Joseph J. Dajcs

Immunization with alpha-toxin toxoid protects the cornea against tissue damage during experimental Staphylococcus aureus keratitis.

ABSTRACT Alpha-toxin is a major virulence factor in Staphylococcus aureus keratitis. Active or passive immunization with alpha-toxin toxoid could protect against corneal damage. Results show that either form of immunization did not kill bacteria but did significantly protect against corneal pathology, especially epithelial erosion.

Lysostaphin is effective in treating methicillin-resistant Staphylococcus aureus endophthalmitis in the rabbit

Purpose. To determine the effectiveness of lysostaphin treatment of experimental endophthalmitis caused by methicillin-resistant Staphylococcus aureus (MRSA). Methods. In one experiment, rabbits were injected in the mid-vitreous with 50 or 200 CFU of S. aureus; untreated groups and groups injected intra-vitreally at 8 or 24 hours postinfection with vehicle or lysostaphin (0.1 mg/ml) were compared in terms of CFU/ml vitreous at 24 or 48 hours postinfection. Histopathology of untreated and treated eyes was also compared. To quantify the potency of lysostaphin, additional rabbits were injected with 50 CFU of S. aureus and untreated eyes and eyes treated at 8 hours with 0.001, 0.01 or 0.05 mg/ml were compared in terms of CFU/ml vitreous at 24 hours postinfection. Results. Vitreous of untreated eyes or vehicle-treated eyes injected with 50 or 200 CFU of S. aureus contained 5–10 million CFU/ml at 24 or 48 hours postinfection. All eyes treated with lysostaphin at 8 hours postinfection had less than 1 log CFU/ml in the vitreous (P = 0.0001). Similarly, eyes treated with lysostaphin at 24 hours postinfection had approximately 1 log of CFU/ml at 48 hours postinfection. None of the untreated eyes were sterile and 88% or 50% of the eyes treated at 8 or 24 hours postinfection, respectively, were sterile. Eyes treated with lysostaphin at 8, but not 24, hours postinfection had less pronounced pathologic changes than the untreated eyes (P = 0.002). A significant reduction in the CFU/ml vitreous at 24 hours postinfection was obtained by treating infected eyes at 8 hours postinfection with lysostaphin at concentrations of =0.001 mg/ml (P = 0.0034). Conclusions. Lysostaphin is effective in treating experimental endophthalmitis mediated by MRSA.

Effectiveness of Ciprofloxacin, Levofloxacin, or Moxifloxacin for Treatment of Experimental Staphylococcus aureus Keratitis

ABSTRACT The purpose of this study was to quantitatively compare, in a rabbit keratitis model, the levels of effectiveness of moxifloxacin, levofloxacin, and ciprofloxacin for the treatment of Staphylococcus aureus isolates of diverse antibiotic susceptibilities. Rabbit eyes were intrastromally injected with approximately 100 CFU of methicillin-sensitive or methicillin-resistant S. aureus (MSSA or MRSA, respectively) organisms that were either sensitive or resistant to ofloxacin. One drop of moxifloxacin (0.5%), levofloxacin (0.5%), or ciprofloxacin (0.3%) was topically applied hourly from 4 to 9 (early) or 10 to 15 (late) h postinfection. At 1 h after cessation of therapy, the corneas were harvested, and the number of CFU per cornea was determined. For the ofloxacin-sensitive strains, early treatment of MSSA or MRSA with moxifloxacin, levofloxacin, or ciprofloxacin produced approximately a 5-log decrease in CFU per cornea relative to that in untreated eyes (P ≤ 0.0001). For late therapy of ofloxacin-sensitive strains, moxifloxacin, levofloxacin, and ciprofloxacin produced approximately 5-, 4-, and 2- to 3-log reductions in CFU per cornea, respectively (P ≤ 0.0001). Early treatment of the ofloxacin-resistant strains with either moxifloxacin or levofloxacin produced a ≥4-log or ≥3-log decrease, respectively, in the MSSA or MRSA strains (P ≤ 0.0001), whereas ciprofloxacin treatment produced a 1-log decrease in CFU per cornea relative to that in untreated eyes (P = 0.1540). For late treatment of ofloxacin-resistant strains, levofloxacin and ciprofloxacin failed to significantly reduce the number of CFU per cornea (P ≥ 0.3627), whereas moxifloxacin produced a significant reduction in CFU per cornea of approximately 1 log (P ≤ 0.0194). Therefore, for three of the four treatments tested, moxifloxacin demonstrated greater effectiveness than either levofloxacin or ciprofloxacin.

Corneal virulence of Staphylococcus aureus in an experimental model of keratitis.

The aim of this study was to determine the pathogenic role of alpha-, beta-, and gamma-toxins in a rabbit model of Staphylococcus aureus keratitis. S. aureus strains 8325-4, Newman, and their isogenic mutants were intrastromally injected into rabbit corneas. Eyes were scored for pathology by slit lamp examination (SLE), histologic examination, and bacterial colony-forming units (CFU) per cornea were determined. Rabbits were immunized against alpha-toxin and subsequently challenged with S. aureus strain 8325-4 or Newman. All strains grew equivalently to approximately 7 log CFU/cornea at 25 h postinfection. SLE scores at 15, 20, and 25 h postinfection revealed that alpha-toxin - producing strains caused greater corneal pathology than strains deficient in alpha-toxin. A beta-toxin - deficient mutant produced significantly less ocular edema than its parent or rescued strains. The gamma-toxin-deficient mutant, relative to its parent strain or genetically rescued strain, had reduced virulence. These results demonstrate that the virulence of S. aureus involves mainly alpha-toxin and to a lesser extent gamma-toxin, with beta-toxin mediating minimal corneal pathology.

Quantitative comparison of fluoroquinolone therapies of experimental Gram-negative bacterial keratitis

Purpose. To determine the effectiveness of topically applied fluoroquinolones for experimental Pseudomonas or Serratia keratitis. Methods. Bacteria were injected intrastromally (10 3 colony forming units [CFU]). From 16 to 22 hours post-infection (PI), a single topical drop of moxifloxacin (Vigamox ®, 0.545%), levofloxacin (Quixin™, 0.5%), ofloxacin (Ocuflox ®, 0.3%) or ciprofloxacin (Ciloxan ®, 0.3%) was applied every 30 minutes. At 23 hours PI, corneas were cultured quantitatively. Results. For Pseudomonas keratitis, untreated eyes contained 7 log CFU/cornea and antibiotic-treated eyes demonstrated a = 5-log reduction in CFU/cornea (p = 0.0001). Moxifloxacin, levofloxacin, or ciprofloxacin therapies were not significantly different from each other (p = 0.67). For Serratia keratitis, untreated eyes contained 7 log CFU/cornea whereas treated eyes had a = 2-log reduction (p = 0.0001). Moxifloxacin therapy proved most effective (p = 0.001). Conclusions. Overall, moxifloxacinwas the most effective of the four fluoroquinolones in reducing CFU/cornea in the rabbit model of Gram-negative keratitis.

Effectiveness of mupirocin and polymyxin B in experimental Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens keratitis.

Purpose. The purpose of this study was to determine the effectiveness of mupirocin and polymyxin B, alone and in combination, in vitro and in vivo using rabbit models of Staphylococcus aureus, Pseudomonas aeruginosa, and Serratia marcescens keratitis. Methods. Rabbit eyes were intrastromally injected with 1,000 colony-forming units (CFUs) of P. aeruginosa or S. marcescens or 100 CFUs of S. aureus. Rabbits were then treated with 2.7 mg/mL mupirocin, 10,000 U/mL polymyxin B, a mupirocin:polymyxin B combination, or 0.3% ciprofloxacin. Vehicle and untreated controls were also included. Treatment schedules depended on the strain injected. The number of CFUs was determined for all eyes after treatment. Results. The mupirocin:polymyxin B combination was effective for all three genera both in vitro and in vivo. For S. aureus keratitis, the mupirocin:polymyxin B combination was more effective than either drug alone and significantly reduced the log number of bacteria in the cornea by more than 3 logs compared with the vehicle or untreated controls (p ≤ 0.0016). For P. aeruginosa, the mupirocin:polymyxin B combination treatment significantly reduced the number of CFUs per cornea relative to the individual drugs, vehicle, or untreated controls (p ≤ 0.016). For S. marcescens, the mupirocin:polymyxin B combination therapy significantly reduced the number of bacteria in rabbit corneas relative to the individual drugs, vehicle, or untreated groups (p ≤ 0.0001). Therapy with the mupirocin:polymyxin B combination was equivalent to ciprofloxacin therapy (p = 0.80). Conclusion. The mupirocin:polymyxin B combination was effective in treating experimental S. aureus, P. aeruginosa, and S. marcescens keratitis.

Staphylococcus petrasii sp. nov. including S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov., isolated from human clinical specimens and human ear infections

Thirteen coagulase-negative, oxidase-negative, and novobiocin-susceptible staphylococci were isolated from human clinical specimens. The isolates were differentiated from known staphylococcal species on the basis of 16S rRNA, hsp60, rpoB, dnaJ, tuf, and gap gene sequencing, automated ribotyping, (GTG)5-PCR fingerprinting, and MALDI-TOF MS analysis. Phylogenetic analysis based on the 16S rRNA gene sequence indicated phylogenetic relatedness of the analyzed strains to Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus devriesei, and Staphylococcus lugdunensis. DNA-DNA hybridization experiments between representative strains CCM 8418(T), CCM 8421(T), and the closest phylogenetic neighbors confirmed that the isolates represent novel Staphylococcus species, for which the name Staphylococcus petrasii sp. nov. is proposed. Genotypic and phenotypic analyses unambiguously split the strains into two closely related subclusters. Based on the results, two novel subspecies S. petrasii subsp. petrasii subsp. nov. and S. petrasii subsp. croceilyticus subsp. nov. are proposed, with type strains CCM 8418(T) (=CCUG 62727(T)) and CCM 8421(T) (=CCUG 62728(T)), respectively.

Host defense against bacterial keratitis.

Purpose: To define factors that protect the eye from Staphylococcus aureus keratitis and limit tissue damage once keratitis occurs. Methods: Rabbit tears were analyzed for bactericidal and phospholipase A 2 (PLA 2 ) activities on S. aureus. Inhibition by spermidine of PLA 2 anti-staphylococcal activity in tears was tested in vitro and in vivo. Rabbits immunized with heat-inactivated alpha-toxin were challenged with intrastromal injection of S. aureus. Results: Arachidonic acid was cleaved from S. aureus by purified PLA 2 or rabbit tears. Spermidine inhibited these reactions in vitro and facilitated keratitis in vivo. PLA 2 activity decreased with advanced age and shortly following sleep, but increased with keratitis. Antibody to alpha-toxin significantly reduced corneal damage and epithelial cell sloughing during keratitis. Conclusions: PLA 2 is a major host-defense component of rabbit tears. Alpha-toxin is a major mediator of corneal damage, and antibody to alpha-toxin reduces pathologic changes during keratitis.

Effectiveness of ciprofloxacin and ofloxacin in a prophylaxis model of Staphylococcus keratitis.

Purpose. To determine the effectiveness of prophylactic fluoroquinolone treatment against staphylococci in a rabbit keratitis model. Methods. Prophylactic ciprofloxacin or ofloxacin was applied as one topical drop 15 minutes before infection or as one drop at three time points (19, 17, and 15 minutes) before infection. In a second experiment, rabbits were treated with two, three, or four drops of ciprofloxacin 1 hour before infection. Approximately 250 colony-forming units (CFUs) of Staphylococcus aureus were injected intrastromally, and CFUs were determined 5 hours after infection. Results. The CFUs per cornea in all treatment groups were significantly less than the 5.6 ± 0.11 log CFUs per cornea in the untreated group (p ≤ 0.0001). Rabbit eyes treated 15 minutes before infection with Ciloxan or Ocuflox had 0.96 ± 0.48 log CFUs per cornea (three of six sterile corneas) or 1.26 ± 0.31 log CFUs per cornea (one of six sterile corneas), respectively (p = 0.5226). Eyes treated with Ciloxan 19, 17, and 15 minutes before infection had 0.0 ± 0.0 log CFUs per cornea, and all eyes were sterile, whereas eyes treated with Ocuflox had 0.98 ± 0.48 log CFUs per cornea and two of six eyes sterile (p = 0.0435). Eyes treated 1 hour before infection with two, three, or four drops of Ciloxan had 2.61 ± 0.69 log CFUs, 1.23 ± 0.32 log CFUs, or 0.85 ± 0.28 log CFUs per cornea, respectively, which was significantly less than untreated eyes (p ≤ 0.0001). Conclusions. Multiple topical drops of a fluoroquinolone administered prophylactically were effective for subsequent staphylococcal ocular infection.

The effectiveness of tobramycin and Ocuflox® in a prophylaxis model of Staphylococcus keratitis

Purpose. To determine the effectiveness of prophylactic antibiotic treatment prior to intra-corneal infection with Staphylococcus aureus. Methods. One topical drop of Tobrex ® (0.3% tobramycin), tobramycin (0.3%) in the Tobrex ® vehicle with 0.05% dodecyl maltoside (DDM)/4.0% hydroxypropylmethycellulose (HPMC), Ocuflox ® (0.3% ofloxacin) or DDM/HPMC vehicle were applied to rabbit eyes at one or five hours prior to injection of bacteria. Approximately 500 colony-forming units (CFU) of S. aureus strain 8325-4 were injected into the corneal stroma. Rabbits were sacrificed five hours after infection and corneal homogenates were cultured to determine the number of colony forming units (CFU) per cornea. Results. Rabbits treated at five hours prior to infection with tobramycin-DDM/HPMC reduced the bacterial load by approximately 2.4 log CFU/cornea as compared to the untreated control (3.47 ± 0.98 vs. 5.71 ± 0.14 log CFU/cornea, respectively; P = 0.0010); however, Ocuflox ®, Tobrex ®, or DDM/HPMC vehicle did not significantly reduce the log CFU (P = 0.4837). Rabbits treated at 1 hour prior to infection with Ocuflox ® or tobramycin-DDM/HPMC had significantly reduced CFU/cornea (1.31 ± 0.86 and 0.48 ± 0.31 log CFU/cornea, respectively) as compared to the untreated group (5.71 ± 0.14 log CFU/cornea; P = 0.0001). Neither Tobrex ® nor the DDM/HPMC vehicle significantly reduced the CFU/cornea compared to the untreated group (P = 0.2312). Conclusions. This pre-treatment model of Staphylococcus keratitis quantitatively measured the prophylactic effectiveness of topical antibiotic formulations. An important finding was that a tobramycin-DDM/HPMC formulation was highly effective as a prophylactic medication.