Demographic characteristics of the patients (age, gender and disease type) were shown in Table 1. There was no significant difference between each group (P>0.05).
OTU stand for operational taxonomic unit (strain, genus, species, grouping, etc.), which is used to classify groups of closely related individuals. Sequences with >97% similarity are grouped into OTUs. Group A had a total OTU number of 973 (369 unique); group B1 had a total OTU number of 760 (213 unique); group B2 had a total OTU number of 668 (166 unique). The number of OTUs with overlap among the three groups was 357. The number of OTUs in Group A was higher than that in Group B1, and the number of OTUs in Group B1 was higher than that in Group B2. These findings indicate that ocular surface microbial community diversity was inversely correlated with the perioperative managements and the number of intravitreal injections administered (Figure 1a). The results of PLS-DA analysis based on OTU abundance revealed the similar changes about the microbiota on ocular surface among Group A, Group B1, and Group B2 (Figure 1b).
Microbial diversity analysis
After obtaining the results of OTU clustering analysis, Ace, Chao, Simpson, and Shannon evaluation indexes were used to analyze the alpha diversity of all samples. There was no statistically significant difference among each sample in microbial community alpha diversity (P>0.05) (Figure 2a), indicating that there was no statistically significant difference in microbial community composition in each group.
Beta diversity analysis was performed to measure differences among multiple groups. The results of beta diversity analysis revealed a significant difference in composition of microbiota on ocular surface between Groups A, B1 and B2 (P=1.84×10-5) (Figure 2b).
Species composition of the conjunctival microbiota
In Figure 3, species abundance was compared among the groups at the phylum, class, order, family, genus, and species levels. All of the microbiota in each group and each level were shown in the left. We selected the top three microbiota in Group A and analyzed their variations in three groups. The results were shown by histogram in the middle. Furthermore, we compared each microbiota in different groups, and found out the top five microbiota that increased (Red) and decreased (Green) significantly. We ranked the microbiota by the results of fold changes (B2/A). These results were shown in the right table.
In Group B1, WPS-2 increased for 54.27 folds and S24-7 increased greatest for 11.1 folds. Furthermore, Streptococcus_euqi dramatically increased for 61088.15 folds. In Group B2，Streptococcus_euqi increased for 298 folds. Instead, Synergistetes decreased for 0.08 fold in Group B1 and 0.12 fold in Group B2. Deinococci, Deinococcales, Deinococcaceae and Deinococcus were all significantly decreased in Group B1 and B2. Overall, microbiota on the ocular surface was obviously changed after perioperative managements. S24-7 and Streptococcus_euqi were gram-negative bacteria. Synergistetes, Deinococci, Deinococcales, Deinococcaceae and Deinococcus were all gram-positive bacteria.
Analysis of species differences
The LefSe histogram shows the microorganisms with significantly differential abundance in Group A, Group B1, and Group B2. Column length represents relative abundance of a given species (Figure 4a)11, 12. The bacteria that were significantly more common in Group B2 were Alphaproteobacteria, Sphingomonadales, Sphingomonadaceae, Rhizobiales, Rhizobiaceae, Trueperaceae, Proteus, Aquabacterium, Oxalobacteraceae, Pelomonas, and Sphingopyxis. Facklamia was significantly more common in Group B1. Mollicutes, Tenericutes, and Moraxella were significantly more common in Group A. At the phylum level, the bacteria with greatest abundance in Group A were Mollicutes, while the main bacteria in Group B2 were Alphaproteobacteria. The results showed that antibiotic treatment altered the dominant and core bacterial species present on the ocular surface. This finding is consistent with the cladogram of species abundance LDA (Figure 4b).