Innovative cold atmospheric plasma (iCAP) decreases corneal ulcer formation and bacterial loads and improves anterior chamber health in methicillin resistant Staphylococcus aureus keratitis.

Bacterial keratitis is a vision-threatening infection of the cornea that is typically treated with antibiotics. However, antibiotics sometimes fail to eradicate the infection and do not prevent or repair the damage caused directly by the bacteria or the host immune response to the infection. Our group previously demonstrated that treatment of Pseudomonas aeruginosa keratitis in rabbits with innovative cold atmospheric plasma (iCAP) resulted in reduced edema, ulcer formation, and bacterial load. In this study, we investigated the efficacy of iCAP treatment in methicillin-resistant Staphylococcus aureus (MRSA). New Zealand white rabbits were infected intrastromally with MRSA then treated with iCAP, moxifloxacin, vancomycin, or combination of iCAP with each antibiotic to assess the safety and efficacy of iCAP treatment compared to untreated controls and antibiotics. iCAP treatment significantly reduced bacterial loads and inflammation, improved anterior chamber clarity, and prevented corneal ulceration compared to untreated controls and antibiotic treatment. Safety assessments of grimace test scores and tear production showed that iCAP was not significantly different from either antibiotic treatment in terms of distress or tear production. Combination iCAP/antibiotic treatment did not appear to provide significant added benefit over iCAP alone. Our findings suggest that the addition of iCAP may be a viable tool in reducing damage to the cornea and anterior chamber of the eye following S. aureus keratitis.

Synthesis and Inhibitory Activity of Machaeridiol-Based Novel Anti-MRSA and Anti-VRE Compounds and Their Profiling for Cancer-Related Signaling Pathways.

Three unique 5,6-seco-hexahydrodibenzopyrans (seco-HHDBP) machaeridiols A−C, reported previously from Machaerium Pers., have displayed potent activities against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium, and E. faecalis (VRE). In order to enrich the pipeline of natural product-derived antimicrobial compounds, a series of novel machaeridiol-based analogs (1−17) were prepared by coupling stemofuran, pinosylvin, and resveratrol legends with monoterpene units R-(−)-α-phellandrene, (−)-p-mentha-2,8-diene-1-ol, and geraniol, and their inhibitory activities were profiled against MRSA ATCC 1708, VRE ATCC 700221, and cancer signaling pathways. Compounds 5 and 11 showed strong in vitro activities with MIC values of 2.5 µg/mL and 1.25 µg/mL against MRSA, respectively, and 2.50 µg/mL against VRE, while geranyl analog 14 was found to be moderately active (MIC 5 µg/mL). The reduction of the double bonds of the monoterpene unit of compound 5 resulted in 17, which had the same antibacterial potency (MIC 1.25 µg/mL and 2.50 µg/mL) as its parent, 5. Furthermore, a combination study between seco-HHDBP 17 and HHDBP machaeriol C displayed a synergistic effect with a fractional inhibitory concentrations (FIC) value of 0.5 against MRSA, showing a four-fold decrease in the MIC values of both 17 and machaeriol C, while no such effect was observed between vancomycin and 17. Compounds 11 and 17 were further tested in vivo against nosocomial MRSA at a single intranasal dose of 30 mg/kg in a murine model, and both compounds were not efficacious under these conditions. Finally, compounds 1−17 were profiled against a panel of luciferase genes that assessed the activity of complex cancer-related signaling pathways (i.e., transcription factors) using T98G glioblastoma multiforme cells. Among the compounds tested, the geranyl-substituted analog 14 exhibited strong inhibition against several signaling pathways, notably Smad, Myc, and Notch, with IC50 values of 2.17 µM, 1.86 µM, and 2.15 µM, respectively. In contrast, the anti-MRSA actives 5 and 17 were found to be inactive (IC50 > 20 µM) across the panel of these cancer-signaling pathways.

Differential bacterial gene expression during experimental pneumococcal endophthalmitis.

Streptococcus pneumoniae (pneumococcus) is a potential cause of bacterial endophthalmitis in humans that can result in ocular morbidity. We sought to identify pneumococcal genes that are differentially expressed during growth in the vitreous humor of the eye in an experimental endophthalmitis model. Microarray analysis was used to identify genes that were differentially expressed when pneumococci replicated in the vitreous of rabbit eyes as compared with bacteria grown in vitro in Todd Hewitt medium. Array results were verified by quantitative real-time PCR analysis of representative genes. Select genes potentially playing a role in virulence during endophthalmitis were deleted, and mutants were tested for reduced eye pathogenesis and altered adhesion to host cells. Array analysis identified 134 genes that were differentially expressed during endophthalmitis; 112 genes demonstrated increased expression during growth in the eye whereas 22 were downregulated. Real-time analysis verified increased expression of neuraminidase A (NanA; SP1693), neuraminidase B (NanB; SP1687) and serine protease (SP1954), and decreased expression of RlrA (SP0461) and choline transporter (SP1861). Mutation of NanA and NanB had no major effect on pathogenesis. Loss of SP1954 led to increased adherence to host cells. S. pneumoniae enhances and represses the expression of a variety of genes during endophthalmitis. While some of these genes reflect changes in metabolic requirements, some appear to play a role in immune evasion and pathogenesis in the eye.

Silver-Doped Titanium Oxide Layers for Improved Photocatalytic Activity and Antibacterial Properties of Titanium Implants.

Titanium has a long history of clinical use, but the naturally forming oxide is not ideal for bacterial resistance. Anodization processes can modify the crystallinity, surface topography, and surface chemistry of titanium oxides. Anatase, rutile, and mixed phase oxides are known to exhibit photocatalytic activity (PCA)-driven bacterial resistance under UVA irradiation. Silver additions are reported to enhance PCA and reduce bacterial attachment. This study investigated the effects of silver-doping additions to three established anodization processes. Silver doping showed no significant influence on oxide crystallinity, surface topography, or surface wettability. Oxides from a sulfuric acid anodization process exhibited significantly enhanced PCA after silver doping, but silver-doped oxides produced from phosphoric-acid-containing electrolytes did not. Staphylococcus aureus attachment was also assessed under dark and UVA-irradiated conditions on each oxide. Each oxide exhibited a photocatalytic antimicrobial effect as indicated by significantly decreased bacterial attachment under UVA irradiation compared to dark conditions. However, only the phosphorus-doped mixed anatase and rutile phase oxide exhibited an additional significant reduction in bacteria attachment under UVA irradiation as a result of silver doping. The antimicrobial success of this oxide was attributed to the combination of the mixed phase oxide and higher silver-doping uptake levels.

Passive immunization with Pneumovax® 23 and pneumolysin in combination with vancomycin for pneumococcal endophthalmitis.

BACKGROUND: Capsule and pneumolysin (PLY) are two major virulence factors of Streptococcus pneumoniae. S. pneumoniae is one of the leading causes of bacterial endophthalmitis. The aim of this study is to determine whether passive immunization with the 23-valent pneumococcal polysaccharide vaccine (Pneumovax® 23; PPSV23) or PLY protects against pneumococcal endophthalmitis. METHODS: New Zealand white rabbits were passively immunized with antiserum to PLY, PPSV23, a mixture of PPSV23/PLY, or PBS (mock). Vitreous was infected with a clinical strain of S. pneumoniae. In a separate group of experiments, vancomycin was injected 4 hours post-infection (PI) for each passively immunized group. Severity of infection, bacterial recovery, myeloperoxidase (MPO) activity and percent loss of retinal function were determined. RESULTS: Passive immunization with each antiserum significantly lowered clinical severity compared to mock immunization (PPSV23 = 9.19, PPSV23/PLY = 10.45, PLY = 8.71, Mock = 16.83; P = 0.0467). A significantly higher bacterial load was recovered from the vitreous of PLY passively immunized rabbits 24 hours PI (7.87 log10 CFU) compared to controls (7.10 log10 CFU; P = 0.0134). Retinas from immunized rabbits were more intact. Vitreous of PLY (2.88 MPO untis/mL) and PPSV23/PLY (2.17) passively immunized rabbits had less MPO activity compared to controls (5.64; P = 0.0480), and both passive immunizations (PLY = 31.34% loss of retinal function, PPSV23/PLY = 27.44%) helped to significantly preserve retinal function compared to controls (64.58%; P = 0.0323). When vancomycin was administered 4 hours PI, all eyes were sterile at 24 hours PI. A significantly lower clinical severity was observed for rabbits administered the combination immunization (5.29) or PPSV23 (5.29) with vancomycin treatment compared to controls (17.68; P = 0.0469). CONCLUSIONS: Passive immunization with antisera to these antigens is effective in reducing clinical severity of pneumococcal endophthalmitis in rabbits. Addition of vancomycin to immunization is effective at eliminating the bacteria.

Absence of Streptococcus pneumoniae Capsule Increases Bacterial Binding, Persistence, and Inflammation in Corneal Infection.

The role of the pneumococcal polysaccharide capsule is largely unclear for Streptococcus pneumoniae keratitis, an ocular inflammatory disease that develops as a result of bacterial infection of the cornea. In this study, capsule-deficient strains were compared to isogenic parent strains in their ability to adhere to human corneal epithelial cells. One isogenic pair was further used in topical ocular infection of mice to assess the contribution of the capsule to keratitis. The results showed that non-encapsulated pneumococci were significantly more adherent to cells, persisted in significantly higher numbers on mouse corneas in vivo, and caused significant increases in murine ocular IL9, IL10, IL12-p70, MIG, and MIP-1-gamma compared to encapsulated S. pneumoniae. These findings indicate that the bacterial capsule impedes virulence and the absence of capsule impacts inflammation following corneal infection.

Radio Frequency Heating of Washable Conductive Textiles for Bacteria and Virus Inactivation.

The ongoing COVID-19 pandemic has increased the use of single-use medical fabrics such as surgical masks, respirators, and other personal protective equipment (PPE), which have faced worldwide supply chain shortages. Reusable PPE is desirable in light of such shortages; however, the use of reusable PPE is largely restricted by the difficulty of rapid sterilization. In this work, we demonstrate successful bacterial and viral inactivation through remote and rapid radio frequency (RF) heating of conductive textiles. The RF heating behavior of conductive polymer-coated fabrics was measured for several different fabrics and coating compositions. Next, to determine the robustness and repeatability of this heating response, we investigated the textile's RF heating response after multiple detergent washes. Finally, we show a rapid reduction of bacteria and virus by RF heating our conductive fabric. 99.9% of methicillin-resistant Staphylococcus aureus (MRSA) was removed from our conductive fabrics after only 10 min of RF heating; human cytomegalovirus (HCMV) was completely sterilized after 5 min of RF heating. These results demonstrate that RF heating conductive polymer-coated fabrics offer new opportunities for applications of conductive textiles in the medical and/or electronic fields.

"Boom" and "Bust" cycles in virus growth suggest multiple selective forces in influenza a evolution.

BACKGROUND: Influenza A virus evolution in humans is driven at least in part by mutations allowing the virus to escape antibody neutralization. Little is known about the evolution of influenza in birds, a major reservoir of influenza A. METHODS: Neutralizing polyclonal antiserum was raised in chicken against reassortant influenza virus, CalX, bearing the hemagglutinin (HA) and neuraminidase (NA) of A/California/7/2004 [H3N2]. CalX was serially passaged in the presence of anti-CalX polyclonal IgY to derive viruses capable of growth in the presence of antibody. RESULTS: Polyclonal chicken antibody neutralized both HA activity and infection by CalX, but had no effect on a strain bearing an earlier human H3 and an irrelevant neuraminidase (A/Memphis/71-Bellamy/42 [H3N1]). Surprisingly, most of the antibody-resistant viruses were still at least partially sensitive to neutralization of HA activity and viral infection. Although mutant HA genes bearing changes that might affect antibody neutralization were identified, the vast majority of HA sequences obtained were identical to wild type, and no individual mutant sequence was found in more than one passage, suggesting that those mutations that were observed did not confer sufficient selective advantage to come to dominate the population. Different passages yielded infectious foci of varying size and plaques of varying size and morphology. Yields of infectious virus and relative frequency of different morphologies changed markedly from passage to passage. Sequences of bulk, uncloned PCR products from antibody-resistant passages indicated changes in the PB2 and PA proteins with respect to the wild type virus. CONCLUSIONS: Each antibody-selected passage consisted of a variety of different cocirculating populations, rather than pure populations of virus able to escape antibody by changes in antibody epitopes. The ability to escape antibody is apparently due to changes in genes encoding the viral polymerase complex, probably resulting in more robust viral replication, allowing the few virus particles not completely neutralized by antibody to rapidly produce large numbers of progeny. Our data suggest that the relative success of an individual variant may depend on both its own gain and loss of fitness, as well as that of its cocirculating variants.

Animal models of bacterial keratitis.

Bacterial keratitis is a disease of the cornea characterized by pain, redness, inflammation, and opacity. Common causes of this disease are Pseudomonas aeruginosa and Staphylococcus aureus. Animal models of keratitis have been used to elucidate both the bacterial factors and the host inflammatory response involved in the disease. Reviewed herein are animal models of bacterial keratitis and some of the key findings in the last several decades.

Pathogenicity and virulence of Streptococcus pneumoniae: Cutting to the chase on proteases.

Bacterial proteases and peptidases are integral to cell physiology and stability, and their necessity in Streptococcus pneumoniae is no exception. Protein cleavage and processing mechanisms within the bacterial cell serve to ensure that the cell lives and functions in its commensal habitat and can respond to new environments presenting stressful conditions. For S. pneumoniae, the human nasopharynx is its natural habitat. In the context of virulence, movement of S. pneumoniae to the lungs, blood, or other sites can instigate responses by the bacteria that result in their proteases serving dual roles of self-protein processors and virulence factors of host protein targets.

Draft Genome Sequences of Viridans Streptococci Causing Bacterial Endophthalmitis in Humans.

The viridans streptococci are a group of bacteria that are commensals of the oral cavity and pharynx. These species tend to cause severe cases of bacterial endophthalmitis with poor prognoses but remain largely uncharacterized in this context. Here, we report the whole-genome sequences of 21 strains of viridans streptococci isolated from endophthalmitis in humans.

Development of a Streptococcus pneumoniae keratitis model in mice.

BACKGROUND: Streptococcus pneumoniae is a common cause of bacterial keratitis, and models to examine the ocular pathogenesis of this bacterium would aid in efforts to treat pneumococcal keratitis. The aim of this study was to establish a murine model of pneumococcal keratitis. METHODS: The corneas of A/J, BALB/c or C57BL/6 mice were scratched and topically infected with a clinical strain of S. pneumoniae. Slitlamp examination (SLE), enumeration of bacteria in the corneas and histology were performed. RESULTS: Bacteria were recovered from the eyes of A/J mice on postinfection (PI) days 1 [1.96 +/- 0.61 log(10) colony-forming units (CFU)] and 3 (1.41 +/- 0.71 log(10) CFU). SLE scores were significantly higher in the infected A/J mice as compared to the BALB/c or C57BL/6 mice on PI day 3 (p < 0.0001) and steadily increased over time, reaching a maximal value of 3.00 +/- 0.35 on PI day 10. Histopathology revealed stromal edema and the influx of polymorphonuclear leukocytes on PI days 7 and 10, and corneal disruption on PI day 7. CONCLUSIONS: S. pneumoniae keratitis was established in A/J mice, but not BALB/c or C57BL/6 mice.

Drug-Loaded Elastin-Like Polypeptide-Collagen Hydrogels with High Modulus for Bone Tissue Engineering.

Emphasizing the role of hydrogel stiffness and cellular differentiation, this study develops collagen and elastin-like polypeptide (ELP)-based bone regenerative hydrogels loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) and doxycycline with mechanical properties suitable for osteogenesis. The drug-incorporated collagen-ELP hydrogels has significantly higher modulus of 35 ± 5 kPa compared to collagen-only hydrogels. Doxycycline shows a bi-phasic release with an initial burst release followed by a gradual release, while rhBMP-2 exhibits a nearly linear release profile for all hydrogels. The released doxycycline shows anti-microbial activity against Pseudomonas aeruginosa, Streptococcus sanguinis, and Escherichia coli. Microscopic observation of the hydrogels reveals their interconnected, macroporous, 3D open architecture with pore diameters between 160 and 400 µm. This architecture supports human adipose-derived stem cell attachment and proliferation from initial days of cell seeding, forming a thick cellular sheath by day 21. Interestingly, in collagen and collagen-ELP hydrogels, cell morphology is elongated with stretched slender lamellipodial formation, while cells assemble as spheroidal aggregates in crosslinked as well as drug-loaded hydrogels. Osteogenic markers, alkaline phosphatase and osteocalcin, are expressed maximally for drug-loaded hydrogels compared to those without drugs. The drug-loaded collagen-ELP hydrogels are thus promising for combating bacterial infection and promoting guided bone regeneration.

A Transcriptional Activator of Ascorbic Acid Transport in Streptococcus pneumoniae Is Required for Optimal Growth in Endophthalmitis in a Strain-Dependent Manner.

Streptococcus pneumoniae is among the top causes of bacterial endophthalmitis, an infectious disease of the intraocular fluids. The mechanisms by which S. pneumoniae grows and thrives in the intraocular cavity are not well understood. We used a bacterial genome-wide assessment tool (transposon insertion site sequencing) to determine genes essential for S. pneumoniae growth in vitreous humor. The results indicated that an ascorbic acid (AA) transport system subunit was important for growth. We created an isogenic gene deletion mutant of the AA transcriptional activator, ulaR2, in 2 strains of S. pneumoniae. Growth curve analysis indicated that ulaR2 deletion caused attenuated growth in vitro for both strains. However, in vivo vitreous humor infection in rabbits with either strain determined that ulaR2 was necessary for growth in one strain but not the other. These results demonstrate that ulaR2 may be important for fitness during S. pneumoniae endophthalmitis depending on the background of the strain.

Innovative Cold Atmospheric Plasma (iCAP) Decreases Mucopurulent Corneal Ulcer Formation and Edema and Reduces Bacterial Load in Pseudomonas Keratitis.

PURPOSE: To evaluate the effect of application of 3% air in helium cold atmospheric plasma jet, using an inexpensive device termed iCAP, in corneal scratch wound closure in vitro and the treatment of Pseudomonas aeruginosa (P. aeruginosa) keratitis in vivo. METHODS: Thermal imaging to measure temperature of surfaces to which iCAP was applied and UV energy density delivered by iCAP were measured. Scratch wounds inflicted on in vitro cultures of a human corneal epithelial cell line were treated with iCAP and wound widths at various times post-application were measured. Rabbit eyes infected with P. aeruginosa were treated with iCAP and slit lamp biomicroscope examination conducted to determine corneal health outcomes 25h post infection. Corneal homogenates were plated on agar and viable bacterial colonies enumerated to determine the effect of iCAP on bacterial load in vivo in P. aeruginosa keratitis. RESULTS: iCAP was shown to operate in the non-thermal regime and also shown to deliver much lower UV energy density than that necessary to cause harmful effects on ocular tissue. iCAP treatment significantly improved the rate of scratch wound gap closure in vitro in a human corneal epithelial cell line compared to controls. In vivo, iCAP treatment of P. aeruginosa keratitis infection in the rabbit eyes (N = 20) significantly reduced the incidence of corneal ulcer (P = 0.003) and corneal edema (P = 0.011) and significantly improved total cornea health (P = 0.034) compared to untreated (N = 10). Finally, in vivo iCAP treatment of P. aeruginosa keratitis infection in the rabbit eyes (N = 19) significantly reduced bacterial loads (P = 0.012) compared to untreated (N = 9). CONCLUSION: Our results strongly suggest that iCAP treatment was effective in improving corneal epithelial defect closure in vitro, reducing ulcer formation and decreasing inflammation in P. aeruginosa infected corneas in vivo and decreasing bacterial loads in P. aeruginosa infected corneas in vivo which led to improved overall cornea health outcomes in vivo. Further studies to investigate iCAP's safety and efficacy against other infectious microbes responsible for causing ulcerative keratitis, with and without co-treatment with antimicrobial therapies are warranted.

Staphylococcus aureus Superantigen-Like Protein SSL1: A Toxic Protease.

Staphylococcus aureus is a major cause of corneal infections that can cause reduced vision, even blindness. Secreted toxins cause tissue damage and inflammation resulting in scars that lead to vision loss. Identifying tissue damaging proteins is a prerequisite to limiting these harmful reactions. The present study characterized a previously unrecognized S. aureus toxin. This secreted toxin was purified from strain Newman ΔhlaΔhlg, the N-terminal sequence determined, the gene cloned, and the purified recombinant protein was tested in the rabbit cornea. The virulence of a toxin deletion mutant was compared to its parent and the mutant after gene restoration (rescue strain). The toxin (23 kDa) had an N-terminal sequence matching the Newman superantigen-like protein SSL1. An SSL1 homodimer (46 kDa) had proteolytic activity as demonstrated by zymography and cleavage of a synthetic substrate, collagens, and cytokines (IL-17A, IFN-γ, and IL-8); the protease was susceptible to serine protease inhibitors. As compared to the parent and rescue strains, the ssl1 mutant had significantly reduced virulence, but not reduced bacterial growth, in vivo. The ocular isolates tested had the ssl1 gene, with allele type 2 being the predominant type. SSL1 is a protease with corneal virulence and activity on host defense and structural proteins.

Protection from Streptococcus pneumoniae keratitis by passive immunization with pneumolysin antiserum.

PURPOSE: To determine whether passive immunization with pneumolysin antiserum can reduce corneal damage associated with pneumococcal keratitis. METHODS: New Zealand White rabbits were intrastromally injected with Streptococcus pneumoniae and then passively immunized with control serum, antiserum against heat-inactivated pneumolysin (HI-PLY), or antiserum against cytotoxin-negative pneumolysin (psiPLY). Slit lamp examinations (SLEs) were performed at 24, 36, and 48 hours after infection. An additional four corneas from rabbits passively immunized with antiserum against psiPLY were examined up to 14 days after infection. Colony forming units (CFUs) were quantitated from corneas extracted at 20 and 48 hours after infection. Histopathology of rabbit eyes was performed at 48 hours after infection. RESULTS: SLE scores at 36 and 48 hours after infection were significantly lower in rabbits passively immunized with HI-PLY antiserum than in control rabbits (P < or = 0.043). SLE scores at 24, 36, and 48 hours after infection were significantly lower in rabbits passively immunized with psiPLY antiserum than in control rabbits (P < or = 0.010). The corneas of passively immunized rabbits that were examined up to 14 days after infection exhibited a sequential decrease in keratitis, with an SLE score average of 2.000 +/- 1.586 at 14 days. CFUs recovered from infected corneas were not significantly different between each experimental group and the respective control group at 20 or 48 hours after infection (P > or = 0.335). Histologic sections showed more corneal edema and polymorphonuclear leukocyte (PMN) infiltration in control rabbits compared with passively immunized rabbits. CONCLUSIONS: HI-PLY and psiPLY both elicit antibodies that provide passive protection against S. pneumoniae keratitis.

The Role of Pneumococcal Virulence Factors in Ocular Infectious Diseases.

Streptococcus pneumoniae is a gram-positive, facultatively anaerobic pathogen that can cause severe infections such as pneumonia, meningitis, septicemia, and middle ear infections. It is also one of the top pathogens contributing to bacterial keratitis and conjunctivitis. Though two pneumococcal vaccines exist for the prevention of nonocular diseases, they do little to fully prevent ocular infections. This pathogen has several virulence factors that wreak havoc on the conjunctiva, cornea, and intraocular system. Polysaccharide capsule aids in the evasion of host complement system. Pneumolysin (PLY) is a cholesterol-dependent cytolysin that acts as pore-forming toxin. Neuraminidases assist in adherence and colonization by exposing cell surface receptors to the pneumococcus. Zinc metalloproteinases contribute to evasion of the immune system and disease severity. The main purpose of this review is to consolidate the multiple studies that have been conducted on several pneumococcal virulence factors and the role each plays in conjunctivitis, keratitis, and endophthalmitis.

Antibiotic susceptibility, cytotoxicity, and protease activity of viridans group streptococci causing endophthalmitis.

The viridans group streptococci comprise multiple species and have gained more recognition in recent years as common etiologic agents of bacterial endophthalmitis. The purpose of this study was to identify the species of human endophthalmitis isolates of viridans streptococci and to characterize their potential virulence attributes. The species of 22 endophthalmitis strains of viridans streptococci were identified by Matrix Assisted Laser Desorption Ionization Time-of-Flight. Susceptibilities to 3 antibiotics commonly used for bacterial endophthalmitis were determined. The extracellular milieu of each strain was tested for cytotoxicity of retinal pigmented epithelial cells, hemolysis of sheep erythrocytes, and protease activity using gelatin zymography. Identified species were Streptococcus mitis/oralis, S. salivarius, S. vestibularis, S. parasanguinis, S. mutans, S. constellatus, and S. gordonii. One strain of S. pseudoporcinus was also identified. All strains were sensitive to vancomycin, 77% were resistant to amikacin, and 27% had intermediate resistance to ceftazidime. Extracellular milieu from all strains except one (S. pseudoporcinus) were largely devoid of toxicity to retinal pigmented epithelial cells and sheep erythrocytes. Twelve strains, 10 of which were S. mitis/oralis, produced protease activity. Interestingly, not all of the S. mitis/oralis strains were proteolytic. These findings highlight the diversity of virulence factor production in ocular strains of the viridans streptococci not only at the group level but also at the species level.

Mechanism of Pseudomonas aeruginosa Small Protease (PASP), a Corneal Virulence Factor.

Purpose: Pseudomonas aeruginosa is the leading cause of contact lens-associated bacterial keratitis. Secreted bacterial proteases have a key role in keratitis, including the P. aeruginosa small protease (PASP), a proven corneal virulence factor. We investigated the mechanism of PASP and its importance to corneal toxicity. Methods: PASP, a serine protease, was tested for activity on various substrates. The catalytic triad of PASP was sought by bioinformatic analysis and site-directed mutagenesis. All mutant constructs were expressed in a P. aeruginosa PASP-deficient strain; the resulting proteins were purified using ion-exchange, gel filtration, or affinity chromatography; and the proteolytic activity was assessed by gelatin zymography and a fluorometric assay. The purified PASP proteins with single amino acid changes were injected into rabbit corneas to determine their pathological effects. Results: PASP substrates were cleaved at arginine or lysine residues. Alanine substitution of PASP residues Asp-29, His-34, or Ser-47 eliminated protease activity, whereas PASP with substitution for Ser-59 (control) retained activity. Computer modeling and Western blot analysis indicated that formation of a catalytic triad required dimer formation, and zymography demonstrated the protease activity of the homodimer, but not the monomer. PASP with the Ser-47 mutation, but not with the control mutation, lacked corneal toxicity, indicating the importance of protease activity. Conclusions: PASP is a secreted serine protease that can cleave proteins at arginine or lysine residues and PASP activity requires dimer or larger aggregates to create a functional active site. Most importantly, proteolytic PASP molecules demonstrated highly significant toxicity for the rabbit cornea.