Michelle C. Callegan

Ocular infections caused by Staphylococcus aureus result in poor visual outcomes due to the expression of numerous virulence factors during infection. We hypothesized that pore-forming toxins (PFTs) contribute to the pathogenesis of S. aureus endophthalmitis, a severe intraocular infection that can result in blindness. We analyzed infection outcomes using wild-type (LAC), PFT-null (LAC ΔΔΔΔΔ), leukocidin-null (LAC Δ leukocidin ), or alpha toxin-deficient (LAC Δ hla ) mutants. These strains exhibited no differences in growth in brain heart infusion (BHI) and explanted rabbit vitreous. The expression of PFT genes was detected in both environments, but transcript levels were less in vitreous relative to that of BHI. Experimental endophthalmitis was induced in C57BL6/J mice by intravitreal injections of 5,000 colony-forming units (CFU) of S. aureus LAC or one of its three mutants. To quantify infection severity, eyes were analyzed for retinal function, intraocular CFU, inflammation via myeloperoxidase (MPO), cytokines and chemokines, neutrophil infiltration, alpha toxin production, and gross pathology via histology and slit-lamp imaging. Infections with LAC ΔΔΔΔΔ or LAC Δ hla resulted in improved retinal function, reduced intraocular CFUs, decreased ocular damage, and a trend toward decreased inflammation compared to infections with LAC. In contrast, infections with LAC Δ leukocidin progressed similarly as infections with LAC. Increasing concentrations of alpha toxin were detected over time in the eyes infected with LAC and LAC Δ leukocidin ; 40%–60% of infiltrating neutrophils were ADAM10 ⁺ , identifying these cells as potential targets for alpha toxin. Collectively, these results suggest that alpha toxin may be the main PFT driving the severity of S. aureus endophthalmitis.

To test the hypothesis that leukocidin ED (LukED) contributes to the pathogenesis of experimental Staphylococcus aureus endophthalmitis. Growth curves were generated for S. aureus strain JE2 and strain JE2 lukE::Tn, the transposon mutant of LukED, in brain heart infusion (BHI) and explanted rabbit vitreous. The expression of leukotoxins (lukSF-PV, lukED, hlgABC, and lukGH) was assessed in 18-hour overnight cultures in BHI, tryptic soy broth, and vitreous. S. aureus endophthalmitis was induced by intravitreal injection of 5000 colony-forming units of JE2 or JE2 lukE::Tn into C57BL/6J mice. At 6, 12, and 24 hours after infection, eyes were assessed for retinal function, intraocular colony-forming units and inflammation, and neutrophil infiltration by flow cytometry. RNA was isolated from infected eyes to assess leukotoxin expression. Strains JE2 and JE2 lukE::Tn grew similarly in BHI and vitreous. Transcript levels of leukotoxin subunits were lower in vitreous compared with laboratory media. In vivo, no differences in retinal function, intraocular growth, intraocular inflation, or neutrophil infiltration were observed in eyes infected with JE2 or JE2 lukE::Tn. During infection, other leukotoxins were expressed in vivo in the absence of LukED. LukED does not seem to be essential for the pathogenesis of experimental S. aureus endophthalmitis. However, other leukotoxins are expressed in vivo, which may compensate for the effects of LukED during infection.

Purpose: The purpose of this study was to explore the therapeutic potential of the novel combination of Bacillus bacteriophage lysin (PlyB) and a synthetic TLR2/4 inhibitor (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, OxPAPC) in the treatment of experimental Bacillus cereus endophthalmitis. Methods: C57BL/6J mice were injected with 100 colony forming units (CFUs) Bacillus cereus to induce endophthalmitis. Two hours postinfection, groups of mice were treated with either PlyB, PlyB with OxPAPC, or the groups were left untreated to serve as a control. A group of uninfected mice was injected with only PlyB to serve as a treatment control. Eight hours post-treatment, infected/treated mice were analyzed for bacterial counts, retinal function, histology, and inflammation. Results: Groups treated with PlyB alone or PlyB/OxPAPC showed significantly reduced bacterial loads compared with untreated eyes. Compared with untreated eyes, PlyB and PlyB/OxPAPC-treated eyes retained significant A-wave and B-wave function. PlyB/OxPAPC-treated eyes retained greater A- and B-wave function compared with eyes treated with PlyB alone. Histology showed that retinal structures were well preserved, and retinal layers were distinguishable in eyes treated with PlyB and PlyB/OxPAPC. Ninety-five percent of infiltrating CD45+ cells in infected untreated eyes were Ly6G+/Ly6C+ neutrophils. Infected eyes treated with PlyB and PlyB/OxPAPC had significantly reduced numbers of CD45+ immune cells compared with untreated eyes. Eyes treated with PlyB/OxPAPC had a significantly lower number of neutrophils than eyes treated with PlyB alone. Conclusions: These results demonstrated that the novel combination of bacteriophage lysin and TLR2/4 inhibitor was a successful treatment option for treating experimental Bacillus cereus endophthalmitis.

Purpose: To test the hypothesis that (C-C motif) ligand 2 (CCL2) and CCL3 impact retinal function decline and inflammation during Staphylococcus aureus endophthalmitis. Methods: Experimental endophthalmitis was initiated by intravitreal injection of 5000 colony-forming units of S. aureus into the eyes of C57BL/6J, CCL2^−/−, or CCL3^−/− mice. At 12 and 24 hours post-infection, retinal function, bacterial load, and myeloperoxidase levels were quantified. Results: During S. aureus endophthalmitis, we observed a significant improvement in retinal function in CCL2^−/− mice relative to C57BL/6J mice at 12 hours but not at 24 hours. In CCL3^−/− mice, retinal function was significantly improved relative to C57BL/6J mice at 12 and 24 hours. The absence of CCL2 did not alter intraocular S. aureus intraocular concentrations. However, CCL3^−/− mice had significantly lower intraocular S. aureus at 12 hours but not at 24 hours. No difference in myeloperoxidase levels was observed between C57BL/6J and CCL2^−/− mice at 12 hours. CCL3^−/− mice had almost no myeloperoxidase at 12 hours. At 24 hours, increased myeloperoxidase was observed in CCL2^−/− and CCL3^−/− mice relative to C57BL/6J mice. Conclusions: Although the absence of CCL2 resulted in improved retinal function retention at 12 hours, CCL3 deficiency resulted in improved retinal function at 12 and 24 hours. CCL3 deficiency, but not CCL2 deficiency, resulted in almost no inflammation at 12 hours. However, at 24 hours, the absence of CCL2 or CCL3 resulted in significantly increased inflammation. These results suggest that, although both CCL2 and CCL3 impact intraocular infection outcomes, CCL3 may have a more significant impact in S. aureus endophthalmitis.

Bacteriophage lytic enzymes (i.e., phage lysins) are a trending alternative for general antibiotics to combat growing antimicrobial resistance. Gram-positive Bacillus cereus causes one of the most severe forms of intraocular infection, often resulting in complete vision loss. It is an inherently β-lactamase-resistant organism that is highly inflammogenic in the eye, and antibiotics are not often beneficial as the sole therapeutic option for these blinding infections. The use of phage lysins as a treatment for B. cereus ocular infection has never been tested or reported. In this study, the phage lysin PlyB was tested in vitro , demonstrating rapid killing of vegetative B. cereus but not its spores. PlyB was also highly group specific and effectively killed the bacteria in various bacterial growth conditions, including ex vivo rabbit vitreous (Vit). Furthermore, PlyB demonstrated no cytotoxic or hemolytic activity toward human retinal cells or erythrocytes and did not trigger innate activation. In in vivo therapeutic experiments, PlyB was effective in killing B. cereus when administered intravitreally in an experimental endophthalmitis model and topically in an experimental keratitis model. In both models of ocular infection, the effective bactericidal property of PlyB prevented pathological damage to ocular tissues. Thus, PlyB was found to be safe and effective in killing B. cereus in the eye, greatly improving an otherwise devastating outcome. Overall, this study demonstrates that PlyB is a promising therapeutic option for B. cereus eye infections. IMPORTANCE: Eye infections from antibiotic-resistant Bacillus cereus are devastating and can result in blindness with few available treatment options. Bacteriophage lysins are an alternative to conventional antibiotics with the potential to control antibiotic-resistant bacteria. This study demonstrates that a lysin called PlyB can effectively kill B. cereus in two models of B. cereus eye infections, thus treating and preventing the blinding effects of these infections.

Ocular infections can be medical emergencies that result in permanent visual impairment or blindness and loss of quality of life. Bacteria are a major cause of ocular infections. Effective treatment of ocular infections requires knowledge of which bacteria are the likely cause of the infection. This survey of ocular bacterial isolates and review of ocular pathogens is based on a survey of a collection of isolates banked over a ten-year span at the Dean McGee Eye Institute in Oklahoma. These findings illustrate the diversity of bacteria isolated from the eye, ranging from common species to rare and unique species. At all sampled sites, staphylococci were the predominant bacteria isolated. Pseudomonads were the most common Gram-negative bacterial isolate, except in vitreous, where Serratia was the most common Gram-negative bacterial isolate. Here, we discuss the range of ocular infections that these species have been documented to cause and treatment options for these infections. Although a highly diverse spectrum of species has been isolated from the eye, the majority of infections are caused by Gram-positive species, and in most infections, empiric treatments are effective.

Purpose: The purpose of this study was to explore the C-X-C chemokines CXCL2 and CXCL10 as potential anti-inflammatory targets for Bacillus endophthalmitis. Methods: Bacillus endophthalmitis was induced in C57BL/6J, CXCL2^−/−, and CXCL10^−/− mice. At specific times postinfection, eyes were analyzed for Bacillus, retinal function, and inflammation. The efficacies of intravitreal anti-CXCL2 and anti-CXCL10 with or without gatifloxacin in B. cereus endophthalmitis were also assessed using the same techniques. Results: Despite similar Bacillus growth in eyes of C57BL/6J, CXCL2^−/−, and CXCL10^−/− mice, retinal function retention was greater in eyes of CXCL2^−/− and CXCL10^−/− mice compared to that of C57BL/6J mice. Neutrophil migration into eyes of CXCL2^−/− and CXCL10^−/− mice was reduced to a greater degree compared to that of eyes of C57BL/6J mice. Infected CXCL2^−/− and CXCL10^−/− mouse eyes had significantly less inflammation compared to that of C57BL/6J eyes. Retinal structures in infected eyes of CXCL2^−/− mice were preserved for a longer time than in CXCL10^−/− eyes. Compared to untreated eyes, there was less inflammation and significant retention of retinal function in eyes treated with anti-CXCL2 and anti-CXCL10 with or without gatifloxacin. Conclusions: For Bacillus endophthalmitis, the absence of CXCL2 or CXCL10 in mice resulted in retained retinal function and less inflammation. The absence of CXCL2 led to a better clinical outcome than the absence of CXCL10. The use of anti-CXCL2 and anti-CXCL10 limited inflammation during B. cereus endophthalmitis. These results highlight the utility of CXCL2 and CXCL10 as potential targets for anti-inflammatory therapy that can be tested in conjunction with antibiotics for improving treating Bacillus endophthalmitis.

Endophthalmitis is a devastating infection that can cause blindness. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Bacillus produces many virulence factors that may contribute to retinal damage and robust inflammation.