumble bees

“…The alpha diversity measures such as the observed ASVs (i.e., the total number of ASVs detected per sample) and Shannon index (i.e., the number of taxa and evenness of their distribution, more influenced by the richness and rare species) were compared (Delbeke et al, 2022). The Kruskal-Wallis test was used to compare the differences among groups as some of the variables were not normally distributed.…”