Correlation of Staphylococcus Epidermidis Phenotype and Its Corneal Virulence.

PURPOSE: S. epidermidis is an ocular pathogen and a leading cause of keratitis. It produces hemolysins and at least 3 proteases. The purpose of the present study is to compare the secretion of hemolysins and proteases between 28 ocular isolates and one non-ocular strain and to determine their relationship to ocular virulence in selected strains using a rabbit model of infection. MATERIALS AND METHODS: Culture supernatants were compared for protease production and hemolysis. Selected strains were injected into rabbit corneas and their virulence and pathology recorded. The major protease activity in a virulent strain was identified and the gene was cloned and expressed as a recombinant protein. The corneal toxicity of this protease was determined. Antibodies to the native protease were generated and tested for neutralizing activity in vivo and in vitro. The corneal pathology of the S. epidermidis protease was compared to the pathology of S. aureus V8 protease. RESULTS: Strains that exhibited the least protease activity in vitro caused significantly less ocular pathology in vivo (p ≤ 0.003). Strains that were hemolytic and secreted a major protease had numerically higher SLE scores. This protease was identified as the serine protease Esp. The recombinant Esp protease caused extensive pathology when injected into the corneal stroma (7.62 ± 0.33). Antibody generated against native Esp did not neutralize the activity of the protease in vivo or in vitro. The antibody reacted with Esp proteases secreted by other S. epidermidis strains. S. epidermidis Esp protease and its homologue in S. aureus caused similar ocular pathology when injected in the rabbit corneal stroma. CONCLUSION: Hemolysins and proteases seem to be important in corneal pathology caused by S. epidermidis infections. The Esp protease mediates significant corneal damage. S. epidermidis Esp and S. aureus V8 protease caused similar and extensive edema in rabbit corneas.

Pseudomonas aeruginosa Keratitis: Protease IV and PASP as Corneal Virulence Mediators.

Pseudomonas aeruginosa is a leading cause of bacterial keratitis, especially in users of contact lenses. These infections are characterized by extensive degradation of the corneal tissue mediated by Pseudomonas protease activities, including both Pseudomonas protease IV (PIV) and the P. aeruginosa small protease (PASP). The virulence role of PIV was determined by the reduced virulence of a PIV-deficient mutant relative to its parent strain and the mutant after genetic complementation (rescue). Additionally, the non-ocular pathogen Pseudomonas putida acquired corneal virulence when it produced active PIV from a plasmid-borne piv gene. The virulence of PIV is not limited to the mammalian cornea, as evidenced by its destruction of respiratory surfactant proteins and the cytokine interleukin-22 (IL-22), the key inducer of anti-bacterial peptides. Furthermore, PIV contributes to the P. aeruginosa infection of both insects and plants. A possible limitation of PIV is its inefficient digestion of collagens; however, PASP, in addition to cleaving multiple soluble proteins, is able to efficiently cleave collagens. A PASP-deficient mutant lacks the corneal virulence of its parent or rescue strain evidencing its contribution to corneal damage, especially epithelial erosion. Pseudomonas-secreted proteases contribute importantly to infections of the cornea, mammalian lung, insects, and plants.

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.

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.

Pseudomonas aeruginosa Protease IV Exacerbates Pneumococcal Pneumonia and Systemic Disease.

Pneumonia is a pulmonary disease affecting people of all ages and is consistently a leading cause of childhood mortality and adult hospitalizations. Streptococcus pneumoniae and Pseudomonas aeruginosa are major lung pathogens commonly associated with community-acquired and nosocomial pneumonia. Additionally, mixed lung infections involving these bacterial pathogens are increasing in prevalence and are frequently more severe than single infections. The cooperative interactions of these two pathogens that impact pulmonary disease severity are understudied. A major secreted virulence factor of P. aeruginosa, protease IV (PIV), cleaves interleukin 22 (IL-22), a cytokine essential for maintaining innate mucosal defenses against extracellular pathogens. Here, we investigate the ability of PIV to augment the virulence of a pneumococcal strain with limited virulence, S. pneumoniae EF3030, in a C57BL/6 murine model of pneumonia. We demonstrate that pulmonary coinfection involving P. aeruginosa 103-29 and S. pneumoniae EF3030 results in pneumococcal bacteremia that is abrogated during pneumococcal coinfection with a PIV-deficient strain. Furthermore, intratracheal administration of exogenous PIV and EF3030 resulted in abundant immune cell infiltration into the lung with large abscess formation, as well as severe bacteremia leading to 100% mortality. Heat-inactivated PIV did not worsen pneumonia or reliably induce bacteremia, suggesting that the specific activity of PIV is required. Our studies also show that PIV depletes IL-22 in vivo Moreover, PIV-mediated enhancement of pneumonia and disease severity was dependent on the expression of pneumolysin (Ply), a prominent virulence factor of S. pneumoniae Altogether, we reveal that PIV and Ply additively potentiate pneumonia in a murine model of lung infection.IMPORTANCES. pneumoniae remains the leading cause of bacterial pneumonia despite widespread use of pneumococcal vaccines, forcing the necessity for appropriate treatment to control pneumococcal infections. Coinfections involving S. pneumoniae with other bacterial pathogens threaten antibiotic treatment strategies and disease outcomes. Currently, there is not an effective treatment for alveolar-capillary barrier dysfunction that precedes bacteremia. An understanding of the dynamics of host-pathogen interactions during single and mixed pulmonary infections could elucidate proper treatment strategies needed to prevent or reduce invasive disease. Antibiotic treatment decreases bacterial burden in the lung but also increases acute pathology due to cytotoxins released via antibiotic-induced bacterial lysis. Therefore, targeted therapeutics that inhibit or counteract the effects of bacterial proteases and toxins are needed in order to limit pathology and disease progression. This study identifies the cooperative effect of PIV and Ply, products of separate lung pathogens that additively alter the lung environment and facilitate invasive disease.

The Pathogenesis of Staphylococcus aureus Eye Infections.

Staphylococcus aureus is a major pathogen of the eye able to infect the tear duct, eyelid, conjunctiva, cornea, anterior and posterior chambers, and the vitreous chamber. Of these infections, those involving the cornea (keratitis) or the inner chambers of the eye (endophthalmitis) are the most threatening because of their potential to cause a loss in visual acuity or even blindness. Each of these ocular sites is protected by the constitutive expression of a variety of antimicrobial factors and these defenses are augmented by a protective host response to the organism. Such infections often involve a predisposing factor that weakens the defenses, such as the use of contact lenses prior to the development of bacterial keratitis or, for endophthalmitis, the trauma caused by cataract surgery or intravitreal injection. The structural carbohydrates of the bacterial surface induce an inflammatory response able to reduce the bacterial load, but contribute to the tissue damage. A variety of bacterial secreted proteins including alpha-toxin, beta-toxin, gamma-toxin, Panton-Valentine leukocidin and other two-component leukocidins mediate tissue damage and contribute to the induction of the inflammatory response. Quantitative animal models of keratitis and endophthalmitis have provided insights into the S. aureus virulence and host factors active in limiting such infections.

Reactions with Antisera and Pathological Effects of Staphylococcus aureus Gamma-Toxin in the Cornea.

PURPOSE: This study analyzed the toxicity of purified gamma-toxin from Staphylococcus aureus and the protectiveness of antisera to gamma-toxin in the rabbit cornea. MATERIALS AND METHODS: Gamma-toxin was purified from cultures of alpha-toxin deficient S. aureus strain Newman Δhla. Antisera to native gamma-toxin (Hlg) were produced in rabbits. These antisera and a commercial polyclonal antibody to recombinant HlgB (rHlgB) were analyzed for specificity and toxin neutralization. Heat-inactivated gamma-toxin, active gamma-toxin either alone or with antisera or with commercial antibody to rHlgB, was injected into the rabbit cornea to observe the pathological effects using slit lamp examination scoring (SLE) and histological analyses. RESULTS: Eyes with intrastromal injection of gamma-toxin developed SLE scores that were significantly higher than eyes injected with heat-inactivated gamma-toxin (p ≤ 0.003). Slit lamp and histological examination of eyes revealed that gamma-toxin injected into the cornea mediated conjunctival injection and chemosis, iritis, fibrin accumulation in the anterior chamber, and polymorphonuclear neutrophil infiltration of the cornea and iris. Also, eyes injected with gamma-toxin plus antisera to native whole gamma-toxin or HlgB, but not with commercial antibody to rHlgB, yielded significantly lower SLE scores than eyes injected with gamma-toxin alone (p ≤ 0.003). CONCLUSIONS: This study illustrates that S. aureus gamma-toxin is capable of causing significant corneal pathology. Furthermore, the use of polyclonal antisera specific for native gamma-toxin was found to inhibit the damaging effects of the toxin in the rabbit cornea.

Pseudomonas aeruginosa proteolytically alters the interleukin 22-dependent lung mucosal defense.

The IL-22 signaling pathway is critical for regulating mucosal defense and limiting bacterial dissemination. IL-22 is unusual among interleukins because it does not directly regulate the function of conventional immune cells, but instead targets cells at outer body barriers, such as respiratory epithelial cells. Consequently, IL-22 signaling participates in the maintenance of the lung mucosal barrier by controlling cell proliferation and tissue repair, and enhancing the production of specific chemokines and anti-microbial peptides. Pseudomonas aeruginosa is a major pathogen of ventilator-associated pneumonia and causes considerable lung tissue damage. A feature underlying the pathogenicity of this bacterium is its capacity to persist and develop in the host, particularly in the clinical context of nosocomial lung infections. We aimed to investigate the ability of P. auruginosa to disrupt immune-epithelial cells cross-talk. We found that P. aeruginosa escapes the host mucosal defenses by degrading IL-22, leading to severe inhibition of IL-22-mediated immune responses. We demonstrated in vitro that, protease IV, a type 2 secretion system-dependent serine protease, is responsible for the degradation of IL-22 by P. aeruginosa. Moreover, the major anti-proteases molecules present in the lungs were unable to inhibit protease IV enzymatic activity. In addition, tracheal aspirates of patients infected by P. aeruginosa contain protease IV activity which further results in IL-22 degradation. This so far undescribed cleavage of IL-22 by a bacterial protease is likely to be an immune-evasion strategy that contributes to P. aeruginosa-triggered respiratory infections.

Strategies for decreasing contamination of homemade nasal saline irrigation solutions.

BACKGROUND: Saline nasal irrigations (SNI) are an important adjunct in the treatment of rhinosinusitis, and many patients prepare and store these solutions in their homes without an awareness of the potential for contamination. The objectives of this study were to determine if such contamination occurs and the effect of preparation methods on contamination. METHODS: Stock solutions of various tonicities and pHs were prepared using boiled, bottled, and distilled water (n = 57). The solutions were stored at ambient temperature or refrigerated for 1 week. Each day, 50 mL of the solutions were decanted to simulate transferring the stock solution into an irrigation vector. Cultures of the stock solutions were taken on days 1, 3, and 7. RESULTS: Overall contamination rate was 35.1%. The boiled water solutions were more likely to demonstrate bacterial growth (p < 0.001), as were those that were hypotonic (p = 0.046). pH had no significant effect (p = 0.127). Growth occurred as early as 24 hours after solution preparation. Pathogenic species isolated were Staphylococcus aureus, Moraxella sp, Sphingomonas paucimobilis, Acinetobacter junii, Methylobacterium sp, and Brevundimonas diminuta. No bacterial growth occurred in refrigerated solutions (p = 0.008). CONCLUSION: Pathogenic bacterial growth can occur in a short period of time in homemade SNI solutions with routine handling. Solutions should be refrigerated if possible. If solutions are to be stored at ambient temperature, they should be either isotonic or hypertonic and prepared from bottled or distilled water.

Staphylococcus Alpha-Toxin Action on the Rabbit Iris: Toxic Effects and Their Inhibition.

PURPOSE: Staphylococcus aureus infection of the anterior chamber can occur after cataract surgery, causing inflammation and extensive damage to the iris. Alpha-toxin, the most potent S. aureus corneal toxin, was tested as a possible mediator of damage to the iris, and alpha-toxin anti-serum and a chemical toxin inhibitor were tested as potential pathology-reducing agents. METHODS: The hemolytic activity of alpha-toxin and its inhibition by a chemical inhibitor or anti-serum were quantified in vitro. Purified alpha-toxin, heat-inactivated toxin, or alpha-toxin plus normal serum, alpha-toxin anti-serum, or the chemical inhibitor, methyl-ß-cyclodextrin-cholesterol (CD-cholesterol), was injected into the rabbit anterior chamber. Pathological changes were photographed, quantified by slit-lamp examination (SLE) scoring, and further documented by histopathological analysis. RESULTS: At five hours post-injection, eyes injected with alpha-toxin or heat-inactivated toxin had a mean SLE score of 7.3 ± 0.59 or 0.84 ± 0.19, respectively. Active toxin caused moderate to severe iris edema, severe erosion of the iris, and mild to moderate fibrin accumulation in the anterior chamber. Alpha-toxin plus anti-serum or CD-cholesterol, in contrast to alpha-toxin alone, caused less iris edema and epithelium sloughing as well as significantly lower SLE scores than eyes receiving alpha-toxin alone (p ≤ 0.019). CONCLUSION: Alpha-toxin caused extensive iris damage and inflammation, and either anti-alpha-toxin anti-serum or CD-cholesterol was able to significantly reduce toxin-mediated damage and inflammation.

Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

PURPOSE: To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. METHODS: A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. RESULTS: LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). CONCLUSIONS: The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

Pseudomonas aeruginosa small protease (PASP), a keratitis virulence factor.

PURPOSE: The virulence contribution of Pseudomonas aeruginosa small protease (PASP) during experimental keratitis was studied by comparing a PASP-deficient mutant with its parent and rescue strains. METHODS: The pasP gene of P. aeruginosa was replaced with the tetracycline resistance gene via allelic exchange. A plasmid carrying the pasP gene was introduced into the PASP-deficient mutant to construct a rescue strain. The PASP protein in the culture supernatants was determined by Western blot analysis. Corneal virulence was evaluated in rabbit and mouse keratitis models by slit lamp examination (SLE), bacterial enumeration, and/or histopathological analysis. Various host proteins and the rabbit tear film were analyzed for their susceptibility to PASP degradation. RESULTS: The PASP-deficient mutant produced a significantly lower mean SLE score when compared with the parent or rescue strain (P ≤ 0.03) at 29 hours postinfection (PI). All of the strains grew equally in the rabbit cornea (P = 0.971). Corneas infected with the PASP-deficient mutant showed moderate histopathology compared with those infected with the parent or rescue strain, which produced severe pathology inclusive of epithelial erosions, corneal edema, and neutrophil infiltration. In the mouse model, eyes inoculated with the PASP-deficient mutant had a significantly lower mean SLE score at 24 hours PI than the eyes inoculated with the parent or rescue strain (P ≤ 0.007). PASP was found to degrade complement C3, fibrinogen, antimicrobial peptide LL-37, and constituents of the tear film. CONCLUSIONS: PASP is a commonly secreted protease of P. aeruginosa that contributes significantly to the pathogenesis of keratitis.

Infectious keratitis: secreted bacterial proteins that mediate corneal damage.

Ocular bacterial infections are universally treated with antibiotics, which can eliminate the organism but cannot reverse the damage caused by bacterial products already present. The three very common causes of bacterial keratitis-Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus pneumoniae-all produce proteins that directly or indirectly cause damage to the cornea that can result in reduced vision despite antibiotic treatment. Most, but not all, of these proteins are secreted toxins and enzymes that mediate host cell death, degradation of stromal collagen, cleavage of host cell surface molecules, or induction of a damaging inflammatory response. Studies of these bacterial pathogens have determined the proteins of interest that could be targets for future therapeutic options for decreasing corneal damage.

Staphylococcus aureus infection of the rabbit cornea following topical administration.

PURPOSE: To determine the ability of diverse S. aureus strains to infect the rabbit cornea following topical inoculation, with special emphasis on a strain of unusual virulence. MATERIALS AND METHODS: S. aureus strains (5 × 10(5) colony forming units; CFU) were topically applied onto scarified rabbit corneas or 100 CFU were intrastromally injected into rabbit corneas. Eyes were scored by slit lamp examination (SLE) and corneas were cultured to determine the log CFU. Polymorphonuclear leukocytes (PMN) were quantified by myeloperoxidase assays and corneas underwent histopathological analysis. Hemolysin titers of S. aureus strains were determined and S. aureus interactions with rabbit tears or human corneal epithelial cells were investigated. RESULTS: All strains injected into the cornea produced high SLE scores and multi-log increases in CFU. Following topical inoculation, four strains produced low SLE scores with no bacterial replication. One strain (UMCR1) topically infected the cornea, causing high SLE scores, extensive PMN infiltration, and multi-log increases in CFU. Histopathologic analysis demonstrated a PMN influx into the UMCR1-infected cornea, destruction of the corneal epithelium, and severe edema. Strain UMCR1 did not demonstrate a high hemolysin titer or resistance to the bactericidal activity of rabbit tears, but did invade human corneal epithelial cells with relatively high efficiency. CONCLUSIONS: One S. aureus strain demonstrated the ability to topically infect the rabbit cornea. This strain was previously found to be unique in its ability to infect the anterior chamber and conjunctiva, suggesting that a key mechanism may be employed to overcome the host defenses of these three ocular sites.

Identification and potency of cyclodextrin-lipid inhibitors of Staphylococcus aureus α-toxin.

PURPOSE: Staphylococcus aureus, a leading cause of bacterial keratitis, secretes α-toxin, a cytotoxin active on the corneal epithelium. This study describes the production and testing of chemical inhibitors of α-toxin action. METHODS: Purified α-toxin was titered by its ability to lyse rabbit erythrocytes in buffered saline (PBS). To prepare potential toxin inhibitors, each of 18 lipids was incorporated into a complex with methyl-ß-cyclodextrin (MßCD) or hydroxypropyl-ß-cyclodextrin (HPßCD). Serial dilutions of each lipid-cyclodextrin (CD-lipid) complex were mixed with α-toxin prior to the addition of rabbit erythrocytes. Select CD-lipid complexes were mixed with 12 hemolytic units (HU) α-toxin and injected into the rabbit corneal stroma so the resulting corneal erosions could be measured at 4 and 8 hours post-injection (PI). Eyes injected with toxin alone, MßCD, or HPßCD alone served as controls. RESULTS: Neither form of CD alone inhibited α-toxin. Of the 36 complexes prepared, 6 lipid-CD complexes were found to inhibit >100 HU of α-toxin. Four lipid complexes able to inhibit >200 HU of α-toxin were tested in toxin-injected corneas; at 4 and 8 hours PI, the complexes of cholesterol or lanosterol with MßCD and squalene or desmosterol with HPßCD caused a significant reduction in the corneal erosion size as compared to eyes injected with α-toxin alone (P ≤ 0.05). CONCLUSIONS: Specific lipid inclusion complexes with either MßCD or HPßCD demonstrated a significant inhibition of α-toxin in both in vitro and in vivo assays. Changes in either the cyclodextrin or lipid of a complex affected the inhibitory activity.

Diverse virulence of Staphylococcus aureus strains for the conjunctiva.

PURPOSE: To determine the virulence of Staphylococcus aureus strains in the rabbit conjunctiva. METHODS: Three strains of methicillin-sensitive S. aureus (8325-4, Newman, and UMCR1) and two strains of methicillin-resistant S. aureus (70490 and MW2) were analyzed. Rabbit bulbar conjunctivas (n ≥ 6 per group) were injected with 10(5) colony forming units (CFU) in 10 µl. Eyes were photographed and analyzed for pathology at 20 hr postinfection (PI) using slit lamp examination (SLE) to measure five parameters on a scale from 0 (normal) to 4 (severe): injection, chemosis, iritis, corneal edema, and pinpoint conjunctival hemorrhages. The parameter grades were added to produce a SLE score. Bacteria were enumerated and histopathological analysis was done at 20 hr PI. Myeloperoxidase assays were performed on conjunctival swabs (n ≥ 3 per strain) at 0 and 20 hr PI. RESULTS: Conjunctivas injected with 8325-4 or Newman had SLE scores of 1.67 ± 0.12 and 0.81 ± 0.16, respectively. Strain 70490 produced an average SLE score of 2.94 ± 0.47, whereas MW2 produced a score of 5.04 ± 0.73. UMCR1 produced severe conjunctivitis having a SLE score of 13.25 ± 0.80. Only strain UMCR1 grew in the conjunctiva showing a 2.7 log increase in CFU; all other strains remained near the inoculated numbers or decreased as much as 1.85 logs. Myeloperoxidase activity was greatest in the tear film of UMCR1 infected eyes with over one million PMN present at 20 hr PI. CONCLUSIONS: Only one S. aureus strain, UMCR1, was able to cause a reproducible severe conjunctivitis. This conjunctival infection could be used to test new antimicrobials and to help understand the pathogenesis of conjunctivitis, especially in terms of overcoming the host defenses.

Penetration and effectiveness of prophylactic fluoroquinolones in experimental methicillin-sensitive or methicillin-resistant Staphylococcus aureus anterior chamber infections.

PURPOSE: To determine the effectiveness of moxifloxacin and besifloxacin prophylactic therapy for experimental Staphylococcus aureus infections originating in the rabbit anterior chamber. SETTING: Microbiology Department, University of Mississippi Medical Center, Jackson, Mississippi, USA. DESIGN: Experimental study. METHODS: Minimum inhibitory concentrations (MICs) of moxifloxacin 0.5% and besifloxacin 0.6% for methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains were determined. Eyes were treated with moxifloxacin, a moxifloxacin alternative formulation 0.5%, or besifloxacin (45 µL) 30 minutes or 60 minutes before anterior chamber infection (10(6) colony-forming units [CFUs]). Aqueous humor was removed 30 minutes after infection for quantification of antibiotic and bacteria. RESULTS: The MIC for both organisms was 0.06 µg/mL for moxifloxacin and 0.03 µg/mL for besifloxacin. In MSSA infections, the untreated eyes contained 5.18 log CFU/mL, which was similar to besifloxacin-treated eyes with either treatment (P≥.1091). Eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly fewer CFUs than untreated controls or besifloxacin-treated eyes with either treatment (P≤.0020). The aqueous humor in eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly more drug than besifloxacin-treated eyes at both prophylactic time points (P≤.0012). In MRSA infections, the untreated eyes contained 4.91 log CFU/mL, which was similar to besifloxacin-treated eyes with either treatment (P≥.5830). Eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly fewer CFUs than untreated controls or besifloxacin-treated eyes at both prophylactic time points (P≤.0008). CONCLUSIONS: Moxifloxacin had greater in vivo effectiveness against MSSA and MRSA than besifloxacin. The aqueous antibiotic concentrations suggest limited penetration by besifloxacin, accounting for its lack of effectiveness.

The effectiveness of an improved combination therapy for experimental Staphylococcus aureus keratitis.

INTRODUCTION: antibiotic and steroid combination therapies, such as tobramycin with dexamethasone, are often used in ophthalmology to treat or prevent infection and inflammation. The purpose of this study was to use a model of Staphylococcus aureus keratitis to quantify and compare the effectiveness of a standard tobramycin and dexamethasone combined therapy, with each drug individually, and with a new formulation of the two drugs in a xanthan gum vehicle. METHODS: rabbit corneas were intrastromally injected with a methicillin-sensitive S. aureus (MSSA) or a methicillin-resistant S. aureus (MRSA) strain. Rabbit eyes were treated every hour from 10 to 15 hours postinfection (PI) with 0.1% dexamethasone, 0.3% tobramycin, 0.3% tobramycin with 0.1% dexamethasone, or 0.3% tobramycin with 0.05% dexamethasone in a xanthan gum vehicle (ST). Slit lamp examinations (SLE) were performed on infected eyes and pathology scored at 15 hours PI. At 16 hours PI, colony forming units (CFUs) per cornea were quantified. RESULTS: the CFUs in eyes treated with dexamethasone alone were similar to untreated control eyes for MSSA or MRSA infections. All other treatment groups had significantly less CFUs per cornea than untreated eyes. The eyes treated with the ST formulation had significantly fewer CFUs per cornea than all other treatment groups when infected with MSSA or MRSA. The SLE scores of MSSA or MRSA infected eyes treated with tobramycin alone were similar to untreated control eyes. All other treatment groups had significantly lower SLE scores than untreated controls eyes, but were not significantly different from each other. CONCLUSION: the results of this study demonstrated that the tobramycin and dexamethasone combination therapy with a xanthan gum vehicle has an improved bactericidal effectiveness compared to the commercially available formulation, and maintains a similar anti-inflammatory effect while containing half the amount of steroid.

Sustained anti-staphylococcal effect of lysostaphin in the rabbit aqueous humor.

PURPOSE: Staphylococcus aureus is an important cause of ocular infections including endophthalmitis. The purpose of this study was to determine the ability of a relatively large molecule, such as lysostaphin, to remain in the rabbit aqueous humor for extended periods while retaining its bactericidal activity. METHODS: Lysostaphin, gatifloxacin, or Tris-buffered saline (TBS) was injected into the rabbit anterior chamber. Aqueous humor was sampled at 0.5, 1, 2, 3, and 4 days after injection, and then assayed for bactericidal activity against S. aureus. The anterior chamber of treated eyes was also challenged 2 days after treatment by an infection of S. aureus. The surviving bacteria were quantified to determine bactericidal effectiveness in the anterior chamber. The aqueous humor of lysostaphin injected eyes was assayed by western blot for the presence of the molecule 2 days post-injection. RESULTS: The bactericidal activity of lysostaphin was confirmed by lysis of S. aureus and sensitivity to zinc. Eyes injected with lysostaphin showed no adverse reactions. Aqueous humor of gatifloxacin injected eyes demonstrated no greater effectiveness than that of TBS injected eyes in vitro at any time point assayed, whereas lysostaphin injected eyes retained potent bactericidal activity for at least 3 days. In an in vivo challenge, the anterior chamber of lysostaphin injected eyes retained significant bactericidal activity for at least 2 days after treatment, whereas gatifloxacin injected eyes demonstrated no significant difference from those injected with TBS. Western blot analysis demonstrated the presence of lysostaphin in the aqueous humor 2 days post-injection. CONCLUSIONS: Lysostaphin demonstrated the ability to remain in the aqueous humor for days while maintaining its bactericidal activity, an indication that a high molecular weight antimicrobial can provide prolonged prophylactic protection of the anterior chamber.