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.

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.

Topical antibiotic therapy for the treatment of experimental Staphylococcus aureus keratitis.

A rabbit model of Staphylococcus aureus keratitis was developed to study the chemotherapeutic efficacy of ciprofloxacin, vancomycin, and cefazolin. Intrastromal injection of 100 colony forming units of log phase S. aureus ATCC strain 25923 resulted in rapid growth in the cornea, peaking at 10(7) cfu/cornea by 12 hr post-infection. Slit-lamp examination revealed that infected eyes reached 30% of maximum inflammation by 10 hr and 60% by 22 hr post-infection. Antibiotic therapy (one drop every 15 min for 5 hr) was initiated at 4 hr post-infection (experiment 1) or 10 hr post-infection (experiment 2). Another group was initiated at 10 hr post-infection and treated for 10 hr (experiment 3). In experiment 1, treatment from 4-9 hr post-infection with 0.3% ciprofloxacin drops decreased the cfu per cornea 6.1 logs, compared to placebo-treated controls (P = 0.0001), and rendered 50% of inoculated eyes sterile. Vancomycin (5.0%) and cefazolin (5.0%) each lowered the cfu per cornea 4.6 logs (P = 0.0187) but did not sterilize any eyes. In experiment 2, therapy from 10-15 hr post-infection with 0.3% ciprofloxacin reduced the cfu per cornea 0.9 logs (P = 0.0001). Vancomycin (5.0%) and cefazolin (5.0%) decreased the cfu per cornea 0.2 logs (P = 0.3973) and 0.3 logs (P = 0.1307), respectively. In experiment 3, therapy from 10-20 hr post-infection with 0.3% ciprofloxacin reduced the cfu per cornea 3.9 logs (P < 0.0001). In this keratitis model, ciprofloxacin was more effective than vancomycin or cefazolin in killing S. aureus.

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.

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.

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.

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.

Methicillin-resistant Staphylococcus aureus keratitis in the rabbit: therapy with ciprofloxacin, vancomycin and cefazolin.

To determine the efficacy of a fluoroquinolone antibiotic in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) keratitis, topical administration of 0.3% ciprofloxacin was compared with topical 5.0% vancomycin or 5.0% cefazolin in experimental infections in the rabbit eye. The infections were established by intrastromal injection of 100 colony forming units (CFU) of MRSA, which resulted in greater than 10(6) CFU per cornea by 12 hr postinfection. Chemotherapy (one drop every 15 min) was given from 4-9, 10-15, or 10-20 hr postinfection. Early therapy (4-9 hr postinfection) with ciprofloxacin rendered all eyes free of bacteria; ciprofloxacin was significantly more effective than vancomycin or cefazolin. When treatment was initiated 6 hr later (10-15 hr postinfection), no corneas became free of bacteria, but ciprofloxacin was again more effective than vancomycin or cefazolin. Bacterial killing by ciprofloxacin after treatment from 10-20 hr postinfection was also significantly greater than that of vancomycin. Overall, the results show that ciprofloxacin is effective in killing methicillin-resistant staphylococcus aureus, and is most effective when applied during the very early stages of infection.

Prednisolone acetate or prednisolone phosphate concurrently administered with ciprofloxacin for the therapy of experimental Pseudomonas aeruginosa keratitis.

This study was conducted to determine the therapeutic efficacy of 3.0 mg/ml ciprofloxacin administered concurrently with one of two salts of prednisolone for the treatment of experimental pseudomonal keratitis. Rabbit corneas were injected intrastromally with Pseudomonas aeruginosa ATCC strain 27853. Sixteen hr after injection, rabbits were randomly divided into four treatment groups (3 rabbits, 6 eyes per group): 1) ciprofloxacin plus prednisolone acetate; 2) ciprofloxacin plus prednisolone phosphate; 3) ciprofloxacin only; 4) untreated. Signs of inflammation were graded in a masked fashion by slit lamp examination (SLE) and by estimating polymorphonuclear leukocyte (PMN) numbers in corneas 27 hr after injection. SLE scores and PMN numbers were significantly lower (P < 0.02) in eyes receiving either salt of prednisolone plus ciprofloxacin compared to the untreated controls. In contrast, SLE scores and PMN numbers were not significantly different in eyes treated with ciprofloxacin alone, compared to untreated controls (P > 0.13). No viable bacteria were recovered from any eye treated with ciprofloxacin (groups 1, 2, and 3). Ciprofloxacin concentrations in the aqueous humor of eyes in groups 1, 2, and 3 were greater than 15-fold higher than the MIC for P. aeruginosa 27853. These results suggest that either salt of prednisolone, when combined with ciprofloxacin, reduces ocular inflammation without affecting the antimicrobial efficacy of the antibiotic.

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.

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)

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.

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.

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.

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.

Bioassays for quantitating ciprofloxacin and tobramycin in aqueous humor.

Development of new therapies for bacterial keratitis requires assays of antibiotics in infected ocular tissues; however, methods for such drug quantitation can be expensive and can lack precision and sensitivity. To develop a more suitable assay for quantitating tobramycin and ciprofloxacin in the aqueous humor, sixteen bioassays were compared. Variables assessed included: the strain of bacteria used as indicator (Staphylococcus aureus ATCC 25923 versus Klebsiella pneumoniae ATCC 10031), the application of bacteria to the agar surface versus inclusion in molten agar, the volume of the agar, and the application of antibiotic on paper discs versus instillation into agar wells. The best results were obtained when the appropriate indicator organism (S. aureus for tobramycin and K. pneumoniae for ciprofloxacin) was applied to the surface of the agar, antibiotic solution was put into agar wells, and the volume of agar was 30 ml per plate. The best sensitivity for assays of tobramycin and ciprofloxacin diluted in water were 0.98 and 0.12 micrograms/ml, respectively. These sensitivities improved as much as fourfold when antibiotics were diluted in aqueous humor or normal saline. The precision of all assays tested was high as determined by regression coefficients (r > 0.90). The sensitivity and precision of these assays indicate their utility for quantitating fluoroquinolones and aminoglycosides in aqueous humor.

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.