The severity of ophthalmic infections ranges widely from self-limiting bacterial conjunctivitis to potentially threatening conditions such as keratitis and endophthalmitis (Grandi et al., 2019). In recent years, the pathogen species that causes ocular infection is constantly changing along with antibiotic resistance rate (Hong, 2021), which will not only increase the difficulty of clinical diagnosis and treatment of diseases, but also prolong the recovery time of patients, thus affecting prognosis (Abebe et al., 2023). Hence, strict monitoring of ocular infection pathogens and antibiotic resistance combining with understanding the clinical characteristics of common pathogens may provide significant value to guide clinicians administrate early treatment for ocular infections. Staphylococcus epidermidis (S. epidermidis) is one of the most common isolates from ocular infections (Asadi-Amoli et al., 2022) since S. epidermidis is normal flora of human skin, including ophthalmic area (Polat et al., 2023). S. epidermidis mainly contributes to foreign body-associated infection, its attachment to the conjunctiva and skin around the eye makes it prone to contaminating medical equipment used in ocular surgery, leading to infections and inflammatory reactions within the eyes (Polat et al., 2023) but a few studies reveal it as infectious pathogen (Belyhun et al., 2018; Liu et al., 2020). Despite high prevalence rate of S. epidermidis in clinical specimens, whether the bacteria act as source of foreign objects or causative paothogen in ocular infection remains uncertain (Lin et al., 2021). For infectious agent perspective, identifying the molecular determinants and biomarkers for S. epidermidis transition from commensal to pathogen may offer potential therapeutic targets (du et al., 2021). According to the literature, the oxygen level in the posterior chamber is very low. In the anterior chamber, there is about a sevenfold oxygen gradient in the human cornea due to oxygen consumption by the corneal cells (Siegfried et al., 2010; Beebe et al., 2014). Therefore, the oxygen content altered when S. epidermidis enters eyes, due to shift from normoxic to microoxic environment. To understand the mechanisms of S. epidermidis adapt oxygen level changes during infection, we analyzed gene expression and metabolic pattern of S. epidermidis in microoxic and normoxic environments, respectively. The total RNA and metabolites have been extracted from S. epidermidis cultured under microoxic and normoxic environment respectively, to perform transcriptome and metabolome. The results indicated pathways and biological processes of bacteria rewired under low oxygen level leading to pathogenic transition from normal flora suggesting environmental oxygen concentration shift may be a key signal for pathogenicity of S. epidermidis.

This comprehensive study analyzes bacterial and fungal profiles of ocular infections, representing a significant contribution to existing literature. The most frequently isolated Gram-positive bacterium was S. epidermidis (25.55%), while P. aeruginosa (2.25%) and Fusarium spp. (11.21%) were the predominant Gram-negative bacterium and fungus, respectively. Notably, S. epidermidis was the most commonly detected microorganism in ocular infections, consistent with prior study (Shams Abadi et al., 2023). The majority of ocular infection specimens (78.06%) originated from corneal and conjunctival secretions, primarily due to the ease of sample collection.

In a retrospective analysis of clinical data from patients with ocular infections, we examined 621 isolates of S. epidermidis, 228 isolates of Fusarium spp., and 47 isolates of P. aeruginosa. Approximately half of the ocular infection cases (48.21%) were attributable to open trauma, predominantly among male patients. This aligns with Qing L et al.’s findings that posttraumatic endophthalmitis accounted for 49.62% of infectious endophthalmitis cases at the Zhongshan Ophthalmic Center (Liu M. et al., 2022; Liu Q. et al., 2022). Ocular surgery is a recognized high-risk factor for ocular infection (Reddy et al., 2015). In our study, S. epidermidis and P. aeruginosa infections occurred frequently during post-surgery, corroborating with other reports of S. epidermidis as a common post-operative endophthalmitis causative agent (Yannuzzi et al., 2017). In an Argentinian tertiary hospital, P. aeruginosa was identified as the primary microorganism in post-cataract surgery endophthalmitis cases (45.4%) (Segretín Gutiérrez et al., 2022). Moreover, Fusarium spp. and P. aeruginosa infections were prevalent among farmers, supporting the association between agricultural work and ocular infection (Toh et al., 2022). Interestingly, about half of S. epidermidis ocular infections persisted for more than 2 weeks, indicating their severity.

The global rise in antibiotic resistance is alarming, partially attributed to excessive and inappropriate antibiotic use (Ghweil et al., 2022; Mitra et al., 2022). In this study, S. epidermidis exhibited high sensitivity to tigacycline, linezolid, vancomycin, quinupristin/dalfopristin, rifampicin, furantoin, and gentamicin. However, clindamycin resistances were > 37.0% annually, surpassing the 25% rate reported elsewhere (Laura et al., 2020). Fluoroquinolones were widely used for the treatment and prevention of ocular bacterial infection, in this study, the resistance rates of S. epidermidis to levofloxacin were >30.0%, and to moxifloxacin were <20.0%, suggesting a decline in levofloxacin’s efficacy as a first-line treatment for empiric endophthalmitis. The cause of the high resistance of S. epidermidis strains to levofloxacin is probably related to the increased use of topical antibiotics, that is, the use of levofloxacin as an ophthalmic antibiotic. It is known that the prescription dose and resistance to antibiotics are closely related (Zeng et al., 2019). As for whether there was horizontal transmission within hospitals of resistant S. epidermidis, further study is needed. In the meantime, the rising trend of the resistance rates of S. epidermidis to levofloxacin and ciprofloxacin suggest that ophthalmologists should pay attention to the changing trends of pathogen distribution and their drug resistance patterns and choose sensitive antibiotics based on the local data.

It is essential to consider whether S. epidermidis isolated from the healthy conjunctiva is causative agent in infection (Kang et al., 2020). Most clinical studies have reported less severe endophthalmitis caused by S. epidermidis compared to S. aureus (Spoto et al., 2022). However, in our study, nearly half of the patients with S. epidermidis ocular infections experienced prolonged symptoms (over 2 weeks), including ocular pain and deteriorating vision. Understanding how S. epidermidis adapts to environmental changes during infection onset and its transition from commensal to pathogen, remains elusive. One significant environmental change is that shift in oxygen concentration from the ocular surface to the intraocular. Our hypothesis posited that microoxic conditions alter S. epidermidis gene expression, transcription, and metabolism, facilitating bacterial pathogenic conversion. To verified this, we analyzed differential transcriptomics and metabolomics under oxic and microoxic conditions, revealing the bacterial responses to environmental oxygen concentration shifts akin to those experienced during intraocular invasion. Bacteria transfer from the ocular surface to the intraocular is faced with changes in many environmental factors, such as pH, oxygen concentration, immune response, availability of nutrients, etc. (Ersan et al., 2015; Agujetas et al., 2018; Braunger et al., 2023), and one change in oxygen concentration is just one of the environmental factors, this condition is not fully representative of the complex ocular surface and intraocular environment. Recent studies also suggest that microaerobic lifestyles are important in host-associated microbial communities and the prevalence of microoxygen environments (Morris and Schmidt, 2013; Pedroza-Dávila et al., 2020), so we selected changes in environmental oxygen concentration as the experimental variable.

S. epidermidis, facultative anaerobic bacteria, possesses a versatile respiratory enzyme system for aerobic oxidation and anaerobic glycolysis. Environmental changes significantly impact microbial biological function (Burian et al., 2022). GO term analysis showed increased anaerobic respiration and decreased aerobic respiration in microoxic conditions. Genes related to anaerobic respiration (narH, pflB) were up-regulated, suggesting an adaptive response for better survival in microoxic environments. Moreover, ATP accumulation and up-regulation of the purine metabolic pathway were observed, indicating higher cellular energy availability in microoxic conditions.

Transcriptome and metabolome analysis revealed that S. epidermidis in a microoxic condition experiences altered gene expression related to environmental alteration. KEGG analysis showed up-regulation gene expression in amino acid metabolism and transmembrane transporter activity, suggesting increase in amino acid and energy metabolism, membrane transport, and metabolism of cofactors and vitamins under oxygen limitation. Additionally, regulatory systems for virulence in S. epidermidis were enhanced compared to oxic conditions. Notably, PhoR and SaeR of the PhoPR and SaeRS Two-Component Systems, which regulate a wide array of cell surface and secreted virulence factors, were up-regulated. PhoPR is involved in phosphate transport under phosphate-limiting conditions, responding to wall teichoic acid metabolism (Devine, 2018). The SaeRS system in S. epidermidis was found to negatively regulate genes involved in competence (comF, murF), cytolysis (lrgA), and autolysis (lytS) (Lou et al., 2016). Additionally, lytS belonging to the LytTR family, involved in cell autolysis regulation, increased in microoxic conditions. The expression of FsrB/D and SprE in S. epidermidis, crucial for gelatinase activity, serine protease production and biofilm formation, was also enhanced (Hashem et al., 2021). AgrB, associated with virulence and biofilm formation, was upregulated under microoxic conditions (Grande et al., 2014). Our findings suggest that S. epidermidis might shift from commensal to pathogen by enhancing nutrient metabolism and increasing virulence genes expression.

Metabolomic and transcriptomic analyses revealed that the valine, leucine, and isoleucine biosynthesis pathway was up-regulated in microoxic models. Branched-chain amino acids (BCAAs) are implicated in numerous physiological processes, including inflammation regulation. Elevated BCAA levels may activate the NF-κB signaling pathway and inflammasome (Ye et al., 2020). In microoxic environments, we estimated L-glutamic acid accumulation, since genes expression related to alanine, aspartate, and glutamate metabolism were up-regulated. L-glutamic acid damage retinal ganglion cells, inhibit axonal growth, elevate inflammatory and glial-cell related genes expression have been reported previously (Liu M. et al., 2022; Liu Q. et al., 2022). ATP and glutamate induce proinflammatory factor release from microglia, including TNF (tumor necrosis factor), IL-1β (interleukin 1 beta), and NO (nitric oxide) (Jesudasan et al., 2021). Moreover, N-Acetyl-D-glucosamine (GlcNAc) and N-Acetyl-D-mannosamine (ManNAc) may accumulate as result of up-regulated murQ, nagB and down-regulated glmS in the amino and nucleotide sugar metabolism pathway. ManNAc and GlcNAc are precursors of N-acetylneuraminic acid (NeuAc), a determinant of bacterial pathogenicity (Revilla-Nuin et al., 2002). These metabolic changes (Figure 2E) under microoxic conditions may contribute to S. epidermidis pathogenic transformation.

In the eye, the innate immune response is triggered by pathogen-associated molecular patterns (PAMPs) of bacteria, fungi, and viruses. Bacterial cell wall components like lipopolysaccharide, peptidoglycan, and teichoic acid activate pattern recognition receptors (PRR), such as Toll-like receptors (TLRs) to initiate signaling cascades that lead to chemokine and cytokine expression (Xia et al., 2021). Various ocular cells, including retinal pigment epithelial (RPE) cells, astrocytes, corneal epithelium, iris epithelium, retinal microglia, and Muller cells, express TLRs (Miller et al., 2019). The clearance of bacteria may cause host-mediated ocular damage when the inflammatory response is vigorous. Metabolites of S. epidermidis in microoxic conditions may also play a significant role in ocular infection-induced cytokine storms. Notably, an increase in cholesterol levels was observed under microoxic conditions. Increased cholesterol amplifies TLR signaling, thereby increasing cytokine and chemokine production and intensifying the inflammatory process (Tabas and Bornfeldt, 2016). Cholesterol also stimulate formation of NLRP3 inflammasome and IL-1β production (Chiu et al., 2021), induce inflammatory cytokine expression in human retinal pigment epithelium cells via the NF-κB pathway (Hu et al., 2014). Therefore, current bacterial endophthalmitis treatment, involving intravitreal antibiotic injections, fails to address inflammation-mediated ocular tissue damage, resulting in partial or complete vision loss (Das et al., 2021, 2023).

Emerging evidences suggest that repolarizing immune cells toward a less inflamed phenotype by manipulating metabolism with small molecules and intermediates (Reithuber et al., 2021). In our study, we noted an increase in glycolysis and gluconeogenesis under microoxic conditions. In cases of bacterial endophthalmitis, both resident and infiltrating cells show heightened glycolysis, interestingly, inhibiting glycolysis in this condition reduces intraocular inflammation (Francis et al., 2020), suggesting a potential therapeutic target for ocular infections. Hyperbaric oxygen therapy, used either as a primary or adjunctive treatment, addresses a range of medical disorders, including ocular conditions (Worley et al., 2020). Additionally, the suppression of the intraocular inflammatory response, which can cause secondary intraocular damage, through the use of intravitreal steroids may consider as complement antibiotic treatment (Jamil et al., 2023).

Therefore, we concluded that in S. epidermidis-related ocular infections, specific metabolites and up-regulated virulence genes may contribute to the bacterial transition from commensal to pathogenic state. However, this study is subject to several limitations. Firstly, the potential for erroneous records or missing data in medical records cannot be disregarded. Secondly, the research was conducted in a single center without including other areas of Yunnan province, the findings might not fully encapsulate the epidemiological profile of ocular infections across China.

In summary, we have isolated 2,712 sample of microorganisms from 2,598 patients with ocular infections with S. epidermidis constituting 25.55%. S. epidermidis ocular infections were predominantly found in men under 60 years of age and non-farmers, which correlate with a history of ocular operations.

Furthermore, our study discovered that the microoxic environment alters the gene expression and metabolism of S. epidermidis, facilitating its transition from commensal to pathogen. This finding underscores the potential of immune metabolism as new therapeutic target for endophthalmitis. We have investigated the risk factors of patients with ocular infections and metabolomics and transcriptomics of S. epidermidis. However, these culture conditions are not representative of the complex ocular surface and intraocular environment. Therefore, some of the pathways and functions altered in our experiments might be tested under specific growth conditions. In addition, the interaction of S. epidermidis microoxic metabolites with host epithelial cells remains unclear. Further research is required to unravel how bacterial metabolites impact on transition from commsal to pathogenic bacteria as well as microbe-host interactions.

Publicly available datasets were analyzed in this study. This data can be found here: The raw data from the transcriptomic experiment have been deposited in the SRA database of NCBI under SRA accession no. PRJNA956543 (https://www.ncbi.nlm. nih.gov/guide/). The raw data from the metabolomic experiment have been deposited into CNGB Sequence Archive (CNSA) of China National GeneBank DataBase (CNGBdb), the accession number of S. epidermidis under oxic and microoxic was CNP0004301.

The studies involving humans were approved by the affiliated hospital of Yunnan University Biomedical Ethics Committee. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.

HL: Conceptualization, Data curation, Project administration, Supervision, Validation, Writing – original draft. WZ: Conceptualization, Investigation, Project administration, Software, Supervision, Writing – review & editing. ZZ: Formal analysis, Investigation, Project administration, Validation, Writing – review & editing. YW: Data curation, Formal analysis, Project administration, Supervision, Validation, Writing – review & editing. ZB: Data curation, Formal analysis, Project administration, Validation, Writing – review & editing. YL: Data curation, Formal analysis, Methodology, Validation, Writing – review & editing. ZH: Formal analysis, Funding acquisition, Resources, Visualization, Writing – review & editing. DD: Funding acquisition, Project administration, Resources, Supervision, Visualization, Writing – review & editing. WY: Funding acquisition, Methodology, Project administration, Resources, Validation, Visualization, Writing – review & editing.