Acanthamoeba species are among the most common free-living amoeba and ubiquitous protozoa, mainly distributed in water and soil, and cause Acanthamoeba keratitis (AK) and severe visual impairment in patients. Although several studies have reported genomic characteristics of Acanthamoeba, limited sample sizes and sources have resulted in an incomplete understanding of the genetic diversity of Acanthamoeba from different sources. While endosymbionts exert a significant influence on the phenotypes of Acanthamoeba, including pathogenicity, virulence, and drug resistance, the species diversity and functional characterization remain largely unexplored. Herein, our study sequenced and analyzed the whole genomes of 19 Acanthamoeba pathogenic strains that cause AK, and by integrating publicly available genomes, we sampled 29 Acanthamoeba strains from ocular, environmental, and other sources. Combined pan-genomic and comparative functional analyses revealed genetic differences and evolutionary relationships among the different sources of Acanthamoeba, as well as classification into multiple functional groups, with ocular isolates in particular showing significant differences that may account for differences in pathogenicity. Phylogenetic and rhizome gene mosaic analyses of ocular Acanthamoeba strains suggested that genomic exchanges between Acanthamoeba and endosymbionts, particularly potential antimicrobial resistance genes trafficking including the adeF, amrA, and amrB genes exchange events, potentially contribute to Acanthamoeba drug resistance. In conclusion, this study elucidated the adaptation of Acanthamoeba to different ecological niches and the influence of gene exchange on the evolution of ocular Acanthamoeba genome, guiding the clinical diagnosis and treatment of AK and laying a theoretical groundwork for developing novel therapeutic approaches. IMPORTANCE Acanthamoeba causes a serious blinding keratopathy, Acanthamoeba keratitis, which is currently under-recognized by clinicians. In this study, we analyzed 48 strains of Acanthamoeba using a whole-genome approach, revealing differences in pathogenicity and function between strains of different origins. Horizontal transfer events of antimicrobial resistance genes can help provide guidance as potential biomarkers for the treatment of specific Acanthamoeba keratitis cases.

Enterococcus faecalis is one of the main pathogens that causes hospital-acquired infections because it is intrinsically resistant to some antibiotics and often is capable of biofilm formation, which plays a critical role in resisting the external environment. Therefore, attacking biofilms is a potential therapeutic strategy for infections caused by E. faecalis . Current research indicates that diacerein used in the treatment of osteoarthritis showed antimicrobial activity on strains of gram-positive cocci in vitro. In this study, we tested the MICs of diacerein using the broth microdilution method, and successive susceptibility testing verified that E. faecalis is unlikely to develop resistance to diacerein. In addition, we obtained a strain of E. faecalis HE01 with strong biofilm-forming ability from an eye hospital environment and demonstrated that diacerein affected the biofilm development of HE01 in a dose-dependent manner. Then, we explored the mechanism by which diacerein inhibits biofilm formation through qRT-PCR, extracellular protein assays, hydrophobicity assays and transcriptomic analysis. The results showed that biofilm formation was inhibited at the initial adhesion stage by inhibition of the expression of the esp gene, synthesis of bacterial surface proteins and reduction in cell hydrophobicity. In addition, transcriptome analysis showed that diacerein not only inhibited bacterial growth by affecting the oxidative phosphorylation process and substance transport but also inhibited biofilm formation by affecting secondary metabolism, biosynthesis, the ribosome pathway and luxS expression. Thus, our findings provide compelling evidence for the substantial therapeutic potential of diacerein against E. faecalis biofilms.

In human medicine, antibiotics have been widely used to treat microbial infections. The extensive use of antibiotics is a leading cause of antibiotic resistance. Currently, the influence of the use of antibiotics on the ocular surface microbiome in the course of keratitis treatment remains to be explored in depth. We performed metagenomic analyses in a cohort of 26 healthy controls (HCs), 28 keratitis patients (KPs) who received antibiotics [KP (abx+) group], and 12 KPs who were antibiotic naive [KP (abx-) group]. We identified that the dissimilarities in microbial community structure (Bray-Curtis and Jaccard analyses) between the KP (abx+) group and the HC group were greater than those between the KP (abx-) group and the HC group. Pseudomonas lactis, P. aeruginosa, Pseudomonas sp. FDAARGOS_380, Pseudomonas sp. J380, Corynebacterium simulans, Streptococcus pyogenes, Finegoldia magna, and Aspergillus oryzae had no statistically significant differences between the KP (abx+) and KP (abx-) groups but did have statistically significant differences between the KP (abx+) and HC groups and between the KP (abx-) and HC groups. Among them, Pseudomonas lactis, P. aeruginosa, Pseudomonas sp. FDAARGOS_380, and Pseudomonas sp. J380 were identified as possible hosts carrying multidrug-resistant genes. The total abundance and number of antibiotic resistance genes (ARGs) were greater in the KP (abx+) group than in the HC and KP (abx-) groups. The functional profile analysis of ocular surface microbiota revealed that pathogenesis-related functional pathways and virulence functions were enriched in KPs. In conclusion, our results show that empirical antibiotic treatment in KPs leads to increases in the antibiotic resistance of ocular surface microbiota. IMPORTANCE Treatment for keratitis is based on appropriate antimicrobial therapy. A direct correlation between antibiotic use and the extent of antibiotic resistance has been reported. Therefore, knowledge of the antibiotic resistance patterns of ocular surface microbial flora in KPs is important for clinical treatment. To the best of our knowledge, this is the first study to use metagenomic approaches to investigate the associations between ophthalmic antibiotic use and the ocular surface microbiome of KPs. Monitoring the microbiota and antibiotic resistome profiles for the ocular surface has huge potential to help ophthalmologists choose the appropriate antibiotics and will thereby improve the efficacy of treatment regimens, which has important implications for reducing the development of antibiotic resistance of the ocular surface to a certain extent.

Acanthamoeba species are among the most ubiquitous protists that are widespread in soil and water and act as both a replicative niche and vectors for dispersal. They are the most important human intracellular pathogens, causing Acanthamoeba keratitis (AK) and severely damaging the human cornea. The sympatric lifestyle within the host and amoeba-resisting microorganisms (ARMs) promotes horizontal gene transfer (HGT). However, the genomic diversity of only A. castellanii and A. polyphaga has been widely studied, and the pathogenic mechanisms remain unknown. Thus, we examined 7 clinically pathogenic strains by comparative genomic, phylogenetic, and rhizome gene mosaicism analyses to explore amoeba-symbiont interactions that possibly contribute to pathogenesis. Genetic characterization and phylogenetic analysis showed differences in functional characteristics between the "open" state of T3 and T4 isolates, which may contribute to the differences in virulence and pathogenicity. Through comparative genomic analysis, we identified potential genes related to virulence, such as metalloprotease, laminin-binding protein, and HSP, that were specific to the genus Acanthamoeba. Then, analysis of putative sequence trafficking between Acanthamoeba and Pandoraviruses or Acanthamoeba castellanii medusaviruses provided the best hits with viral genes; among bacteria, Pseudomonas had the most significant numbers. The most parsimonious evolutionary scenarios were between Acanthamoeba and endosymbionts; nevertheless, in most cases, the scenarios are more complex. In addition, the differences in exchanged genes were limited to the same family. In brief, this study provided extensive data to suggest the existence of HGT between Acanthamoeba and ARMs, explaining the occurrence of diseases and challenging Darwin's concept of eukaryotic evolution. IMPORTANCEAcanthamoeba has the ability to cause serious blinding keratitis. Although the prevalence of this phenomenon has increased in recent years, our knowledge of the underlying opportunistic pathogenic mechanism maybe remains incomplete. In this study, we highlighted the importance of Pseudomonas in the pathogenesis pathway using comprehensive a whole genomics approach of clinical isolates. The horizontal gene transfer events help to explain how endosymbionts contribute Acanthamoeba to act as an opportunistic pathogen. Our study opens up several potential avenues for future research on the differences in pathogenicity and interactions among clinical strains.

Coagulase-negative Staphylococcus (CoNS) is the most common pathogen causing traumatic endophthalmitis. Among which, Staphylococcus epidermidis is the most common species that colonizes human skin, eye surfaces, and nasal cavity. It is also the main cause of nosocomial infection, specially foreign body-related bloodstream infections (FBR-BSIs). Although some studies have reported the genome characteristics of S. epidermidis, the genome of ocular trauma-sourced S. epidermidis strain and a comprehensive understanding of its pathogenicity are still lacking. Our study sequenced, analyzed, and reported the whole genomes of 11 ocular trauma-sourced samples of S. epidermidis that caused traumatic endophthalmitis. By integrating publicly available genomes, we obtained a total of 187 S. epidermidis samples from healthy and diseased eyes, skin, respiratory tract, and blood. Combined with pan-genome, phylogenetic, and comparative genomic analyses, our study showed that S. epidermidis, regardless of niche source, exhibits two founder lineages with different pathogenicity. Moreover, we identified several potential biomarkers associated with the virulence of S. epidermidis, including essD, uhpt, sdrF, sdrG, fbe, and icaABCDR. EssD and uhpt have high homology with esaD and hpt in Staphylococcus aureus, showing that the genomes of S. epidermidis and S. aureus may have communicated during evolution. SdrF, sdrG, fbe, and icaABCDR are related to biofilm formation. Compared to S. epidermidis from blood sources, ocular-sourced strains causing intraocular infection had no direct relationship with biofilm formation. In conclusion, this study provided additional data resources for studies on S. epidermidis and improved our understanding of the evolution and pathogenicity among strains of different sources.