Pathogenesis of gram-positive bacterial endophthalmitis

Abstract

The severity of endophthalmitis has been associated generally with the virulence of the offending pathogen. However, precisely what constitutes the virulence in intraocular infections remains ill defined. We therefore sought to identify the basis for virulence for three common ocular pathogens (Bacillus cereus, Enterococcus faecalis, and Staphylococcus aureus) in terms of intraocular growth rates, bacterial localization patterns, and the contribution of cell walls and secreted products to the pathogenesis of endophthalmitis. Rabbit eyes were injected intravitreally with (i) viable B. cereus, E. faecalis, or S. aureus, (ii) metabolically inactive B. cereus, E. faecalis, or S. aureus, (iii) sacculus preparations from each strain, or (iv) culture fluid containing products secreted by each strain. Eyes were assessed at various times following injection by slit lamp biomicroscopy, electroretinography (ERG), bacterial and inflammatory cell enumeration, and histology. B. cereus endophthalmitis followed a more rapid and virulent course than E. faecalis or S. aureus endophthalmitis, eliminating retinal responsiveness, as measured by ERG, by 12 h. Analysis of bacterial localization revealed that B. cereus uniquely migrated rapidly from posterior to anterior segment during infection. Although injection of neither metabolically inactive bacteria nor cell wall sacculi greatly affected ERG, significant intraocular inflammation was observed. Injection of B. cereus or S. aureus culture fluids caused both significant reductions in retinal responsiveness and significant intraocular inflammation, paralleling that seen in natural infections. The results demonstrate that toxins, intraocular localization, and, to a lesser extent, the intraocular host response to cell walls all contribute to the pathogenesis of B. cereus, S. aureus, and E. faecalis endophthalmitis in a pathogen-specific manner. The key pathophysiologic differences in these intraocular diseases highlight opportunities for optimizing conventional therapies and deriving new ones.

FIG. 1

Quantification of viable bacteria per eye (A), ERG (B), and enumeration of inflammatory cells in the aqueous humor (C) during experimental bacterial endophthalmitis. Eyes were injected midvitreally with 10² CFU of B. cereus, S. aureus, or E. faecalis. (A) Ocular tissues were recovered at various times postinfection for quantification of cumulative bacteria per eye. (B) ERG was performed at various times postinfection for quantification of B-wave activity. (C) Aqueous humor was aspirated at various times postinfection for enumeration of inflammatory cells per milliliter of aqueous humor. All values represent the mean ± SEM for ≥4 eyes per group.

FIG. 2

Ocular tissue distribution of B. cereus during experimental endophthalmitis. Ocular tissues (cornea, iris, lens, vitreous, and outer tunic) were recovered at various times postinfection for quantification of B. cereus. The charted values represent the percentage of organisms recovered from each tissue compared with the cumulative number of organisms recovered per eye (listed at the top; data from Fig. 1A). Because E. faecalis and S. aureus were recovered exclusively from posterior segment tissues during all stages of infection, those data are not included.

FIG. 3

Histological analysis of experimental B. cereus endophthalmitis. B. cereus is designated by arrows, unless otherwise indicated. All sections were stained with hematoxylin and eosin. (A) B. cereus residing between the outer limiting membrane and retinal pigment epithelium of the retina at 9 h postinfection. Magnification, ×1,000. INL, inner limiting membrane; ONL, outer nuclear layer; OLM, outer limiting membrane; RPE, retinal pigment epithelium. (B) B. cereus migrating toward the iris at 9 h postinfection. Magnification, ×400. LC, lens capsule; AHM, anterior hyaloid membrane; VC, vitreous chamber. (C) B. cereus in the anterior chamber at 12 h postinfection. Magnification, ×400. CO, cornea; ic, inflammatory cells; Bc, B. cereus; AC, anterior chamber. (D) Photoreceptor layer folding of the retina 6 h following injection of B. cereus supernatant. Magnification, ×200. VC, vitreous chamber; ic, inflammatory cells; R, retina; CH, choroid.

FIG. 4

Histological analysis of experimental E. faecalis (A) and S. aureus (B) endophthalmitis. (A) E. faecalis adhering to the anterior hyaloid membrane at 24 h postinfection. Brown & Hopps tissue Gram’s stain; magnification, ×1,000. AHM, anterior hyaloid membrane; VC, vitreous chamber; Ef, E. faecalis. (B) Clusters of inflammatory cells in the vitreous during S. aureus endophthalmitis (48 h). Hematoxylin-and-eosin stain; magnification, ×400. VC, vitreous chamber; SaA, staphylococcal abscess; ic, inflammatory cells.

FIG. 5

Comparative retinal toxicity of the natural infection, metabolically inactive organisms, cell wall sacculi, and bacterial supernatants for the rabbit eye. ERG was performed at various times following midvitreal injection of the following preparations of B. cereus (A), E. faecalis (B), and S. aureus (C): live organisms (⧫), cell-free supernatant (●), metabolically inactive organisms (■), and sacculus preparations (▴). Values represent the mean ± SEM of the percent retinal responsiveness retained for ≥4 eyes per group. Individual data sets are designated by bracketed numbers that correspond to text in Results.

FIG. 6

Comparative intraocular inflammogenicity of the natural infection, metabolically inactive organisms, cell wall sacculi, and bacterial supernatants for the rabbit eye. Anterior chamber paracentesis and enumeration of inflammatory cells per milliliter of aqueous humor was performed at various times following midvitreal injection of the following preparations of B. cereus (A), E. faecalis (B), and S. aureus (C): live organisms (⧫), cell-free supernatant (●), metabolically inactive organisms (■), and sacculus preparations (▴). Values represent the mean ± SEM of the log₁₀ inflammatory cells per milliliter of aqueous humor for ≥4 eyes per group. Individual data sets are designated by bracketed numbers that correspond to text in Results.