Bacillus cereus induces permeability of an in vitro blood-retina barrier.

Most Bacillus cereus toxin production is controlled by the quorum-sensing-dependent, pleiotropic global regulator plcR, which contributes to the organism's virulence in the eye. The purpose of this study was to analyze the effects of B. cereus infection and plcR-regulated toxins on the barrier function of retinal pigment epithelium (RPE) cells, the primary cells of the blood-retina barrier. Human ARPE-19 cells were apically inoculated with wild-type or quorum-sensing-deficient B. cereus, and cytotoxicity was analyzed. plcR-regulated toxins were not required for B. cereus-induced RPE cytotoxicity, but these toxins did increase the rate of cell death, primarily by necrosis. B. cereus infection of polarized RPE cell monolayers resulted in increased barrier permeability, independent of plcR-regulated toxins. Loss of both occludin and ZO-1 expression occurred by 8 h postinfection, but alterations in tight junctions appeared to precede cytotoxicity. Of the several proinflammatory cytokines analyzed, only interleukin-6 was produced in response to B. cereus infection. These results demonstrate the deleterious effects of B. cereus infection on RPE barrier function and suggest that plcR-regulated toxins may not contribute significantly to RPE barrier permeability during infection.

Clonal associations among Staphylococcus aureus isolates from various sites of infection.

A molecular epidemiological analysis was undertaken to identify lineages of Staphylococcus aureus that may be disproportionately associated with infection. Pulsed-field gel electrophoresis analysis of 405 S. aureus clinical isolates collected from various infection types and geographic locations was performed. Five distinct S. aureus lineages (SALs 1, 2, 4, 5, and 6) were identified, which accounted for 19.01, 9.14, 22.72, 10.12, and 4.69% of isolates, respectively. In addition, 85 lineages which occurred with frequencies of <2.5% were identified and were termed "sporadic." The most prevalent lineage was methicillin-resistant S. aureus (SAL 4). The second most prevalent lineage, SAL 1, was also isolated at a high frequency from the anterior nares of healthy volunteers, suggesting that its prevalence among clinical isolates may be a consequence of high carriage rates in humans. Gene-specific PCR was carried out to detect genes for a number of staphylococcal virulence traits. tst and cna were found to be significantly associated with prevalent lineages compared to sporadic lineages. When specific infection sites were examined, SAL 4 was significantly associated with respiratory tract infection, while SAL 2 was enriched among blood isolates. SAL 1 and SAL 5 were clonally related to SALs shown by others to be widespread in the clinical isolate population. We conclude from this study that at least five phylogenetic lineages of S. aureus are highly prevalent and widely distributed among clinical isolates. The traits that confer on these lineages a propensity to infect may suggest novel approaches to antistaphylococcal therapy.

In vitro pharmacodynamics of ofloxacin and ciprofloxacin against common ocular pathogens.

PURPOSE: Time-kill curve methodology was used to assess the pharmacodynamics of two fluoroquinolones, ofloxacin and ciprofloxacin, against six strains representing the most common ocular pathogens: Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Serratia marcescens, and Haemophilus influenzae. METHODS: For time-kill studies, ofloxacin and ciprofloxacin solutions were prepared at concentrations of 0.5 x, 1.0 x, 2.0 x, and 3.0 x the MIC (minimal inhibitory concentration) for each respective strain. Inocula were prepared by diluting overnight cultures to final concentrations of 10(2), 10(3), 10(4), and 10(5) cfu (colony-forming units)/mL in each antibiotic solution. Growth controls were included. Viability counts of antibiotic-containing and control bacterial suspensions were performed at 0, 10, 20, 30, 60, 90, 120, and 180 minutes. RESULTS: In general, the kill rates of ofloxacin at 1.0 x, 2.0 x, and 3.0 x the MIC were significantly faster than the kill rates of ciprofloxacin by approximately 30 minutes, regardless of the bacterial concentration tested. At 0.5 x MIC, the kill kinetics of ciprofloxacin and ofloxacin were similar, regardless of the strain tested. At 1.0 x MIC, ofloxacin achieved 99.9% killing of P. aeruginosa and S. aureus within 30 minutes, and S. epidermidis and S. marcescens within 90 minutes. Overall, the kill kinetics of both quinolones for H. influenzae were similar, while neither quinolone achieved 99.9% killing of S. pneumoniae, regardless of the antibiotic concentration tested. CONCLUSION: Time-kill curve analyses in the present study demonstrate that ofloxacin achieved killing of the majority of ocular pathogens tested at rates equivalent to or faster than that of ciprofloxacin. Both fluoroquinolones were more effective against nonencapsulated bacteria than against encapsulated bacteria.

Role of hemolysin BL in the pathogenesis of extraintestinal Bacillus cereus infection assessed in an endophthalmitis model.

Bacillus cereus is a rare cause of serious human infection but, paradoxically, causes one of the most severe posttraumatic or endogenous infections of the eye, endophthalmitis, which frequently results in blindness. The virulence of B. cereus endophthalmitis historically has been attributed to toxin production. We therefore sought to examine the contribution of the dermonecrotic toxin, hemolysin BL, to the pathogenesis of B. cereus infection in an endophthalmitis system that is highly amenable to study. The pathogenesis of infection resulting from intravitreal injection of 10(2) CFU of either a clinical ocular isolate of B. cereus producing hemolysin BL (HBL+) or an isogenic mutant in this trait (HBL-) was assessed bacteriologically and by slit lamp biomicroscopy, electroretinography, histology, and inflammatory cell enumeration. Both HBL+ and HBL- strains evoked severe intraocular inflammatory responses as early as 12 h postinfection, with complete loss of retinal responsiveness by 12 h. The infections caused by both strains spread of the infection to adjacent tissues by 18 h. No significant differences in intraocular bacterial growth (P >/= 0.21) or inflammatory changes (P >/= 0.21) were observed in eyes infected with either HBL+ or HBL- strains during the course of infection. The level of retinal responsiveness was greater in HBL- infected eyes than in HBL+-infected eyes at 6 h only (P = 0.01). These results indicate that hemolysin BL makes no essential contribution to the severe and rapid course of infection in the endophthalmitis model.

Pathogenesis of gram-positive bacterial endophthalmitis.

The severity of endophthalmitis has been associated generally with the virulence of the offending pathogen. However, precisely what constitutes the virulence in intraocular infections remains ill defined. We therefore sought to identify the basis for virulence for three common ocular pathogens (Bacillus cereus, Enterococcus faecalis, and Staphylococcus aureus) in terms of intraocular growth rates, bacterial localization patterns, and the contribution of cell walls and secreted products to the pathogenesis of endophthalmitis. Rabbit eyes were injected intravitreally with (i) viable B. cereus, E. faecalis, or S. aureus, (ii) metabolically inactive B. cereus, E. faecalis, or S. aureus, (iii) sacculus preparations from each strain, or (iv) culture fluid containing products secreted by each strain. Eyes were assessed at various times following injection by slit lamp biomicroscopy, electroretinography (ERG), bacterial and inflammatory cell enumeration, and histology. B. cereus endophthalmitis followed a more rapid and virulent course than E. faecalis or S. aureus endophthalmitis, eliminating retinal responsiveness, as measured by ERG, by 12 h. Analysis of bacterial localization revealed that B. cereus uniquely migrated rapidly from posterior to anterior segment during infection. Although injection of neither metabolically inactive bacteria nor cell wall sacculi greatly affected ERG, significant intraocular inflammation was observed. Injection of B. cereus or S. aureus culture fluids caused both significant reductions in retinal responsiveness and significant intraocular inflammation, paralleling that seen in natural infections. The results demonstrate that toxins, intraocular localization, and, to a lesser extent, the intraocular host response to cell walls all contribute to the pathogenesis of B. cereus, S. aureus, and E. faecalis endophthalmitis in a pathogen-specific manner. The key pathophysiologic differences in these intraocular diseases highlight opportunities for optimizing conventional therapies and deriving new ones.

Molecular epidemiology of Staphylococcus aureus and Enterococcus faecalis in endophthalmitis.

Genomic DNA fingerprint analysis was performed on 39 Staphylococcus aureus and 28 Enterococcus faecalis endophthalmitis isolates collected from multiple clinical centers. Among 21 S. aureus genomic DNA fingerprint patterns identified, five clonotypes were recovered from multiple unrelated patients and accounted for 58.9% (23 of 39) of the isolates analyzed. Compared with strains having unique genomic DNA fingerprint patterns, the S. aureus clonotypes occurring more than once were more likely to result in visual acuities of 20/200 or worse (P = 0.036 [chi2 test]). In contrast to the S. aureus isolates, the E. faecalis endophthalmitis isolates were a clonally diverse population, enriched for the expression of a known toxin, cytolysin, which is plasmid encoded.

Pseudomonas deficient in protease IV has significantly reduced corneal virulence.

PURPOSE: The role of protease IV in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated by comparing a mutant strain completely deficient in protease IV activity with its protease IV activity-producing parent. METHODS: A protease IV-deficient Pseudomonas strain PA103-29::Tn9 was generated by mutagenesis of strain PA103-29, which produces protease IV, through transposon insertion. Protease IV activity was determined by a casein agar assay, zymography, and cleavage of the chromogenic substrate, Chromozym PL. Corneal virulence was evaluated by slit lamp examination and bacterial cultures in both a rabbit intrastromal model and a mouse topical model of keratitis. RESULTS: The protease IV-deficient strain PA103-29::Tn9 had significantly reduced corneal virulence relative to its parent strain PA103-29 in both a rabbit intrastromal model and a mouse topical model of infection. In the rabbit model, ocular damage (slit lamp examination score) mediated by the parent strain was severe at 32 hours after infection, whereas damage mediated by the mutant was minimal at both 32 and 55 hours after infection. This difference in virulence was not a result of differences in growth in vivo, because both strains grew equally. In the mouse model, eyes inoculated with the protease IV-producing parent strain had significant corneal damage as early as 24 hours after infection, whereas the protease IV-deficient mutant strain produced no significant corneal damage during 6 days of infection. CONCLUSIONS: The ability to produce active protease IV was the determining factor in the severity of corneal virulence. Protease IV appears to mediate corneal virulence and should be considered as a target in the development of medications designed to minimize corneal damage during Pseudomonas keratitis.

Specific roles of alpha-toxin and beta-toxin during Staphylococcus aureus corneal infection.

Staphylococcus aureus corneal infection results in extensive inflammation and tissue damage. Our previous studies of bacterial mutants have demonstrated a role for alpha-toxin in corneal virulence. This study analyzes, by genetic rescue experiments, the virulence of mutants affecting alpha-toxin and beta-toxin activity and demonstrates the ocular toxicity of these purified staphylococcal proteins. Three types of isogenic mutants were analyzed: (i) mutants specifically deficient in alpha-toxin (Hla) or beta-toxin (Hlb), (ii) a mutant deficient in both Hla and Hlb, and (iii) a regulatory mutant, deficient in the accessory gene regulator (agr), that produces reduced quantities of multiple exoproteins, including alpha- and beta-toxins. Plasmids coding for Hla and Hlb (pDU1212 and pCU1hlb, respectively) were used to restore toxin activity to mutants specifically deficient in each of these toxins. Either corneas were injected intrastromally with logarithmic-phase S. aureus or purified alpha- or beta-toxins were administered to normal eyes. Ocular pathology was evaluated by slit lamp examination and myeloperoxidase activity of infiltrating polymorphonuclear leukocytes. Corneal homogenates were cultured to determine the CFU per cornea. Eyes infected with the wild-type strain developed significantly greater corneal damage than eyes infected with Agr-, Hlb-, or Hla- strains. Epithelial erosions produced by parent strains were not produced by Agr- or Hla- strains. Hlb+ strains, unlike Hlb- strains, caused scleral edema. Plasmid pDU1212 restored corneal virulence to strain DU1090 (Hla-), and plasmid pCU1hlb restored corneal virulence to strain DU5719 (Hlb-). Application of purified alpha-toxin produced corneal epithelial erosions and iritis, while application of beta-toxin caused scleral inflammation. These studies confirm the role of alpha-toxin as a major virulence factor during S. aureus keratitis and implicate beta-toxin, a mediator of edema, as a lesser contributor to ocular damage.

Staphylococcal accessory regulator (sar) in conjunction with agr contributes to Staphylococcus aureus virulence in endophthalmitis.

Previous studies showed that an agr mutant strain of Staphylococcus aureus was partially attenuated in virulence compared to a parental strain in experimental endophthalmitis. The purpose of this study was to determine whether the sar locus, either alone or through interactions with agr, contributes to the regulation of virulence in S. aureus endophthalmitis. Experimental endophthalmitis was established by the midvitreous injection of approximately 30 CFU of S. aureus RN6390 or the isogenic attenuated strains RN6911 (agr mutant), ALC136 (sar mutant), and ALC135 (agr sar double mutant). Unexpectedly, the rate of reduction in electroretinographic B-wave amplitude in eyes infected with strain ALC136 (sar mutant) was not significantly different from the parental strain on postinfection day (PID) 5 (10% retention). In contrast, ALC135 (agr sar double mutant)-infected eyes retained 73% of preoperative B-wave amplitude on PID 5. Therefore, unlike agr, a mutation in the sar locus alone does not alter the overall virulence of wild-type S. aureus in experimental endophthalmitis. However, the combined effect of insertional mutations in both the sar and agr global regulators leads to near-complete attenuation of virulence.

Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence.

PURPOSE: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV. METHODS: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent. Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria. RESULTS: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate. Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02). CONCLUSIONS: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface. Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.

The effectiveness of two ciprofloxacin formulations for experimental Pseudomonas and Staphylococcus keratitis.

Ciprofloxacin is a fluoroquinolone antibiotic with broad spectrum bactericidal activity. A commercially available form of ciprofloxacin contains benzalkonium chloride (BAC) (0.006%) and EDTA (0.05%) as preservatives. Since BAC has been shown to cause adverse changes in corneal epithelial cells, a formulation of ciprofloxacin devoid of BAC and EDTA but with the same effectiveness would be valuable. We present here the results of experiments designed to assess the efficacy of a BAC-free and EDTA-free formulation of ciprofloxacin, Ciprofloxacin-polystyrene sulfonate (PSS), in experimental models of Pseudomonas aeruginosa and Staphylococcus aureus keratitis. Both formulations of ciprofloxacin sterilized corneas infected with P aeruginosa, and both formulations showed equal bactericidal activity for S aureus. Normal eyes treated with either formulation showed mild conjunctival irritation compared to untreated normal eyes. The bactericidal activities of both formulations of ciprofloxacin were excellent. Therefore, the Ciprofloxacin-PSS formulation could serve as an effective single drug therapy for ocular infections.

Pharmacokinetics of topically applied ciprofloxacin in rabbit tears.

Fluoroquinolones provide an important single antibiotic therapy for bacterial keratitis caused by Staphylococcus aureus and numerous gram-negative bacteria, including Pseudomonas aeruginosa. The pharmacokinetics of three ocular fluoroquinolones, norfloxacin (CHIBOXIN, Merck, Sharp & Dohme), ciprofloxacin (CILOXAN, Alcon Laboratories), and ofloxacin (OCUFLOX, Allergan Pharmaceuticals), have been studied in the human eye and in both the normal rabbit eye and rabbit models of keratitis. However, the pharmacokinetics of ciprofloxacin have not been previously studied in the tear film of rabbits. This study was done to determine the pharmacokinetics of topical ciprofloxacin in the rabbit tear film. Two drops of CILOXAN were applied to the eyes of normal rabbits. Tear samples were collected at 5, 10 and 30 minutes, and 1, 2, 4 and 6 hours after topical drug application. Tear samples were analyzed for ciprofloxacin concentrations by HPLC. Ciprofloxacin concentrations reached a peak at 5 minutes, then declined in a manner similar to that reported for norfloxacin and ofloxacin. The ciprofloxacin concentrations in tears were substantially higher throughout the length of the study than the MIC90 for most ocular pathogens including Staphylococcus aureus and Pseudomonas aeruginosa.

Growth and virulence of a complement-activation-negative mutant of Streptococcus pneumoniae in the rabbit cornea.

Our previous work has demonstrated the importance of pneumolysin in the virulence of S. pneumoniae in a rabbit intracorneal model. This was accomplished by showing that deletion of the gene encoding pneumolysin resulted in reduced virulence, whereas restoration of the wild-type gene resulted in restoration of the virulent phenotype. To assess the importance of a particular domain in the pneumolysin molecule, we have now constructed a strain which produces a pneumolysin molecule which is hemolytic but which bears a site-specific mutation in the domain known to be associated with the complement-activating properties of this molecule. Comparison of the virulence of this strain with that of a strain bearing the wild-type gene showed statistically significantly lower total slit lamp examination (SLE) scores at 12, 18, 24, and 36 h (particularly with respect to fibrin formation), but no difference at 48 h. Determination of colony forming units (CFU) in eyes infected with the two strains showed approximately 10(6) bacteria per cornea until 36 h. Between 36 and 48 h, the bacteria were almost completely cleared with very few bacteria recoverable at the later time point. The loss of virulence observed with this mutation in the complement-activation domain of pneumolysin, though less than that observed with the gene deletion mutant, suggests that complement activation by pneumolysin has a significant role in the pathology observed in this model of corneal infection.

Effectiveness of specific antibiotic/steroid combinations for therapy of experimental Pseudomonas aeruginosa keratitis.

Ciprofloxacin and prednisolone, but not an aminoglycoside and dexamethasone, were previously found to be effective in killing bacteria and reducing inflammation for the treatment of Pseudomonas keratitis. We investigated the therapeutic effectiveness of tobramycin/prednisolone and ciprofloxacin/dexamethasone in a rabbit model of experimental keratitis to increase our understanding of the effectiveness of antibiotic/steroid combinations. To our knowledge, this is the first analysis of the effectiveness of a combination of ciprofloxacin and dexamethasone for experimental keratitis. Two experiments were conducted. In the first experiment, 36 rabbits were divided into six groups: 1) untreated; 2) prednisolone acetate, 1.0%; 3) prednisolone phosphate, 1.0%; 4) tobramycin, 1.36%; 5) tobramycin plus prednisolone acetate; 6) tobramycin plus prednisolone phosphate. In the second experiment, 23 rabbits were divided into four groups: 1) untreated; 2) ciprofloxacin, 0.3%, plus dexamethasone alcohol, 0.1%; 3) ciprofloxacin; 4) dexamethasone alcohol. Topical antibiotic and/or steroid was given for 10 h, from 16 to 26 h postinfection, one drop every 15 min for the first hour and then every 30 min for the remaining 9 h. At 27 h postinfection, eyes were evaluated by slit lamp examination (SLE) and assayed for the presence of bacteria in terms of colony forming units (CFU) per cornea. Both prednisolone acetate and prednisolone phosphate reduced ocular inflammation (as determined by SLE), compared with no treatment (P < or = 0.036); the phosphate was more effective (P = 0.005). Tobramycin alone and in combination with prednisolone also significantly reduced SLE, compared with no treatment (P < or = 0.006). The bactericidal activity of tobramycin was not affected by either steroid formulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Efficacy of tobramycin drops applied to collagen shields for experimental staphylococcal keratitis.

Treatment of staphylococcal keratitis includes tobramycin drops at repeated intervals, a prolonged therapy that is disruptive to the patient. To identify a regimen involving less frequent drug application, we compared the efficacy of fortified tobramycin (1.36%) administered by collagen shields or in topical drop form to rabbit corneas intrastromally infected with staphylococci. Eyes were treated with shields hydrated in and supplemented with fortified tobramycin drops (1.36%) applied every 1, 2, 5, or 10 h, from 10 to 20 h postinfection. For topical drop treatment alone, tobramycin was applied following the identical regimen. Untreated corneas contained 10(6) colony forming units. Shields supplemented with tobramycin drops applied every 1, 2, or 5 h sterilized 100% of the corneas. Shields supplemented with tobramycin drops applied at 10 h sterilized 58% of the corneas. Topical delivery of tobramycin every h sterilized all corneas; drops alone applied at longer intervals, such as 2, 5, or 10 h, sterilized 83%, 17%, and 0% of the corneas, respectively. Collagen shield delivery of tobramycin with supplemental topical drops can eradicate staphylococci in this model with less frequent dosing intervals than are required with topical therapy alone.

Pharmacokinetic considerations in the treatment of bacterial keratitis.

The eye is relatively impermeable to micro-organisms and other environmental elements. However, if corneal integrity is breached by trauma, a sight-threatening bacterial infection can result. Staphylococcus aureus, Pseudomonas aeruginosa, and Streptococcus pneumoniae are the most common bacterial pathogens associated with infection of compromised corneas. Bacterial enzymes and toxins, as well as factors associated with the host immune response, can lead to tissue destruction during corneal infection. For successful therapy, an antibacterial agent must be active against the pathogen and must be able to overcome the permeability barrier of the cornea. Topical application of antibacterial agents adequately delivers drugs to the cornea and aqueous humour. However, drug concentrations at the site of infection are not always sufficient to rapidly kill infective organisms. Infections with antibiotic-resistant strains present an even greater therapeutic challenge. In addition, sterilisation of the cornea by antibacterial agents does not eliminate inflammation and corneal scarring that accompany infection. Steroidal and non-steroidal antiinflammatory agents limit corneal scarring during experimentally induced bacterial keratitis. However, although promising, concomitant use of these drugs with antibacterial agents remains controversial. Two ocular drug delivery systems that provide high and sustained concentrations of drug to ocular tissues are corneal collagen shields and transcorneal iontophoresis. The collagen shield, originally designed as a bandage lens, prolongs drug contact with the cornea. Chemotherapeutic studies of experimental bacterial keratitis demonstrate that shields hydrated with antibacterial agents reduce bacteria in the cornea as well as or better than frequent applications of fortified antibacterial drops. Transcorneal iontophoresis uses an electric current to drive charged drugs into the cornea. In experimentally induced bacterial keratitis, transcorneal iontophoresis of antibacterial agents is superior to topically administered ocular drops for reducing the numbers of bacteria in the cornea. Although both drug delivery systems appear to be well tolerated and nontoxic in animal models, clinical trials in patients are required to determine the usefulness of these drug delivery systems in clinical trials. Based on present experimental results, future therapy of bacterial keratitis will involve efficient drug delivery devices, the use of new antibacterial agents or combinations of presently available antibacterial agents, and careful use of adjuvant anti-inflammatory agents.

Corneal virulence of Staphylococcus aureus: roles of alpha-toxin and protein A in pathogenesis.

Staphylococcus aureus produces a variety of proteins, including alpha-toxin and protein A, that could contribute to corneal tissue damage during keratitis. We examined corneal infections produced by intrastromal injection of four S. aureus strains--three isogenic mutants, one lacking alpha-toxin (Hly- Spa+), one lacking protein A (Hly+ Spa-), and one lacking both alpha-toxin and protein A (Hly- Spa-), and the wild type (Hly+ Spa+)--in a rabbit model of experimental keratitis. Rabbit corneas were injected intrastromally with 100 CFU of one of the four strains, and the eyes were examined by slit lamp biomicroscopy over a 25-h period. Corneal homogenates were used for determination of CFU and neutrophil myeloperoxidase activity at 5-h intervals. All strains had the same logarithmic growth curve from 0 to 10 h postinfection, after which CFU remained constant at 10(7) CFU per cornea. By 15 h postinfection, slit lamp examination scores were significantly higher for eyes infected with Hly+ strains than for Hly(-)-infected eyes. At this time, distinct epithelial erosions were seen in Hly(+)-infected eyes but not in Hly(-)-infected eyes. Myeloperoxidase activity was significantly greater for Hly(+)-infected corneas than for Hly(-)-infected corneas at both 20 and 25 h postinfection. Spa(+)- and Spa(-)-infected eyes showed no differences in slit lamp examination scores or myeloperoxidase activities. These results suggest that alpha-toxin, but not protein A, is a major virulence factor in staphylococcal keratitis, mediating the destruction of corneal tissue in eyes infected with this bacterial pathogen.

Ciprofloxacin versus tobramycin for the treatment of staphylococcal keratitis.

PURPOSE: To compare the chemotherapeutic efficacies of ciprofloxacin (0.3%) and fortified (1.36%) tobramycin for the treatment of methicillin-sensitive and methicillin-resistant Staphylococcus aureus keratitis during early and late stages of infection. METHODS: Rabbit corneas were intrastromally injected with 10(2) colony-forming units (CFU) of methicillin-sensitive S. aureus (MSSA) or methicillin-resistant S. aureus (MRSA). Topical therapy was initiated at either 4 hours postinfection (early stage) or at 10 hours postinfection (late stage). Drops were administered every 15 minutes for 5 hours. Corneal bacterial counts and aqueous humor antibiotic concentrations were determined. RESULTS: Early administration of ciprofloxacin sterilized all MSSA-infected corneas and 83% of MRSA-infected corneas. Late administration of ciprofloxacin reduced the numbers of viable MSSA and MRSA to 3.6 and 3.7 log10 CFU per cornea, respectively, but did not sterilize any corneas. Early administration of fortified (1.36%) tobramycin sterilized all MSSA-infected corneas but none of the MRSA-infected corneas. Late administration of tobramycin reduced the viable MSSA to very low numbers (0.5 and 0.0 log10, respectively) and sterilized 33% of MSSA-infected corneas, but had little effect on MRSA-infected corneas. CONCLUSIONS: Early in infection, ciprofloxacin was highly effective against MSSA and MRSA, whereas tobramycin was effective only against MSSA. During later stages of infection, tobramycin was more effective than ciprofloxacin against MSSA, and neither antibiotic was effective against MRSA. Thus, ciprofloxacin is limited by the time of application and tobramycin is limited by the resistance of the MRSA strain.

Pseudomonas aeruginosa keratitis in leukopenic rabbits.

To study the role of the host inflammatory response in Pseudomonas aeruginosa keratitis, rabbits were made leukopenic with intravenous injections of cyclophosphamide and dexamethasone. Twenty-four hr later, keratitis was initiated in all rabbits with an intrastromal injection of 1,000 log phase P. aeruginosa strain 27853. Slit lamp examination of eyes showed that leukopenic rabbits had significantly less (P < 0.0001) ocular pathology at 16, 22, and 27 hr postinfection. The number of viable bacteria recovered from corneas of leukopenic rabbits was the same as the number recovered from nonleukopenic rabbits (P = 0.95). These results suggest that the host inflammatory response significantly contributes to the overall ocular pathology associated with P. aeruginosa keratitis, but does not influence the survival of the infecting organism in the cornea at the height of the infection.