September 2005
Volume 46, Issue 9
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Immunology and Microbiology  |   September 2005
Bacillus Endophthalmitis: Roles of Bacterial Toxins and Motility during Infection
Author Affiliations
  • Michelle C. Callegan
    From the Departments of Ophthalmology,
    Microbiology and Immunology, University of Oklahoma Health Sciences Center;
    Molecular Pathogenesis of Eye Infections Research Center, the Dean A. McGee Eye Institute, Oklahoma City, Oklahoma;
  • Scott T. Kane
    From the Departments of Ophthalmology,
  • Daniel C. Cochran
    From the Departments of Ophthalmology,
  • Billy Novosad
    From the Departments of Ophthalmology,
  • Michael S. Gilmore
    From the Departments of Ophthalmology,
    Microbiology and Immunology, University of Oklahoma Health Sciences Center;
    Molecular Pathogenesis of Eye Infections Research Center, the Dean A. McGee Eye Institute, Oklahoma City, Oklahoma;
  • Myriam Gominet
    Genetique et Physiologie des Bacillus Pathogenes, Institut Pasteur, Paris, France; and the
  • Didier Lereclus
    Genetique et Physiologie des Bacillus Pathogenes, Institut Pasteur, Paris, France; and the
    Unite Genetique Microbienne et Environnement, Institut National de la Recherche Agronomique (INRA), Guyancourt, France.
Investigative Ophthalmology & Visual Science September 2005, Vol.46, 3233-3238. doi:10.1167/iovs.05-0410
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      Michelle C. Callegan, Scott T. Kane, Daniel C. Cochran, Billy Novosad, Michael S. Gilmore, Myriam Gominet, Didier Lereclus; Bacillus Endophthalmitis: Roles of Bacterial Toxins and Motility during Infection. Invest. Ophthalmol. Vis. Sci. 2005;46(9):3233-3238. doi: 10.1167/iovs.05-0410.

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      © 2017 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Bacillus endophthalmitis is a highly explosive infection of the eye that commonly results in rapid inflammation and vision loss, if not loss of the eye itself, within a few days. Quorum-sensing–controlled toxins are essential to virulence during infection. Another unique characteristic of this disease is the ability of Bacillus to replicate rapidly and migrate to all parts of the eye. This study was conducted to determine the combined roles of toxins and motility during Bacillus endophthalmitis.

methods. Rabbit eyes were injected intravitreally with approximately 100 cfu of wild type, nonmotile, or nonmotile/quorum-sensing–deficient Bacillus thuringiensis. Infection courses were analyzed by biomicroscopy, histology, electroretinography, and quantitation of bacteria and inflammatory cells.

results. Infection with wild type B. thuringiensis resulted in complete retinal function loss by 18 hours after infection, whereas nonmotile B. thuringiensis infections required 30 hours to achieve a reduction of >90% in retinal function. Further attenuation of infection resulted from infection with the nonmotile/quorum-sensing–deficient B. thuringiensis strain, with approximately 90% retinal function loss occurring at 36 hours. Overall, the nonmotile and nonmotile/quorum-sensing–deficient mutants were significantly less virulent than wild-type B. thuringiensis.

conclusions. The results demonstrate that, in addition to quorum-sensing-controlled toxin production, bacterial migration within the eye contributed to the rapidly fulminant and destructive course of Bacillus endophthalmitis. Motility and quorum-sensing may therefore represent possible targets for the development of therapies designed to attenuate the devastating effects of Bacillus in the eye during endophthalmitis.

Endophthalmitis is a severe infection caused by the introduction of bacteria into the eye after trauma or surgery. Bacillus cereus causes a highly explosive form of endophthalmitis that, despite aggressive therapeutic and surgical measures, commonly results in significant loss of vision, if not loss of the eye itself, within 24 to 48 hours. Bacillus thuringiensis, an organism genetically and phenotypically similar to B. cereus, is not generally considered a significant ocular pathogen, but has also been isolated from severe cases of endophthalmitis (Kane ST et al. IOVS, 2002;43:ARVO E-Abstract 1598). B. cereus and B. thuringiensis produce several similar virulence factors that have the potential to contribute to disease—namely hemolysins, lipases, enterotoxins, and proteases. 1 The unique virulence of B. cereus and B. thuringiensis endophthalmitis has been attributed to toxin production during intraocular growth. Individual toxins, such as hemolysin BL, phosphatidylinositol-specific phospholipase C, and phosphatidylcholine-specific phospholipase C, contribute little to the overall intraocular virulence of these organisms. 2 3 However, several toxins, including enterotoxins, phospholipases, hemolysins, and proteases, have been found, as a group, to contribute significantly to virulence. The production of these toxins by both B. cereus and B. thuringiensis depends on a quorum-sensing system involving the transcriptional activator PlcR and a small diffusible peptide, PapR. 4 Isogenic mutants of B. cereus and B. thuringiensis that are deficient in quorum-sensing cause severe endophthalmitis, but at a significantly slower rate than do wild-type Bacillus. 5  
An additional aspect of Bacillus endophthalmitis that may contribute to pathogenesis is the organism’s unique behavior in the eye. Comparative analyses of the intraocular localization of Gram-positive organisms during endophthalmitis showed that Bacillus migrate rapidly throughout all parts of the eye, from the initial site of injection in the midvitreous into the anterior segment within 6 to 12 hours. 6 The correlation between the ability of a bacterium to migrate within the eye and its virulence potential has not been addressed in detail, although numerous reports have related motility to virulence in several nonocular in vivo models. Attenuation of virulence has been reported for motility-defective isogenic mutants of Listeria, 7 Pseudomonas, 8 9 Salmonella, 10 11 Campylobacter, 12 13 Helicobacter, 14 and Vibrio. 15 16 17 18 In addition, attenuation of virulence by the anti-flagella monoclonal antibody has been demonstrated for Pseudomonas aeruginosa and Campylobacter. 19 20 21 22 Bacterial motility contributes to the virulence of ocular pathogens such as Pseudomonas and Serratia. 8 9 19 23 However, a direct link between the motility of these organisms and ocular disease has not been made. 
In this study, we analyzed the intraocular virulence of quorum-sensing- and motility-deficient Bacillus mutant strains in an experimental model of endophthalmitis. Our results demonstrated that production of toxins, as well as and bacterial migration within the eye, contributed significantly to the fulminant and destructive course of Bacillus endophthalmitis. 
Methods
Bacterial Strains and Culture Media
The B. thuringiensis strain used in these studies (BT407 Cry−) is an acrystalliferous strain that produces several of the same toxins as B. cereus laboratory and ocular isolates, including hemolysin BL, cereolysin O, PI-PLC, and PC-PLC. 1 24 B. thuringiensis BT407 Cry− has been used in an in vivo model of endophthalmitis, causing a fulminant intraocular infection similar to that of B. cereus within approximately 18 hours. 3 5  
The nonmotile mutant of BT407 Cry− harbors a mini-Tn10 insertion in flhA gene and is designated MP02. 25 Briefly, the thermosensitive plasmid pIC333 26 carrying mini-Tn10 was introduced into B. thuringiensis strain BT407 Cry− by electroporation, and spectinomycin-resistant colonies were selected and screened for lack of motility on semisolid (0.25%) brain heart infusion (BHI; Difco, Detroit, MI) agar. Insertion of mini-Tn10 was confirmed by sequencing. 
The nonmotile/quorum-sensing–deficient mutant of strain MP02 was constructed by insertional inactivation of plcR with the kanamycin resistance cassette aphA3, as previously described. 27 This strain was designated MP02plcR::kan R
For cultivation of Bacillus, BHI was used, with or without antibiotic selection. For transformation studies, B. thuringiensis was cultured in Luria-Bertani (LB) medium. All antibiotics were purchased from Sigma and were used at the following concentrations: spectinomycin (250 μg/mL) or kanamycin (200 μg/mL). 
Phenotypic Analysis of Bacillus Strains
Phenotypic analysis involved assays of hemolysis, proteolysis, toxin activities, and motility, as described previously. 3 5 Hemolytic activity was determined by incubating twofold serial dilutions of culture supernatants with an equal volume of 4% (vol/vol) sheep or rabbit erythrocytes in phosphate-buffered saline for 30 minutes. The hemolytic titer was determined as the lowest dilution of supernatant exhibiting 50% hemolysis at OD540. Proteolytic activity was determined by incubating culture supernatants with 10 mg hide azure powder (Sigma-Aldrich, St. Louis, MO) in assay buffer (10 mM Tris-HCl, 10 mM CaCl2 [pH 8.0]) for 2 hours. The proteolytic activity (in U/mL) was expressed as an increase in absorbance of 1 OD562 U/h at 37°C. Phosphatidylinositol-specific phospholipase C (PI-PLC) activity was determined by a colorimetric assay that quantified acetylcholinesterase release. Phosphatidylcholine-specific phospholipase C (PC-PLC) activity was determined by an egg yolk agar turbidity assay. Sphingomyelinase activity was determined by a colorimetric assay that quantified trinitrophenylaminolauroyl-sphingomyelin hydrolysis. Motility was determined on semisolid (0.25%) BHI agar by measuring the diameter of colonies during 37°C incubations for 12 and 24 hours. 
Experimental Bacillus Endophthalmitis
Experimental Bacillus endophthalmitis was induced in New Zealand White rabbits, as previously described. 2 3 5 6 28 Rabbits were maintained in accordance with Institutional Animal Care and Use Committee guidelines and the Association for Research in Vision and Ophthalmology Statement for the Use of Laboratory Animals in Ophthalmic and Vision Research. Briefly, rabbits were anesthetized by intramuscular injection of ketamine (Ketaved, 35 mg/kg of body weight; Phoenix Scientific, Inc., St. Joseph, MO) and xylazine (Rompun, 5 mg/kg of body weight; Bayer Corp., Shawnee Mission, KS). Topical anesthetic (0.5% proparacaine HCl, Ophthetic; Allergan, Hormigueros, Puerto Rico) was applied to each eye before injection. Aqueous humor (100-μL) was withdrawn by paracentesis before intravitreal injection. Approximately 100 cfu of Bacillus were injected into the midvitreous, and contralateral eyes were injected with either BHI (surgical control) or were left undisturbed (absolute control). At various times after injection, eyes were analyzed by biomicroscopy, electroretinography (ERG), histopathology, and bacterial and inflammatory cell quantitation, as described in the following sections. 
Analysis of Bacillus Endophthalmitis
Electroretinography.
ERG was used to measure retinal responsiveness to a single-light flash (one per second). After dilation and a 30-minute dark adaptation, b-wave amplitudes were recorded for each eye, using scotopic bright-flash ERG (EPIC2000 and UTAS3000; LKC Technologies, Inc., Gaithersburg, MD). The b-wave amplitudes for each time point represent the average of 14 repeated measures. The percentage retinal function retained was calculated as [100 − (1 − (experimental b-wave amplitude/absolute control b-wave amplitude)] × 100. 2 3 5  
Bacterial Quantitation.
Quantitation of B. thuringiensis in ocular tissues has been described. 2 3 5 6 Briefly, globes were enucleated, and the aqueous and vitreous removed and homogenized. Bacteria were quantified by plating serial 10-fold dilutions onto BHI agar. Retention of mutant phenotypes was confirmed by replica plating onto 2.5% sheep erythrocyte agar, skim milk agar, and motility agar. 
Thin-Section Histology.
Globes recovered for histologic analysis were fixed in 10% formalin for 24 hours. Eyes (n = 4 per group) were sectioned and stained with hematoxylin and eosin. 2 3 5 6  
Statistical Analysis
Student’s t-test was used for statistical comparison of phenotypic data. Values represented the mean ± SD for n ≥ 3 samples per strain, unless otherwise specified. P ≤ 0.05 was considered significant. Values for parameters used to analyze progressive infection represented the mean ± SEM for n ≥ 4 eyes per time point, unless otherwise specified. Wilcoxon’s rank sum test was used for statistical comparison between infection groups. 
Results
Phenotypic Analysis of Bacillus Mutants
The in vitro growth and hemolytic and proteolytic profiles of wild-type B. thuringiensis BT407 Cry− and its isogenic mutants MP02 and MP02plcR::kan R appeared similar after overnight growth on solid agar (data not shown). However, the phenotypic characteristics of these strains were different in liquid medium, particularly early in growth (Table 1) . The hemolytic titer of the wild-type strain was greater than that of the nonmotile and nonmotile/quorum-sensing–deficient isogenic mutants. The PI-PLC activity of the wild-type strain was greater than that of the mutants at 10 hours (P ≤ 0.0001). The PC-PLC activities of the wild-type, nonmotile, and nonmotile/quorum-sensing–deficient mutants were not significantly different (P ≥ 0.052). On motility agar, the diameters of single wild-type B. thuringiensis colonies were approximately 10 times greater than that of colonies of the isogenic mutants. 
Experimental Bacillus Endophthalmitis
Reproducible endophthalmitis was achieved by intravitreal injection of either 2.01 ± 0.04 log10 cfu strain B. thuringiensis BT407 Cry−, 1.96 ± 0.06 log10 CFU strain MP02, or 2.04 ± 0.04 log10 cfu strain MP02plcR::kan R. Although the intravitreal growth rates of wild-type B. thuringiensis BT407 Cry− and its isogenic mutants MP02 and MP02plcR::kan R were similar (Fig. 1) , bacilli were recovered from the aqueous humor of eyes infected with the wild-type strain only (2.15 ± 0.13 log10 cfu at 18 hours after infection). Endophthalmitis induced by the nonmotile and nonmotile/quorum-sensing–deficient mutants progressed at a considerably slower rate. The explosive course of B. thuringiensis endophthalmitis has been described. 3 5 Briefly, experimental B. thuringiensis endophthalmitis caused by B. thuringiensis BT407 Cry− evolved rapidly, leading to significant inflammation by 6 to 12 hours and complete loss of retinal function by 18 hours. 
During infections resulting from nonmotile B. thuringiensis MP02, inflammatory signs (anterior chamber inflammatory cell influx, increasing vitreous haze, decreased fundus reflex) did not occur until 12 hours after infection. Before that time, most eyes infected with nonmotile Bacillus lacked detectable inflammation. The remaining eyes exhibited a slight infiltration of inflammatory cells into the anterior chamber and a normal fundus reflex. In these eyes, the inflammatory response also evolved at a slower rate than that in the wild-type strain. Between 18 and 30 hours, these eyes exhibited corneal ring infiltrates, severe iritis, vitreous opacities, significant inflammatory cell influx into the cornea and anterior chamber, and a decreasing fundus reflex. Because of the impending panophthalmitis, these infections were not allowed to progress past 30 hours. 
In eyes infected with the nonmotile/quorum-sensing–deficient B. thuringiensis mutant, inflammation was undetectable until approximately 18 hours after infection. Signs of intraocular inflammation were similar to that of the nonmotile mutant, but increased in severity at a slower rate. In these eyes, severe inflammation was not present until 30 hours after infection. Because of the impending panophthalmitis, these infections were not allowed to progress past 36 hours. 
No pathologic changes were observed in surgical or absolute control eyes during these experiments. 
Electroretinography
ERG of eyes infected with wild-type B. thuringiensis or its isogenic mutants is summarized in Figure 2 . The retinal function of all surgical and absolute control eyes was similar to the preoperative retinal function throughout the duration of the experiment. The rapid retinal function loss in eyes infected with wild-type B. thuringiensis BT407 Cry− was similar to that observed in previous studies. 3 5 By 18 hours, Retinal function was completely lost in eyes infected with wild-type B. thuringiensis
Lose of retinal function in eyes infected with nonmotile B. thuringiensis strain MP02 was less than that in eyes infected with wild-type B. thuringiensis, throughout the course of infection (P ≤ 0.034). B. thuringiensis MP02-infected eyes exhibited supernormal ERG responses at 6 hours, but retinal function declined steadily thereafter, reaching <10% retinal function by 30 hours after infection. The loss of retinal function in eyes infected with the nonmotile/quorum-sensing–deficient B. thuringiensis mutant was significantly less than that of eyes infected with the wild-type (P ≤ 0.009) and the non-motile mutant (P ≤ 0.028) bacilli throughout the infection course. A supernormal ERG response was noted for the first 12 hours, followed by a gradual decline in retinal function to approximately 13% by 36 hours after infection. 
Histologic Analysis
Whole-organ and retinal histology of Bacillus endophthalmitis are summarized in Figures 3 and 4 , respectively. Immediately after intravitreal injection, eyes in all infection groups had intact retinal layers, no anterior or posterior segment inflammation, and few bacilli in the vitreous. 
Histologic analysis of B. thuringiensis BT407 Cry− endophthalmitis has been described. 3 5 Briefly, these eyes exhibit mild to moderate inflammatory cell influx into the posterior segment at 6 hours after infection, with bacilli in the vitreous and detectable disruption of retinal layers. Inflammatory cells were present in most parts of the eye by 12 hours after infection, with retinal photoreceptor layer folding and partial retinal detachments. At 18 hours after infection, retinal layers in most eyes infected with wild-type B. thuringiensis were completely destroyed, and the eyes and periocular tissues were severely inflamed. 
Eyes infected with the nonmotile B. thuringiensis mutant MP02 were less inflamed than eyes infected with the wild-type strain throughout 18 hours. In these eyes, inflammatory cells were first seen in the vitreous in close proximity to the optic nerve head as early as 4 to 6 hours after infection. By 12 hours after infection, inflammatory cells were observed close to the iris and ciliary body. Areas of retinal photoreceptor layer folding were also noted. Overall, inflammatory changes evolved slowly from 12 to 24 hours after infection. By 24 hours after infection, inflammatory cells began to invade the corneal stroma from the limbus, and corneal edema increased. At 30 hours, significant inflammation in the anterior and posterior segments was noted. By this time, photoreceptor layers were indistinguishable. Throughout the infection course, bacilli remained within the vitreous. No bacilli were observed in the anterior segment or associated with the retina. 
Eyes infected with the nonmotile/quorum-sensing–deficient B. thuringiensis mutant exhibited hallmarks of inflammation similar to those observed in eyes infected with nonmotile strain MP02, but at a slightly slower rate. Inflammation and retinal layer disruption in these eyes reached moderate to severe levels at 36 hours after infection. No bacilli were recovered from tissues other than the vitreous throughout the course of infection. 
Discussion
For endophthalmitis caused by B. cereus, B. thuringiensis, and other types of virulent bacterial pathogens, treatment failures are common despite aggressive surgical and therapeutic intervention. These devastating outcomes highlight the need to understand the pathogenic mechanisms involved and to recognize the specific virulence factors associated with disease. One virulence trait important to B. cereus and B. thuringiensis may be their ability to migrate within the eye during infection. The intraocular motility of B. cereus and B. thuringiensis during endophthalmitis is a unique characteristic of the disease that had not been investigated in detail. In this study, loss of motility prevented movement of B. thuringiensis within the eye, a mutation that appeared to attenuate the rapid course of endophthalmitis. Nonmotile B. thuringiensis grew in the eye at rates comparable to wild-type strains, but did not evoke notable inflammatory responses at the same rate as did the wild-type strain. Most important, nonmotile B. thuringiensis did not migrate into the anterior chamber, where significant inflammation was notably absent. Although nonmotile B. thuringiensis did not migrate into retinal layers, retinal function was ultimately lost in these eyes. The rate of loss of retinal function in eyes infected with nonmotile B. thuringiensis was significantly reduced compared with that in eyes infected with the wild-type strain. These results suggest that intravitreal growth hours, tissue invasion, loss of retinal function, and anterior segment inflammation may be delayed if migration into surrounding ocular tissues is prevented. 
In addition to defective motility, deficiencies in toxin production in the nonmotile mutant may have also contributed to attenuation of virulence. Ghelardi et al. 25 demonstrated that in B. thuringiensis strain MP02, motility and toxin production are intimately associated. A mutation in flhA abolishes motility, but also reduces toxin production, a finding that was confirmed in our study. In a previous study, we analyzed the intraocular virulence of a nonmotile B. cereus Tn917 transposon mutant that was also unable to produce some toxins at concentrations equivalent to that of the wild-type parental strain. 28 Sequence analysis 29 30 of this strain revealed the transposon insertion into methylisocitrate dehydratase, an aconitase 31 32 that oxidizes propionate to pyruvate (Callegan MC, unpublished observations, 2003). Further analysis of this B. cereus mutant has revealed that detectable flagella are absent (Fall R, unpublished observations, 2004). Endophthalmitis caused by this nonmotile/toxin production-defective B. cereus mutant was also highly attenuated. 28 Together, these results demonstrate an additive decrease in ocular virulence associated with defects in toxin production and motility. However, because these nonmotile Bacillus strains are also defective in toxin production, the precise contribution of motility to endophthalmitis remains in question. 
We have also recently demonstrated that a deficiency in quorum-sensing due to mutation in plcR significantly attenuates the intraocular virulence of both B. cereus and B. thuringiensis. 5 In the present study, mutations in both motility and quorum-sensing resulted in similar attenuation of endophthalmitis compared to infection caused by a strain harboring a mutation in quorum-sensing alone. Significant retinal function was retained for a much longer period in eyes infected with the nonmotile/quorum-sensing–deficient B. thuringiensis mutant compared with eyes infected with nonmotile B. thuringiensis and nonmotile B. cereus 28 and to a similar degree as in eyes infected with quorum-sensing–deficient plcR mutants. 5  
Taken together, these results show that in addition to the regulation and/or production of toxins in the eye, the ability of B. cereus and B. thuringiensis to migrate within the eye may contribute to endophthalmitis pathogenesis. These results highlight toxin production and motility as potential therapeutic targets. This information, combined with results from ongoing studies analyzing the host response to specific toxins and cell wall components, will further our understanding of the explosive virulence of this disease. Understanding the primary mechanisms of pathogenesis is essential for the development of successful target-based strategies focused on the prevention of vision loss during Bacillus endophthalmitis. 
 
Table 1.
 
Phenotypic Analysis of B. thuringiensis Wild-Type Strain and Isogenic Mutants
Table 1.
 
Phenotypic Analysis of B. thuringiensis Wild-Type Strain and Isogenic Mutants
B. thuringiensis Strain Hemolytic Titer* PI-PLC (μg/mL) PC-PLC (μg/mL) Protease, ‡ (U/mL) Motility, § (mm)
4 h 10 h 4 h 10 h 4 h 10 h 4 h 10 h 12 h 24 h
BT 407 Cry− 128 256 ND 12.9 ± 0.3, † 0.08 ± 0.01 4.33 ± 1.18 0.70 ± 0.04 0.90 ± 0.02 16.5 28.5
MP02 8 128 ND 2.7 ± 0.7 0.05 ± 0.01 2.33 ± 0.99 0.64 ± 0.02 0.77 ± 0.01 1.0 2.5
MP02plcR::kan R 8 64 ND 2.5 ± 0.3 0.05 ± 0.02 2.42 ± 0.68 0.61 ± 0.02 0.79 ± 0.02 1.0 2.5
Figure 1.
 
Intraocular growth of B. thuringiensis and its nonmotile and nonmotile/quorum-sensing–deficient mutants. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). Approximately 100 cfu of each Bacillus strain was injected intravitreally. Wild-type Bacillus was quantified every 6 hours throughout 18 hours, whereas nonmotile and nonmotile/quorum-sensing–deficient strains were quantified every 6 hours throughout 30 or 36 hours of intraocular growth, respectively. Data represent the mean ± SEM of results in four or more eyes per group.
Figure 1.
 
Intraocular growth of B. thuringiensis and its nonmotile and nonmotile/quorum-sensing–deficient mutants. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). Approximately 100 cfu of each Bacillus strain was injected intravitreally. Wild-type Bacillus was quantified every 6 hours throughout 18 hours, whereas nonmotile and nonmotile/quorum-sensing–deficient strains were quantified every 6 hours throughout 30 or 36 hours of intraocular growth, respectively. Data represent the mean ± SEM of results in four or more eyes per group.
Figure 2.
 
Retinal function analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). An ERG was performed every 6 hours throughout the infection course. Rapid decreases in b-wave amplitude were observed in eyes infected with wild-type B. thuringiensis throughout 18 hours, whereas gradual decreases were observed over a 30- or 36-hour period in eyes infected with mutant strains. All data represent the mean ± SEM of results in four or more eyes per group.
Figure 2.
 
Retinal function analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). An ERG was performed every 6 hours throughout the infection course. Rapid decreases in b-wave amplitude were observed in eyes infected with wild-type B. thuringiensis throughout 18 hours, whereas gradual decreases were observed over a 30- or 36-hour period in eyes infected with mutant strains. All data represent the mean ± SEM of results in four or more eyes per group.
Figure 3.
 
Whole-organ histologic analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). In eyes infected with wild-type B. thuringiensis, severe inflammation was observed, and retinal layers were difficult to differentiate at 18 hours. In eyes infected with nonmotile or nonmotile/quorum-sensing–deficient B. thuringiensis strains, inflammatory changes were relatively mild until 24 hours of infection. From 30 to 36 hours, photoreceptor folding, random retinal detachments, and severe inflammation were observed. All representative histologic sections were stained with hematoxylin and eosin. Each section is representative of four eyes per group. Magnification, ×10.
Figure 3.
 
Whole-organ histologic analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). In eyes infected with wild-type B. thuringiensis, severe inflammation was observed, and retinal layers were difficult to differentiate at 18 hours. In eyes infected with nonmotile or nonmotile/quorum-sensing–deficient B. thuringiensis strains, inflammatory changes were relatively mild until 24 hours of infection. From 30 to 36 hours, photoreceptor folding, random retinal detachments, and severe inflammation were observed. All representative histologic sections were stained with hematoxylin and eosin. Each section is representative of four eyes per group. Magnification, ×10.
Figure 4.
 
Retinal histologic analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). By 18 hours after infection, specific retinal cell layers of eyes infected with wild-type B. thuringiensis were virtually indistinguishable, whereas retinas of eyes infected with nonmotile or nonmotile/quorum-sensing–deficient B. thuringiensis were mildly inflamed and essentially intact. By 24 hours, significant inflammation and photoreceptor layer folding were apparent in eyes infected with the mutant strains; and, by 30 hours, retinal layers were severely disrupted. Each section is representative of four eyes per group. V, vitreous; ILM, inner limiting membrane; PL, photoreceptor cell layer; OLM, outer limiting membrane; CH, choriocapillaris; S, sclera. Magnification, ×200.
Figure 4.
 
Retinal histologic analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). By 18 hours after infection, specific retinal cell layers of eyes infected with wild-type B. thuringiensis were virtually indistinguishable, whereas retinas of eyes infected with nonmotile or nonmotile/quorum-sensing–deficient B. thuringiensis were mildly inflamed and essentially intact. By 24 hours, significant inflammation and photoreceptor layer folding were apparent in eyes infected with the mutant strains; and, by 30 hours, retinal layers were severely disrupted. Each section is representative of four eyes per group. V, vitreous; ILM, inner limiting membrane; PL, photoreceptor cell layer; OLM, outer limiting membrane; CH, choriocapillaris; S, sclera. Magnification, ×200.
The authors thank Ray Fall (University of Colorado, Boulder, CO) for flagellar analysis of B. cereus strains, Mark Dittmar and Andrea Mauer (University of Oklahoma Health Sciences Center [OUHSC] Animal Facility) for technical assistance, and Paula Pierce (Dean A. McGee Eye Institute [DMEI] Pathology Laboratory) for preparation of histology specimens. 
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Figure 1.
 
Intraocular growth of B. thuringiensis and its nonmotile and nonmotile/quorum-sensing–deficient mutants. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). Approximately 100 cfu of each Bacillus strain was injected intravitreally. Wild-type Bacillus was quantified every 6 hours throughout 18 hours, whereas nonmotile and nonmotile/quorum-sensing–deficient strains were quantified every 6 hours throughout 30 or 36 hours of intraocular growth, respectively. Data represent the mean ± SEM of results in four or more eyes per group.
Figure 1.
 
Intraocular growth of B. thuringiensis and its nonmotile and nonmotile/quorum-sensing–deficient mutants. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). Approximately 100 cfu of each Bacillus strain was injected intravitreally. Wild-type Bacillus was quantified every 6 hours throughout 18 hours, whereas nonmotile and nonmotile/quorum-sensing–deficient strains were quantified every 6 hours throughout 30 or 36 hours of intraocular growth, respectively. Data represent the mean ± SEM of results in four or more eyes per group.
Figure 2.
 
Retinal function analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). An ERG was performed every 6 hours throughout the infection course. Rapid decreases in b-wave amplitude were observed in eyes infected with wild-type B. thuringiensis throughout 18 hours, whereas gradual decreases were observed over a 30- or 36-hour period in eyes infected with mutant strains. All data represent the mean ± SEM of results in four or more eyes per group.
Figure 2.
 
Retinal function analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). An ERG was performed every 6 hours throughout the infection course. Rapid decreases in b-wave amplitude were observed in eyes infected with wild-type B. thuringiensis throughout 18 hours, whereas gradual decreases were observed over a 30- or 36-hour period in eyes infected with mutant strains. All data represent the mean ± SEM of results in four or more eyes per group.
Figure 3.
 
Whole-organ histologic analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). In eyes infected with wild-type B. thuringiensis, severe inflammation was observed, and retinal layers were difficult to differentiate at 18 hours. In eyes infected with nonmotile or nonmotile/quorum-sensing–deficient B. thuringiensis strains, inflammatory changes were relatively mild until 24 hours of infection. From 30 to 36 hours, photoreceptor folding, random retinal detachments, and severe inflammation were observed. All representative histologic sections were stained with hematoxylin and eosin. Each section is representative of four eyes per group. Magnification, ×10.
Figure 3.
 
Whole-organ histologic analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). In eyes infected with wild-type B. thuringiensis, severe inflammation was observed, and retinal layers were difficult to differentiate at 18 hours. In eyes infected with nonmotile or nonmotile/quorum-sensing–deficient B. thuringiensis strains, inflammatory changes were relatively mild until 24 hours of infection. From 30 to 36 hours, photoreceptor folding, random retinal detachments, and severe inflammation were observed. All representative histologic sections were stained with hematoxylin and eosin. Each section is representative of four eyes per group. Magnification, ×10.
Figure 4.
 
Retinal histologic analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). By 18 hours after infection, specific retinal cell layers of eyes infected with wild-type B. thuringiensis were virtually indistinguishable, whereas retinas of eyes infected with nonmotile or nonmotile/quorum-sensing–deficient B. thuringiensis were mildly inflamed and essentially intact. By 24 hours, significant inflammation and photoreceptor layer folding were apparent in eyes infected with the mutant strains; and, by 30 hours, retinal layers were severely disrupted. Each section is representative of four eyes per group. V, vitreous; ILM, inner limiting membrane; PL, photoreceptor cell layer; OLM, outer limiting membrane; CH, choriocapillaris; S, sclera. Magnification, ×200.
Figure 4.
 
Retinal histologic analysis of wild-type, nonmotile, and nonmotile/quorum-sensing–deficient Bacillus endophthalmitis. Strains analyzed were B. thuringiensis BT407 Cry− (wild-type), strain MP02 (nonmotile), and strain MP02plcR::kan R (nonmotile/quorum-sensing–deficient). By 18 hours after infection, specific retinal cell layers of eyes infected with wild-type B. thuringiensis were virtually indistinguishable, whereas retinas of eyes infected with nonmotile or nonmotile/quorum-sensing–deficient B. thuringiensis were mildly inflamed and essentially intact. By 24 hours, significant inflammation and photoreceptor layer folding were apparent in eyes infected with the mutant strains; and, by 30 hours, retinal layers were severely disrupted. Each section is representative of four eyes per group. V, vitreous; ILM, inner limiting membrane; PL, photoreceptor cell layer; OLM, outer limiting membrane; CH, choriocapillaris; S, sclera. Magnification, ×200.
Table 1.
 
Phenotypic Analysis of B. thuringiensis Wild-Type Strain and Isogenic Mutants
Table 1.
 
Phenotypic Analysis of B. thuringiensis Wild-Type Strain and Isogenic Mutants
B. thuringiensis Strain Hemolytic Titer* PI-PLC (μg/mL) PC-PLC (μg/mL) Protease, ‡ (U/mL) Motility, § (mm)
4 h 10 h 4 h 10 h 4 h 10 h 4 h 10 h 12 h 24 h
BT 407 Cry− 128 256 ND 12.9 ± 0.3, † 0.08 ± 0.01 4.33 ± 1.18 0.70 ± 0.04 0.90 ± 0.02 16.5 28.5
MP02 8 128 ND 2.7 ± 0.7 0.05 ± 0.01 2.33 ± 0.99 0.64 ± 0.02 0.77 ± 0.01 1.0 2.5
MP02plcR::kan R 8 64 ND 2.5 ± 0.3 0.05 ± 0.02 2.42 ± 0.68 0.61 ± 0.02 0.79 ± 0.02 1.0 2.5
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