Composition and Diversity of the Ocular Surface Microbiota in Patients With Blepharitis in Northwestern China

Abstract

Purpose: To investigate the composition and diversity of the microbiota on the ocular surface of patients with blepharitis in northwestern China via 16S rDNA amplicon sequencing.Methods: Thirty-seven patients with blepharitis divided into groups of anterior, posterior and mixed blepharitis and twenty healthy controls from northwestern China were enrolled in the study. Samples were collected from the eyelid margin and conjunctival sac of each participant. The V3–V4 region of bacterial 16S rDNA in each sample was amplified and sequenced on the Illumina HiSeq 2500 sequencing platform, and the differences in taxonomy and diversity among different groups were compared.Results: The composition of the ocular surface microbiota of patients with blepharitis was similar to that of healthy subjects, but there were differences in the relative abundance of each bacterium. At the phylum level, the abundances of Actinobacteria, Cyanobacteria, Verrucomicrobia, Acidobacteria, Chloroflexi, and Atribacteria were significantly higher in the blepharitis group than in the healthy control group, while the relative abundance of Firmicutes was significantly lower (p < 0.05, Mann-Whitney U). At the genus level, the abundances of Lactobacillus, Ralstonia, Bacteroides, Akkermansia, Bifidobacterium, Escherichia-Shigella, Faecalibacterium, and Brevibacterium were significantly higher in the blepharitis group than in the healthy control group, while the relative abundances of Bacillus, Staphylococcus, Streptococcus, and Acinetobacter were significantly lower in the blepharitis group (p < 0.05, Mann-Whitney U). The microbiota of anterior blepharitis was similar to that of mixed blepharitis but different from that of posterior blepharitis. Lactobacillus and Bifidobacterium are biomarkers of posterior blepharitis, and Ralstonia is a biomarker of mixed blepharitis. There was no significant difference in the ocular surface microbiota between the eyelid margin and conjunctival sac with or without blepharitis.Conclusion: The ocular surface microbiota of patients with blepharitis varied among different study groups, according to 16S rDNA amplicon sequencing analysis. The reason might be due to the participants being from different environments and having different lifestyles. Lactobacillus, Bifidobacterium, Akkermansia, Ralstonia, and Bacteroides may play important roles in the pathogenesis of blepharitis.

DNA extraction protocol impacts ocular surface microbiome profile

“…Bacillus could not be extracted with all DNA extraction protocols, which is in line with our earlier publication (Delbeke et al, 2021), stating that Bacillus was present with a high abundance in only certain publications (Zhou et al, 2014;Ozkan et al, 2017;Ozkan et al, 2019;Wang et al, 2021). We extracted Bacillus with NucleoSpin, Blood & Tissue and to a far lesser extent with PowerSoil; which is consistent with Ozkan et al and Wang et al retrieving Bacillus using different Macherey -Nagel NucleoSpin protocols (Ozkan et al, 2017(Ozkan et al, , 2019Wang et al, 2021) and Zhou et al (2014) using the PowerSoil extraction protocol. Interestingly and as briefly stated above, there is a large drop in relative abundance of Bacillus in the NucleoSpin (45-1%) and Blood & Tissue (8-2%) analysed samples after removing all ASVs retrieved with decontam (NucleoSpin n = 246, Blood & Tissue n = 203).…”

“…Those 18 genera were Cutibacterium, Staphylococcus, Streptococcus, Corynebacterium_1, Acinetobacter, Pseudomonas, Lawsonella, Finegoldia, Anaerococcus, Ezakiella, Peptoniphilus, Actinomyces, Enhydrobacter, Haemophilus, Corynebacterium, Neisseria, Lactobacillus, Micrococcus ; with the first 6 being the core bacteria ( Delbeke et al, 2021 ). Bacillus could not be extracted with all DNA extraction protocols, which is in line with our earlier publication ( Delbeke et al, 2021 ), stating that Bacillus was present with a high abundance in only certain publications ( Zhou et al, 2014 ; Ozkan et al, 2017 ; Li Z. et al, 2019 ; Li S. et al, 2019 ; Ozkan et al, 2019 ; Wang et al, 2021 ). We extracted Bacillus with NucleoSpin, Blood & Tissue and to a far lesser extent with PowerSoil; which is consistent with Ozkan et al and Wang et al retrieving Bacillus using different Macherey – Nagel NucleoSpin protocols ( Ozkan et al, 2017 , 2019 ; Wang et al, 2021 ) and Zhou et al (2014) using the PowerSoil extraction protocol.…”

“…Based on both abundance levels ( Figure 6 and Table 5 ) and results of the LEfSe analyses ( Table 1 ); Bacillus has a high abundance in the NucleoSpin (relative abundance 45%), and Blood & Tissue (relative abundance 8%) extracted samples. Extrapolating our observation with published literature confirmed a Bacillus tendency of different Macherey – Nagel NucleoSpin protocols (6%: Ozkan et al, 2019 ; 2%: Wang et al, 2021 ). Ozkan et al (2019) and Wang et al (2021) used the NucleoSpin Tissue XS kit and NucleoSpin 96 Soil DNA Isolation Kit, respectively.…”

“…Extrapolating our observation with published literature confirmed a Bacillus tendency of different Macherey – Nagel NucleoSpin protocols (6%: Ozkan et al, 2019 ; 2%: Wang et al, 2021 ). Ozkan et al (2019) and Wang et al (2021) used the NucleoSpin Tissue XS kit and NucleoSpin 96 Soil DNA Isolation Kit, respectively. The NucleoSpin Tissue and the NucleoSpin Tissue XS kit have the same buffer chemistry and workflow; the only difference lays in the elution buffer [which we have lowered in our set-up to the same amount as used in the XS kit ( Supplementary 1 )] and the reduced surface of the membrane.…”

“…Microbial DNA was extracted from frozen samples. Commercially available DNA extraction protocols were selected based on previous publications in the field of the ocular surface microbiome: DNeasy PowerSoil kit (QIAGEN, Hilden, Germany) (PowerSoil) ( Dong et al, 2011 ; Zhou et al, 2014 ; Shin et al, 2016 ; Cavuoto et al, 2018 ), DNeasy Blood and Tissue kit (QIAGEN, Hilden, Germany) (Blood & Tissue) ( Doan et al, 2016 ; Chao et al, 2018 ), NucleoSpin Tissue (MACHEREY NAGEL, Dueren, Germany) (NucleoSpin) ( Ozkan et al, 2017 , 2018 , 2019 ; Wang et al, 2021 ; Yang et al, 2022 ), RNeasy PowerMicrobiome Kit (QIAGEN, Hilden, Germany) (RNeasy) ( Cavuoto et al, 2018 ), FastDNA TM Spin Kit for Soil (MP, Illkirch-Graffenstaden, France) (FastDNA) ( Graham et al, 2007 ). Two host DNA depletion protocols were added based on their reported effective host DNA depletion without significant reduction in bacterial DNA concentration (QIAamp DNA microbiome kit (QIAGEN, Hilden, Germany) (QIAamp) ( Heravi et al, 2020 ) and HostZERO Microbial DNA kit [ZYMO research, California, United States of America) (HostZERO)] ( Ahannach et al, 2021 ).…”