Virulence-related genotypic differences among Bacillus cereus ocular and gastrointestinal isolates and the relationship to endophthalmitis pathogenesis

BackgroundBacillus cereus (Bc) can cause self-limiting gastrointestinal infections, but when infecting the eye, can cause rapid and irreversible blindness. This study investigated whether clinical ocular and gastrointestinal Bc isolates differed in terms of virulence-related genotypes and endophthalmitis virulence.MethodsTwenty-eight Bc ocular, gastrointestinal, and laboratory reference isolates were evaluated. Hemolysis assays were performed to assess potential differences in hemolytic activity. The presence of twenty Bc virulence-related genes was assessed by PCR. A subset of ocular and gastrointestinal isolates differing in PCR positivity for 5 virulence genes was compared to strain ATCC14579 in an experimental murine model of endophthalmitis. At 8 hours post infection, retinal function was evaluated by electroretinography, and intraocular bacterial concentrations were determined by plate counts.ResultsGastrointestinal Bc isolates were more hemolytic than the Bc ocular isolates and ATCC14579 (p < 0.0001). Bc ocular isolates were more frequently PCR-positive for capK, cytK, hblA, hblC, and plcR compared to the gastrointestinal isolates (p ≤ 0.0002). In the endophthalmitis model, mean A-wave retention did not differ significantly between eyes infected with ATCC14579 and eyes infected with the selected ocular or gastrointestinal isolates (p ≥ 0.3528). Similar results were observed for mean B-wave retention (p ≥ 0.0640). Only one diarrheal isolate showed significantly greater B-wave retention when compared to ATCC14579 (p = 0.0303). No significant differences in mean A-wave (p ≥ 0.1535) or B-wave (p ≥ 0.0727) retention between the selected ocular and gastrointestinal isolates were observed. Intraocular concentrations of ATCC14579 were significantly higher than the selected ocular isolate and 3 of the gastrointestinal isolates (p ≤ 0.0303). Intraocular concentrations of the selected ocular isolate were not significantly different from the gastrointestinal isolates (p ≥ 0.1923).ConclusionsAmong the subset of virulence-related genes assessed, 5 were significantly enriched among the ocular isolates compared to gastrointestinal isolates. While hemolytic activity was higher among gastrointestinal isolates, retinal function retention and intraocular growth was not significantly different between the selected ocular and gastrointestinal isolates. These results suggest that Bc strains causing gastrointestinal infections, while differing from ocular isolates in hemolytic activity and virulence-related gene profile, are similarly virulent in endophthalmitis

Role of swarming migration in the pathogenesis of bacillus endophthalmitis.

PURPOSE. Bacillus cereus causes one of the most rapidly blinding forms of bacterial endophthalmitis. Migration of B. cereus throughout the eye during endophthalmitis is a unique aspect of this disease that may contribute to intraocular virulence. This study was conducted to analyze the contribution of swarming and intraocular migration to the pathogenesis of experimental endophthalmitis. METHODS. Eyes were injected intravitreally with 100 colonyforming units (CFU) of either wild-type, nonswarming, or swarming-complemented strains of B. cereus. Pathogenicity was compared throughout the course of infection by biomicroscopy, histology, electroretinography, and bacterial and inflammatory cell quantitation. RESULTS. Wild-type, nonswarming, and swarming-complemented B. cereus strains grew to a similar number in the vitreous throughout the course of infection. Unlike the wild-type and swarming-complemented strains, the nonswarming mutant did not migrate to the anterior segment during infection. The rate of decrease in retinal responses of eyes infected with the all strains was similar, resulting in near complete elimination of retinal function by 12 hours. All Bacillus strains caused similar degrees of posterior segment inflammation and retinal destruction. However, the accumulation of inflammatory cells in the anterior chamber, hyphemae, and corneal ring abscesses did not occur in eyes infected with the nonswarming mutant. CONCLUSIONS. The deficiency in swarming had little effect on retinal function loss or the overall course or severity of experimental B. cereus endophthalmitis. However, a deficiency in swarming prevented Bacillus from migrating to the anterior segment, leading to less severe anterior segment disease

Role of Toll-Like Receptor (TLR) 2 in Experimental Bacillus cereus Endophthalmitis

Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known Gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2-/- mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to108 CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2-/- eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2-/- eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2-/- eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial ocular response to B. cereus endophthalmitis

Bloodstream-To-Eye Infections Are Facilitated by Outer Blood-Retinal Barrier Dysfunction

This work was funded by National Institutes of Health (NIH; http://www.nih.gov) Grants R01EY024140 and R21EY022466 (to M.C.C.) and R01EY019494 (to M.H.E.). Our research is also funded in part by NIH Core Grant P30EY021725 (to Robert E. Anderson, OUHSC) and an unrestricted grant from Research to Prevent Blindness Inc. (http://www.rpbusa.org) to the Dean A. McGee Eye Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.We thank Bolanle Adebayo (Cameron University, Lawton OK), Craig Land (Oklahoma State University, Stillwater OK), Nathan Jia (Oklahoma Christian University, Edmond OK), Kobbe Wiafe (Oklahoma School of Science and Mathematics, Oklahoma City OK), and Amanda Roehrkasse and Madhu Parkunan (OUHSC) for intellectual discussions and technical assistance. The authors also acknowledge thank Nanette Wheatley, Dr. Feng Li, and Mark Dittmar (OUHSC Live Animal Imaging Core, P30EY021725) for their invaluable technical assistance.This work was presented in part at the 2014 Association for Research in Vision and Ophthalmology Annual Conference in Orlando FL.The blood-retinal barrier (BRB) functions to maintain the immune privilege of the eye, which is necessary for normal vision. The outer BRB is formed by tightly-associated retinal pigment epithelial (RPE) cells which limit transport within the retinal environment, maintaining retinal function and viability. Retinal microvascular complications and RPE dysfunction resulting from diabetes and diabetic retinopathy cause permeability changes in the BRB that compromise barrier function. Diabetes is the major predisposing condition underlying endogenous bacterial endophthalmitis (EBE), a blinding intraocular infection resulting from bacterial invasion of the eye from the bloodstream. However, significant numbers of EBE cases occur in non-diabetics. In this work, we hypothesized that dysfunction of the outer BRB may be associated with EBE development. To disrupt the RPE component of the outer BRB in vivo, sodium iodate (NaIO3) was administered to C57BL/6J mice. NaIO3-treated and untreated mice were intravenously injected with 108 colony forming units (cfu) of Staphylococcus aureus or Klebsiella pneumoniae. At 4 and 6 days postinfection, EBE was observed in NaIO3-treated mice after infection with K. pneumoniae and S. aureus, although the incidence was higher following S. aureus infection. Invasion of the eye was observed in control mice following S. aureus infection, but not in control mice following K. pneumoniae infection. Immunohistochemistry and FITC-dextran conjugate transmigration assays of human RPE barriers after infection with an exoprotein-deficient agr/sar mutant of S. aureus suggested that S. aureus exoproteins may be required for the loss of the tight junction protein, ZO-1, and for permeability of this in vitro barrier. Our results support the clinical findings that for both pathogens, complications which result in BRB permeability increase the likelihood of bacterial transmigration from the bloodstream into the eye. For S. aureus, however, BRB permeability is not required for the development of EBE, but toxin production may facilitate EBE pathogenesis.Yeshttp://www.plosone.org/static/editorial#pee

The SAG1 Toxoplasma gondii Surface Protein Is Not Required for Acute Ocular Toxoplasmosis in Mice

The SAG1 Toxoplasma gondii surface protein stimulates acute ileitis. To determine whether SAG1 is also important in the eye, wild-type or SAG1 knockout parasites were injected intravitreally into mice. No differences in retinal damage or parasite growth were observed, indicating that unlike the case for the intestine, factors besides SAG1 are important for retinal damage

Contribution of Mucoviscosity-Associated Gene A (magA) to Virulence in Experimental Klebsiella pneumoniae Endophthalmitis

Klebsiella pneumoniae endogenous endophthalmitis is a uniformly blinding infection. The authors report that the K1 capsule of invasive K. pneumoniae significantly contributes to disruption of retinal function, intraocular growth to a high density, and persistence despite immune cell recruitment

A Role for Tumor Necrosis Factor-␣ in Experimental Bacillus cereus Endophthalmitis Pathogenesis

PURPOSE. To determine the contribution of tumor necrosis factor-alpha (TNF␣) in the pathogenesis of experimental Bacillus cereus endophthalmitis. METHODS. Experimental B. cereus endophthalmitis was induced in wild-type control (B6.129F1) and age-matched homozygous TNF␣ knockout mice (TNF␣ Ϫ/Ϫ , B6.129S6-Tnf tm1Gk1 /J). At various times after infection, eyes were analyzed by electroretinography and were harvested for quantitation of bacteria, myeloperoxidase, proinflammatory cytokines and chemokines, and histologic analysis. RESULTS. B. cereus replicated more rapidly in the eyes of TNF␣ Ϫ/Ϫ mice than in the eyes of B6.129F1 mice. Retinal function decreased more rapidly in TNF␣ Ϫ/Ϫ mice than in B6.129F1 mice. Retinal layers were not as structurally intact at 6 and 12 hours after infection in TNF␣ Ϫ/Ϫ eyes as in B6.129F1 eyes. Histologic analysis suggested less polymorphonuclear leukocyte (PMN) infiltration into the vitreous of TNF␣ Ϫ/Ϫ mice than of B6.129F1 mice. B6.129F1 eyes also had greater myeloperoxidase concentrations than did eyes of TNF␣ Ϫ/Ϫ mice. In general, concentrations of proinflammatory cytokines and chemokines (IL-1␤, KC, IL-6, and MIP-1␣) were greater in eyes of TNF␣ Ϫ/Ϫ mice than of B6.129F1 mice. CONCLUSIONS. TNF␣ is important to intraocular pathogen containment by PMNs during experimental B. cereus endophthalmitis. In the absence of TNF␣, fewer PMNs migrated into the eye, facilitating faster bacterial replication and retinal function loss. Although greater concentrations of proinflammatory cytokines were synthesized in the absence of TNF␣, the resultant inflammation was diminished, and an equally devastating course of infection occurred. (Invest Ophthalmol Vis Sci