Overview of microbiome in all patients:
To our knowledge, this is the first study trying to identify bacterial composition in human conjunctivitis using 16S rRNA metagenome.
Culture-independent approach by 16S rRNA metagenome sequencing has been used to identify our microbiota during both healthy condition and illness.
In a smaller study using only 4 healthy volunteers, Dong et al. identified a core conjunctival microbiome of five bacterial phyla using 16S rRNA sequencing, three of which [Proteobacteria (64%), Actinobacteria (19.6%), and Firmicutes (3.9%)] accounted for > 87.9% of all sequences . The other two phyla Cyanobacteria and Bacteroidetes were found in contamination-level quantities (0.21% and 0.16%, respectively) . In another larger study (31 subjects), Huang et al. using the 16S rRNA gene sequencing reads classified the conjunctival microbiome into 25 bacterial phyla. Most sequences (98.88%) were affiliated predominantly with five phyla, which included Proteobacteria (46.50%), Actinobacteria (33.89%), Firmicutes (15.50%), Bacteroidetes (2.28%), and DeinococcuseThermus (0.71%) .
Although Firmicutes constituted the least abundance in their studies among the main first three phyla (Proteobacteria, Actinobacteria, Firmicutes), but in our study is the highest one (one of most significant results in this study).
In another study, the conjunctiva of 45 healthy subjects were sampled at three time points over three months with the aim of understanding whether the microbial communities of the ocular surface (OS) change over time. They determined that the majority of phyla at each time point consisted of Proteobacteria (range 52–73%), Firmicutes (13–20%) and Actinobacteria (8–22%) . Thus, our study is the first to identify that Firmicutes is the main predominant phylum in conjunctival microbiome in conjunctivitis cases. It is difficult to explain the reason why Phylum Firmicutes is predominant in the patients with conjunctivitis. However, the consumption of oxygen by growth of causative bacteria including facultative aerobes may provide anaerobic condition, which has the advantage for Phylum Firmicutes.
In comparison between our study and other previous normal studies, as regards to predominant genera (> 1%), we determined three groups. The first group includes the predominant genera shared with other normal studies [11–14]. This group includes Streptococcus, Cutibacterium, Staphylococcus, Corynebacterium, and Acinetobacter. The second group includes genera only present in our study and not present in normal previous studies. This group includes Nocardioides, Anoxybacillus, Janibacter, Porphyromonas, Bacillus, Clostridium sensu stricto 7, and Haemophilus. Last group includes predominant genus which is present in normal studies and absent in our study as Pseudomonas [11–13, 15].
Advances in next-generation sequencing and bioinformatics tools have revealed an expansive and diverse microbial community inhabiting the human conjunctiva. The most abundant genera identified using 16S rRNA sequencing were Pseudomonas, Bradyrhizobium, Cutibacterium, Acinetobacter, and Corynebacterium [15, 16].
It has been suggested that these “normal bacteria” serve a protective role under most circumstances by directly inhibiting colonization of more pathogenic species . Many previous studies confirmed that Pseudomonas represented a major genus in healthy conjunctiva [11–13]. Lee et al. compared between the ocular microbial communities with and without blepharitis . They confirmed that the relative proportions of Staphylococcus, Streptophyta, Corynebacterium, and Enhydrobacter were higher in subjects with blepharitis than in healthy subjects . However, the proportions of Pseudomonas clearly was lower in subjects with blepharitis than in healthy subjects, suggesting that Pseudomonas might be an important as resident commensals microbiota for the prevention of blepharitis .
In our study, we confirmed that Pseudomonas was present in scanty percentage in cases of conjunctivitis and agreed with previous study in their suggestion about the importance of Pseudomonas as resident commensals microbiota for prevention of infection.
For the ocular microbiota during purulent conjunctivitis, studies using previous methods such as the denaturing gradient gel electrophoresis (DGGE) and the clone library methods have revealed that genera Staphylococcus, Corynebacterium, and Cutibacterium were commonly observed [19, 20]. However, there is a bias in these studies because the findings were basically based on the database of clinically-known bacteria. Thus, our study provided the composition of ocular microbiota including uncultured bacteria with reducing the methodological selection bias.
The diversity of microbial communities in the subjects was assessed with alpha diversity analysis. The observed genesis and Shannon index were used to evaluate the richness and biodiversity of the microbiota. Beta diversity refers to species diversity among different groups. Beta diversity and alpha diversity together constitute the biological heterogeneity of overall diversity or a certain community or group. The beta diversities in different groups were calculated using the Unweighted UniFrac distances of 16S rRNA genes between microbial communities or groups.
In our study, there were no difference in Beta diversity analysis for sex (P = 0.087), chemosis (P = 0.064), unclassified eye drops (P = 0.431), pain (P = 0.315) and itching (P = 0.133). There was a statistical significant difference between bilateral versus unilateral conjunctivitis (P = 0.017) and for antibiotics (P = 0.020) (Fig. 3A, B), although there were no statistical significant differences in these factors using Shannon index (Alpha diversity analysis). These results, to our knowledge, considered the first diversity analysis for subjects with conjunctivitis in the literatures.
The rate for the culture method has been reported to be between 47.5% and 97.8% in eyes with bacterial conjunctivitis [19–21] and 9.0–90.6% [6, 7, 12, 22–24] in normal conjunctival sacs.
To overcome culture limitations, there has been an increase in the number of studies using molecular methods, for example, PCR with species-specific primers [25–27], amplification of the 16S rRNA gene by PCR using universal primer sets followed by direct sequencing [28–30], DGGE , and pyrosequencing .
16S rRNA sequencing has been used for bacterial identification and discovery of novel genera leading to extending our knowledge about OS microbial diversity [4, 11, 12, 14].
In our study, we had only 4 (8.5%) positive conjunctival cultures and all of them were congruous with abundant genera with 16S rRNA sequencing analysis. From our point of view, if the result of 16S rRNA sequencing analysis denoting abundance of one or more genera in the conjunctivitis sample, most probably it is/are the causative pathogen especially if the culture is congruous with it.