Phillip S. Coburn

Background: Bacillus 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.Methods: Twenty-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.Results: Gastrointestinal 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).Conclusions: Among 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.

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: To test the hypothesis that the C-X-C chemokines CXCL1, CXCL2, and CXCL10 contribute to inflammation during Staphylococcus aureus endophthalmitis. Methods: S. aureus endophthalmitis was induced by intravitreal injection of 5000 colony forming units of S. aureus into the eyes of C57BL/6J, CXCL1^−/−, CXCL2^−/−, or CXCL10^−/− mice. At 12, 24, and 36 hours postinfection, bacterial counts, intraocular inflammation, and retinal function were assessed. Based on these results, the effectiveness of intravitreal administration of anti-CXCL1 in reducing inflammation and improving retinal function was evaluated in S. aureus–infected C57BL/6J mice. Results: We observed significant attenuation of inflammation and improvement in retinal function in CXCL1^−/− mice relative to C57BL/6J at 12 hours but not at 24 or 36 hours postinfection with S. aureus. Co-administration of anti-CXCL1 antibodies with S. aureus, however, did not improve retinal function or reduce inflammation at 12 hours postinfection. In CXCL2^−/− and CXCL10^−/− mice, retinal function and intraocular inflammation were not significantly different from those of C57BL/6J mice at 12 and 24 hours postinfection. At 12, 24, or 36 hours, an absence of CXCL1, CXCL2, or CXCL10 did not alter intraocular S. aureus concentrations. Conclusions: CXCL1 appears to contribute to the early host innate response to S. aureus endophthalmitis, but treatment with anti-CXCL1 did not effectively limit inflammation in this infection. CXCL2 and CXCL10 did not seem to play an integral role in inflammation during the early stages of S. aureus 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.

Bacillus cereus is recognized as a causative agent of gastrointestinal syndromes, but can also cause a devastating form of intraocular infection known as endophthalmitis. We have previously reported that the PlcR/PapR master virulence factor regulator system regulates intraocular virulence, and that the S-layer protein (SlpA) contributes to the severity of B. cereus endophthalmitis. To better understand the role of other B. cereus virulence genes in endophthalmitis, expression of a subset of factors was measured at the midpoint of disease progression in a murine model of endophthalmitis by RNA-Seq. Several cytolytic toxins were expressed at significantly higher levels in vivo than in BHI. The virulence regulators codY, gntR, and nprR were also expressed in vivo. However, at this timepoint, plcR/papR was not detectable, although we previously reported that a B. cereus mutant deficient in PlcR was attenuated in the eye. The motility-related genes fla, fliF, and motB, and the chemotaxis-related gene cheA were detected during infection. We have shown previously that motility and chemotaxis phenotypes are important in B. cereus endophthalmitis. The sodA2 variant of manganese superoxide dismutase was the most highly expressed gene in vivo. Expression of the surface layer protein gene, slpA, an activator of Toll-like receptors (TLR)−2 and −4, was also detected during infection, albeit at low levels. Genes expressed in a mouse model of Bacillus endophthalmitis might play crucial roles in the unique virulence of B. cereus endophthalmitis, and serve as candidates for novel therapies designed to attenuate the severity of this often blinding infection.

Pseudomonas aeruginosa is a significant opportunistic pathogen responsible for a variety of human infections. Its high pathogenicity resides in a diverse array of virulence factors and an ability to adapt to hostile environments. We report that these factors are tied to the activity of condensins, SMC and MksBEF, which primarily function in structural chromosome maintenance. This study revealed that both proteins are required for P. aeruginosa virulence during corneal infection. The reduction in virulence was traced to broad changes in gene expression. Transcriptional signatures of smc and mksB mutants were largely dissimilar and non-additive, with the double mutant displaying a distinct gene expression profile. Affected regulons included those responsible for lifestyle control, primary metabolism, surface adhesion and biofilm growth, iron and sulfur assimilation, and denitrification. Additionally, numerous virulence factors were affected, including type 3 and type 6 secretion systems, hemagglutinin, pyocin and macroglobulin production, and a host of virulence regulators. in vitro properties of condensin mutants mirrored their transcriptional profiles. MksB-deficient cells were impaired in pyocyanin, c-di-GMP production, and sessile growth whereas smc mutants mildly upregulated c-di-GMP, secreted fewer proteases and were growth deficient under nutrient-limiting conditions. Moreover, condensin mutants displayed an abnormal regulation upon transition to stationary phase. These data reveal that condensins are integrated into the control of multiple genetic programs related to epigenetic and virulent behavior, establishing condensins as an essential factor in P. aeruginosa ocular infections. Author Summary: Bacterial pathogenicity is a complex phenomenon dependent on the ability of a bacterium to thrive in a hostile environment while combating the host using an array of virulence factors. This study reports that pathogenicity is also tied to structural chromosome maintenance through condensins, proteins that are responsible for the global organization of the chromosome. We show that the two Pseudomonas aeruginosa condensins, SMC and MksB, act as global regulators of gene expression. The inactivation of SMC and MksB induces opposite regulatory programs in the cell that resemble those observed during the acute and chronic phases of infection. A substantial portion of this regulation is mediated by the intracellular signaling network of P. aeruginosa . Accordingly, virulence regulation is altered in condensin mutants. The results were validated by genetic, phenotypic and virulence studies of condensin mutants. Overall, these data establish condensins as an essential factor during ocular P. aeruginosa infections revealing their involvement in the regulatory virulence network and the control of the bacterial lifestyle.

Bacterial endophthalmitis is a devastating infection that can cause blindness following the introduction of organisms into the posterior segment of the eye. Over half of Bacillus endophthalmitis cases result in significant loss of useful vision. Often, these eyes have to be enucleated. Bacillus produces many virulence factors in the eye that may contribute to retinal damage and robust inflammation. This study analyzed Bacillus immune inhibitor A (InhA) metalloproteases, which digest extracellular matrix, tight junction proteins, and antimicrobial proteins. We hypothesized that InhAs contribute to Bacillus intraocular virulence and inflammation. We analyzed phenotypes and infectivity of wild type (WT), InhA1-deficient (Δ inhA1 ), InhA2-deficient (Δ inhA2 ), or InhA1, A2, and A3-deficient (Δ inhA1-3 ) Bacillus thuringiensis. In vitro analysis of growth, proteolysis, and cytotoxicity were compared between B. thuringiensis strains. WT and InhA mutants were similarly cytotoxic to retinal cells. Mutant Δ inhA1 and Δ inhA2 entered log phase growth earlier than WT. Proteolysis of the Δ inhA1-3 mutant was decreased, but this strain grew similar to WT in vitro . Experimental endophthalmitis was initiated by intravitreally infecting C57BL/6J mice with 200 CFU of B. thuringiensis WT or InhA mutants. Intraocular Bacillus and retinal function loss were quantified. Intraocular myeloperoxidase concentrations were quantified and histology was analyzed. Eyes infected with Δ inhA1 or Δ inhA2 strains contained greater numbers of bacteria than eyes infected with WT throughout the course of infection. Eyes infected with single mutants had inflammation and retinal function loss similar to eyes infected with WT strain. Eyes infected with Δ inhA1-3 cleared the infection, with less retinal function loss and inflammation compared to eyes infected with the WT strain. RT-PCR results suggested that single InhA mutant results may be explained by compensatory expression of the other InhAs in these mutants. These results indicate that together, the InhA metalloproteases contribute to the severity of infection and inflammation in Bacillus endophthalmitis. Author summary: Bacterial endophthalmitis is an infection of the eye, which can follow accidental contamination of the posterior segment following ocular surgery (postoperative), a penetrating wound (post-traumatic), or during spread of bacteria into the eye from the bloodstream (endogenous). During bacterial endophthalmitis, virulent pathogens such as Bacillus cause ocular damage via the activities of an array of virulence factors, including proteases. A class of proteases that are expressed by Bacillus during ocular infection are the immune inhibitor A metalloproteases. Here, we used a mouse model of endophthalmitis to test mutant Bacillus that lack single or multiple InhAs to determine if these metalloproteases contributed to the virulence during the disease. In the absence of the production of all InhAs, Bacillus could not cause severe infection. Our study provides new insights into the virulence of Bacillus in the eye, and the contribution of its InhA metalloproteases to establishing infection.

Bacillus cereus is recognized as a causative agent of gastrointestinal syndromes, but can also cause a devastating form of intraocular infection known as endophthalmitis. We have previously reported that the PlcR/PapR master virulence factor regulator system regulates intraocular virulence, and that the S-layer protein (SlpA) contributes to the severity of B. cereus endophthalmitis. To begin to better understand the role of other B. cereus virulence genes in endophthalmitis, expression levels of a subset of factors was measured at the midpoint of disease progression in a murine model of experimental endophthalmitis by RNA-Seq. Several cytolytic toxins were expressed at significantly higher levels in vivo than in BHI. The virulence regulators codY, gntR, and nprR were also expressed in vivo. However, at this timepoint, plcR/papR was not detectable, we previously reported that a B. cereus mutant deficient in PlcR was attenuated in the eye. The motility-related genes fla, fliF, and motB, and the chemotaxis-related gene cheA were detected during infection. We have shown previously that motility and chemotaxis phenotypes are important in B. cereus endophthalmitis. The sodA2 variant of manganese superoxide dismutase was the most highly expression gene in vivo , suggesting that this gene is criticial for intraocular survival, potentially through inhibition of neutrophil activity. Expression of the surface layer protein gene, slpA, an activator of Toll-like receptors (TLR) −2 and −4, and a potent contributor to intraocular inflammation and disease severvity, was also detected during infection, albeit at low levels. In summary, genes expressed in a mouse model of Bacillus endophthalmitis might prove to play crucial roles in the unique virulence of B. cereus endophthalmitis, and serve as candidates for novel therapies designed attenuate the severity of this often blinding infection. Impact statement: B. cereus causes a potent and rapid infection of the eye that usually results in blindness or enucleation, even with the utilization of current treatment modalities. This necessitates the development of new treatment modalities based on new targets. To begin to better define those B. cereus factors with roles in intraocular infection, we analyzed the expression of genes with both known and hypothesized roles in intraocular infection at the midpoint of infection using a murine model of Bacillus endophthalmitis. Potentially targetable candidate genes were demonstrated to be expressed in vivo , which suggests that these genes might contribute to the unique virulence of B. cereus endophthalmitis. Importantly, our results begin to define the virulome of B. cereus in intraocular infections and identify previously uncharacterized factors with potential roles in the severity and outcome of Bacillus endophthalmitis.

Bacillus cereus produces many factors linked to pathogenesis and is recognized for causing gastrointestinal toxemia and infections. B. cereus also causes a fulminant and often blinding intraocular infection called endophthalmitis. We reported that the PlcR/PapR system regulates intraocular virulence, but the specific factors that contribute to B. cereus virulence in the eye remain elusive. Here, we compared gene expression in ex vivo vitreous humor with expression in Luria Bertani (LB) and Brain Heart Infusion (BHI) broth by RNA-Seq. The expression of several cytolytic toxins in vitreous was less than or similar to levels observed in BHI or LB. Regulators of virulence genes, including PlcR/PapR, were expressed in vitreous. PlcR/PapR was expressed at low levels, though we reported that PlcR-deficient B. cereus was attenuated in the eye. Chemotaxis and motility genes were expressed at similar levels in LB and BHI, but at low to undetectable levels in vitreous, although motility is an important phenotype for B. cereus in the eye. Superoxide dismutase, a potential inhibitor of neutrophil activity in the eye during infection, was the most highly expressed gene in vitreous. Genes previously reported to be important to intraocular virulence were expressed at low levels in vitreous under these conditions, possibly because in vivo cues are required for higher level expression. Genes expressed in vitreous may contribute to the unique virulence of B. cereus endophthalmitis, and future analysis of the B. cereus virulome in the eye will identify those expressed in vivo, which could potentially be targeted to arrest virulence.