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.

Metallo-beta-lactamase production by Pseudomonas otitidis: a species-related trait.

Susceptibility to several ß-lactams and ß-lactamase production was investigated in a collection of 20 strains of Pseudomonas otitidis, a new Pseudomonas species that has been recently recognized in association with otic infections in humans. All strains appeared to be susceptible to piperacillin, cefotaxime, ceftazidime, and aztreonam, while resistance or decreased susceptibility to carbapenems was occasionally observed. All strains were found to express metallo-ß-lactamase (MBL) activity and to carry a new subclass B3 MBL gene, named bla(POM), that appeared to be highly conserved in this species. P. otitidis, therefore, is the first example of a pathogenic Pseudomonas species endowed with a resident MBL. The POM-1 protein from P. otitidis type strain MCC10330 exhibits the closest similarity (60 to 64%) to the L1 MBL of Stenotrophomonas maltophilia. Expression in Escherichia coli and Pseudomonas aeruginosa revealed that, similar to L1 and other subclass B3 MBLs, POM-1 confers decreased susceptibility or resistance to carbapenems, penicillins, and cephalosporins but not to aztreonam. Expression of the POM MBL in P. otitidis is apparently constitutive and, in most strains, does not confer a carbapenem-resistant phenotype. However, a strong inoculum size effect was observed for carbapenem MICs, and carbapenem-resistant mutants could be readily selected upon exposure to imipenem, suggesting that carbapenem-based regimens should be considered with caution for P. otitidis infections.

A highly virulent Staphylococcus aureus: rabbit anterior chamber infection, characterization, and genetic analysis.

PURPOSE: To describe and characterize a Staphylococcus aureus strain with unique virulence that overcomes host defenses of the rabbit anterior chamber and mimics clinical cases of postcataract surgery endophthalmitis. METHODS: Nine isolates of S. aureus were tested to determine their viability in the rabbit anterior chamber. Growth of UMCR1 in the anterior chamber was established and expressed as log colony-forming units per milliliter of aqueous humor. Pathologic changes produced by UMCR1 were documented by photographs, slit lamp examination, histopathologic analysis, and quantification of neutrophils. UMCR1 was characterized by antibiotic susceptibility, biochemical tests, ribotyping, genome restriction mapping, and multilocus sequence typing (MLST). RESULTS: UMCR1 was the only S. aureus strain that grew within the anterior chamber, reaching log 6.97 ± 0.18 CFU/mL by 16 hours after infection. Pathologic changes included conjunctival injection, chemosis, corneal edema, severe iritis, fibrin accumulation, and a 193-fold increase in neutrophils by 16 hours after infection. UMCR1 was only resistant to sulfamethoxazole and, like other S. aureus isolates, polymyxin B. UMCR1 also had biochemical reactions and a ribotype pattern typical of S. aureus. The genomic reconstruction analysis of UMCR1 was most similar to strains MW2 and MSSA476. MLST revealed a 1 in 3198 nucleotide difference between UMCR1 and strains MW2 and MSSA476. CONCLUSIONS: This study describes a unique S. aureus strain that overcomes host defenses and replicates in the anterior chamber. The survival and growth of this organism could be used for studies of S. aureus pathogenesis, host defenses, and effectiveness of antibiotics within the anterior chamber.

Kinetics of kill of bacterial conjunctivitis isolates with moxifloxacin, a fluoroquinolone, compared with the aminoglycosides tobramycin and gentamicin.

PURPOSE: To compare the kinetics and speed of kill of Streptococcus pneumoniae and Haemophilus influenzae on exposure to three topical ophthalmic antibiotic solutions. MATERIALS AND METHODS: Bacterial conjunctivitis isolates of S. pneumoniae and H. influenzae were exposed to 1:1000 dilutions of moxifloxacin 0.5%, tobramycin 0.3%, gentamicin 0.3%, and water (control). At 15, 30, 60, 120, and 180 minutes after exposure, aliquots were collected, cells were cultured, and viable cell counts were determined using standard microbiological methods. RESULTS: Moxifloxacin achieved 99.9% kill (3-log reduction) at approximately 2 hours for S. pneumoniae and at 15 minutes for H. influenzae. Tobramycin and gentamicin did not achieve 3-log reduction of S. pneumoniae during the 180-minute study period. An increase in bacterial growth was noted for these isolates. Gentamicin took more than 120 minutes to achieve the 3-log reduction of H. influenzae and tobramycin did not reach the 3-log reduction of this pathogen during the 180-minute study period. CONCLUSION: Moxifloxacin killed S. pneumoniae and H. influenzae in vitro faster than tobramycin and gentamicin, suggesting its potential clinical benefit as a first-line treatment for bacterial conjunctivitis to minimize patient symptoms and to limit the contagiousness of the disease.

Comparison of fluoroquinolone kinetics of kill in susceptible and resistant gram-positive conjunctival pathogens.

INTRODUCTION: The purpose of this study was to compare moxifloxacin's rate of kill of susceptible and resistant Gram-positive organisms with that of ciprofloxacin and ofloxacin, using concentrations found in human conjunctiva after instillation of one drop. METHODS: Staphylococcus aureus (S. aureus) and Streptococcus pneumoniae (S. pneumoniae) isolates were exposed to moxifloxacin, ciprofloxacin, or ofloxacin diluted to human conjunctival concentrations achieved after instillation of one drop. These treated isolates were cultured on blood agar plates at 0, 15, 30, and 60 minutes after exposure, and incubated to observe the number of surviving colony-forming units/mL. RESULTS: In susceptible S. pneumoniae, moxifloxacin showed the most rapid reduction of colonies at 15 and 30 minutes, with the fewest colonies at 60 minutes compared with ciprofloxacin and ofloxacin. In S. pneumoniae resistant to ciprofloxacin and ofloxacin, moxifloxacin had rapid reduction in colonies at each time point and near-eradication at 60 minutes, while ciprofloxacin and ofloxacin had an increase in colonies at 60 minutes. In susceptible S. aureus, moxifloxacin had a rapid decrease in colonies at 15 and 30 minutes, compared with a slight reduction in colonies at these intervals for the other antibiotics. In methicillin-resistant S. aureus with cross-resistance to fluoroquinolones and other antibiotics, moxifloxacin had a decrease in colonies at each time point compared with an increase at each time point for ciprofloxacin and ofloxacin. CONCLUSION: Moxifloxacin showed an increased speed of kill against both of the common susceptible Gram-positive conjunctival pathogens, compared with the inconsistency of killing activity of two other fluoroquinolones tested. In addition, at the concentration level achieved in the conjunctiva after the instillation of one drop, moxifloxacin effectively and rapidly killed resistant Gram-positive conjunctival pathogens, while ciprofloxacin and ofloxacin had no effect against these organisms.

DNA sequencing by Microseq kit targeting 16S rRNA gene for species level identification of mycobacteria.

BACKGROUND & OBJECTIVES: Identification of mycobacteria to the species level is of therapeutic significance. Conventional methods are laborious and time consuming so we did 16S rRNA sequencing using a commercial MicroSeq sequencing kit, which includes DNA sequencing with software package for identification and phylogenetic analysis of clinical mycobacterial isolates. METHODS: A total of 47 mycobacteria were tested by both conventional and genotypic method using commercially available MicroSeq 500 amplification kit assay. The identification was determined by comparing the 500 bp amplified product of 16S rDNA sequence to the MicroSeq database. RESULTS: The phenotypic identification was concordant with genotypic identification in 33 (70.2%) isolates of 14 Mycobacterium tuberculosis, 11 M. fortuitum, 7 M. abscessus and 1 M. duvalii. For the discrepant isolates, identification was possible only by DNA sequencing in 14 (29.7%) isolates. The 14 discrepant isolates were 5 M. farcinogenes, 3 M. genavense, 2 M. species. nov and 1 each of M. fortuitum, M. immuogenum, M. simiae and M. wolinskyi. Of these, five were uncommon species that were difficult to identify by phenotypic method. INTERPRETATION & CONCLUSION: The MicroSeq DNA sequencing is an excellent tool for species identification of mycobacteria, which reduces the turn around time, makes repeat analysis easy as compared to phenotypic identification specially for mycobacterial isolates with ambiguous biochemical profiles.

Pseudomonas otitidis sp. nov., isolated from patients with otic infections.

A novel Pseudomonas species, for which the name Pseudomonas otitidis sp. nov. is proposed, was identified from clinical specimens of infected human ears. Forty-one pseudomonads (34 from patients with acute otitis externa, six from patients with acute otitis media with otorrhoea and one from a patient with chronic suppurative otitis media) were recovered that did not match any known species. On the basis of genetic analyses and biochemical characterization, these isolates were shown to belong to the genus Pseudomonas. 16S rRNA gene sequence analysis and DNA-DNA hybridization studies indicated that this novel bacterium is closely related to, but different from, Pseudomonas aeruginosa. A description of this species is based on polyphasic studies of 11 clinical isolates. The type strain of Pseudomonas otitidis is MCC10330T (=ATCC BAA-1130T = DSM 17224T).

The effectiveness of lysostaphin therapy for experimental coagulase-negative Staphylococcus endophthalmitis.

PURPOSE: To quantitatively determine the effectiveness of lysostaphin therapy for experimental endophthalmitis mediated by coagulase-negative Staphylococcus species, the leading cause of postsurgical endophthalmitis. METHODS: Minimal inhibitory concentrations (MIC) of lysostaphin were determined for 54 isolates representing the following species: S. epidermidis, S. warneri, S. haemolyticus, S. cohnii, S. simulans, and S. capitis. The effectiveness of lysostaphin therapy was tested in an experimental model of endophthalmitis by intravitreally injecting log phase bacteria (100 colony-forming units; cfu) into rabbit eyes (n = 3 eyes per group). At 8 hr postinfection (PI), lysostaphin (250 microg) was injected intravitreally, and the number of cfu/ml of vitreous was determined at 24 hr PI. RESULTS: Average MIC for S. epidermidis was 0.7 microg/ml for 90% of the 33 strains tested. Six methicillin-resistant strains of S. epidermidis (MRSE) had an average MIC of 0.74 micro g/ml. All other species had MIC values of =1.1 microg/ml, except for one strain of S. capitis (MIC = 15.6 microg/ml) and one S. haemolyticus (MIC = 1.41 microg/ml). At 24 hr PI, all untreated eyes had between 5.7 and 8.08 log cfu/ml vitreous humor. Treatment with lysostaphin significantly reduced the cfu/ml as compared with untreated eyes for 13 strains tested in vivo (p = 0.020), but not for two S. haemolyticus strains (p = 0.13), two MRSE strains (p = 0.544), or one S. cohnii strain (p = 0.1366). Treatment with lysostaphin reduced the cfu/ml of methicillin-sensitive S. epidermidis strains by 6 logs; for S. warneri, there was a 2 log reduction; and for the other species a 4 log reduction in cfu/ml relative to untreated eyes. CONCLUSIONS: Lysostaphin was mostly effective in treating coagulase-negative staphylococcal experimental endophthalmitis.

In vitro and in vivo potency of moxifloxacin and moxifloxacin ophthalmic solution 0.5%, a new topical fluoroquinolone.

Fluoroquinolones are a class of synthetic antibacterial agents that were approved for ocular therapy in 1991 and have become popular therapy for the treatment and prevention of various ocular infections. These agents are synthetic, broad-spectrum, rapidly bactericidal, and have good penetration into ocular tissues. Their main mechanism of action is the inhibition of bacterial enzymes needed for bacterial DNA synthesis. However, antibiotic resistance occurred swiftly to the earlier fluoroquinolones and better fluoroquinolones were needed. The fourth-generation fluoroquinolones, such as moxifloxacin and gatifloxacin, have enhanced activity against gram-positive bacteria while retaining potent activity against most gram-negative bacteria. These fourth-generation fluoroquinolones have improved penetration into the anterior chamber and have also demonstrated increased in vivo efficacy in several animal models of ocular infections. In addition, topical ophthalmic antibiotic products can deliver antibiotic concentrations directly to the eye that are thousands of times higher than their MICs. This article reviews published data describing the in vitro potency of moxifloxacin and its in vivo activity for treating and preventing experimental ocular infections.

Pseudomonas aeruginosa protease IV: a corneal virulence factor of low immunogenicity.

PURPOSE: To study antibody production to Pseudomonas aeruginosa protease IV (PIV) for immunoassay development and to assess the possible role of antibody in arresting corneal damage. METHODS: Rabbits were immunized with PIV, urea-soluble recombinant PIV (rPIV), or precipitated rPIV. Antibody was analyzed by ELISA and Western blotting. Antibody-mediated inhibition of PIV activity was tested by colorimetric assay and during keratitis by slit-lamp examination of infected eyes. RESULTS: Antibody was not produced after PIV immunization but was induced by rPIV. Rabbits immunized first with soluble and then precipitated rPIV produced high titers (log(10)) to rPIV (4.28 +/- 0.09) and significantly higher titers to PIV (3.90 +/- 0.06) compared to the other immunized groups. Antibody to rPIV reacted with PIV, but neither neutralized enzyme activity in vitro nor protected infected rabbits in vivo. CONCLUSIONS: The present study demonstrates that PIV is a virulence factor which can escape a protective immune response.

Pathogenesis of Staphylococcus in the rabbit anterior chamber.

PURPOSE: To investigate the host defense against Staphylococcus in the rabbit anterior chamber. METHODS: The bactericidal activity of rabbit aqueous humor was investigated in vitro. Rabbit anterior chambers were injected with viable Staphylococcus aureus or Staphylococcus epidermidis (1,000 or 500,000 colony-forming units [CFU]), killed bacteria, culture supernatants of either organism, or purified S. aureus alpha-toxin. CFU as well as phospholipase (PLA(2)) and myeloperoxidase (MPO) activities of aqueous humor were determined up to 25 hours postinfection (PI). RESULTS: The number of viable S. aureus or S. epidermidis was significantly reduced when incubated with aqueous humor for 30 minutes (P

Calcium and magnesium enhance the production of Pseudomonas aeruginosa protease IV, a corneal virulence factor.

The effect of calcium and magnesium on protease IV production during the growth of Pseudomonas aeruginosa was investigated. Strain PA103 was grown to stationary phase in medium containing various concentrations of either calcium or magnesium. Culture supernatants were concentrated, standardized relative to cell density, and the pyoverdine concentrations were measured. Overall extracellular protease activity and specific protease IV (lysine endoproteinase) activity were measured with or without TLCK, a serine protease inhibitor effective against protease IV activity. Protease IV activity was also observed by casein zymography. Calcium and magnesium were quantified in the corneas and aqueous humor of rabbits that were inoculated intrastromally with strain PA103. Pyoverdine production was not significantly different in cultures grown in medium with added calcium or magnesium, but extracellular caseinase activity increased in these cultures. Susceptibility of caseinase activity to TLCK inhibition and a specific assay for protease IV indicated that protease IV activity increased in cultures grown in calcium or magnesium. Casein zymography supported the observation that protease IV activity increased in the cultures with added calcium and magnesium. Addition of calcium or magnesium to the protease IV-specific assay had no effect on the catalytic activity of pure protease IV. Infection of rabbit corneas with PA103 did not change the magnesium concentration in either corneas or aqueous humor, but significantly increased the concentration of calcium in corneas. These results indicate that calcium and magnesium enhance the production of protease IV, but not pyoverdine production. Calcium increases in the cornea following infection with P. aeruginosa could favor production of protease IV.

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 > or = 5-log reduction in CFU/cornea (p < or = 0.0001). Moxifloxacin, levofloxacin, or ciprofloxacin therapies were not significantly different from each other (p > or = 0.67). For Serratia keratitis, untreated eyes contained 7 logCFU/cornea whereas treated eyes had a > or = 2-log reduction (p < or = 0.0001). Moxifloxacin therapy proved most effective (p < or = 0.001). CONCLUSIONS: Overall, moxifloxacin was the most effective of the four fluoroquinolones in reducing CFU/cornea in the rabbit model of gram-negative keratitis.

Effectiveness of ciprofloxacin, levofloxacin, or moxifloxacin for treatment of experimental Staphylococcus aureus keratitis.

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 /=4-log or >/=3-log decrease, respectively, in the MSSA or MRSA strains (P /= 0.3627), whereas moxifloxacin produced a significant reduction in CFU per cornea of approximately 1 log (P

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.

Phospholipase A2 activity in normal and Staphylococcus aureus-infected rabbit eyes.

PURPOSE: To quantify phospholipase A(2) (PLA(2)) activity in normal rabbit eyes and in eyes with Staphylococcus aureus keratitis. METHODS: PLA(2) was assayed by the killing of S. aureus at 33 degrees C or by the release of arachidonic acid from S. aureus labeled with radioactive oleic acid. Rabbit corneas were intrastromally injected with 100 log phase colony-forming units (CFU) of S. aureus 8325-4. The activity of myeloperoxidase (MPO) and PLA(2) were quantified in ocular tissues. RESULTS: The PLA(2)-mediated killing of S. aureus by normal rabbit tears decreased by more than 70% as the rabbits aged from 10 to 28 weeks and by nearly 50% from early morning to afternoon. In rabbits with S. aureus keratitis, the activity of PLA(2) and MPO increased proportionally with time from 5 to 25 hours postinfection (PI), as measured in ocular tissues. PLA(2) activity increased fivefold in tears from infected eyes collected at 25 hours PI compared with normal tears (P < or = 0.0001), whereas a ninefold increase was found in aqueous humor of infected eyes at 25 hours PI (P < or = 0.0001). Infected eyes demonstrated a significant increase in MPO activity compared with uninfected eyes beginning at 10 hours PI for the aqueous humor (P = 0.03), at 16 hours PI for the tear film (P = 0.0024) and at 22 hours PI for the corneal homogenate (P = 0.0007). CONCLUSIONS: The decrease in PLA(2) activity in the rabbit eye with age or after sleep and its increase during sleep or with the progression of infection are consistent with its role as an innate host defense factor.

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, and keratitis. METHODS: Rabbit eyes were intrastromally injected with 1,000 colony-forming units (CFUs) of or or 100 CFUs of 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 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

Immunity to lysostaphin and its therapeutic value for ocular MRSA infections in the rabbit.

PURPOSE: To determine the effects of immunization against lysostaphin on the bactericidal action of lysostaphin in ocular tissue and the possible induction of allergic reactions. METHODS: Rabbits were immunized against lysostaphin by subcutaneous, intranasal, or topical routes. Anti-lysostaphin antibody titers were determined by ELISA and by neutralization of lysostaphin. Methicillin-resistant Staphylococcus aureus was intrastromally or intravitreously injected into rabbit eyes. Eyes were treated either topically with drops of lysostaphin (0.3%) or with a single intravitreous injection (0.1 mL) of lysostaphin (0.1%). At the time of death, corneas or vitreous humors were cultured to determine the number of colony forming units (CFU). RESULTS: Rabbits in keratitis experiments that were immunized subcutaneously, intranasally, or topically had serum antibody titers of 10,240, 187, and 1,867, respectively, and neutralization titers of 8 or less. In both normal and immunized rabbits with keratitis, lysostaphin significantly reduced the log CFU to less than 1 log, whereas the untreated eyes contained more than 10(6) CFU/cornea (P < or = 0.0001). Rabbits that were subcutaneously or topically immunized for endophthalmitis experiments had serum antibody titers of 1636 or 137, respectively, and neutralization titers of 2 or less. A single intravitreous injection of lysostaphin (0.1%) sterilized all eyes of immunized and nonimmune rabbits with endophthalmitis. No adverse effects were observed with the administration of lysostaphin to either normal or immunized rabbit eyes. CONCLUSIONS: Lysostaphin treatment of immunized rabbits was effective in treating S. aureus-infected eyes, despite the presence of anti-lysostaphin antibody. No adverse reactions were produced by administration of lysostaphin to immunized rabbits.

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.

Corneal pathogenesis of Staphylococcus aureus strain Newman.

PURPOSE: To determine the pathogenic role of gamma- and alpha-toxin in a rabbit model of Staphylococcus aureus keratitis. METHODS: S. aureus strains Newman (expressing gamma-toxin), Newman Delta(hlg) (deficient in gamma-toxin), Newman Delta(hlg)/pCU1 hlg(+) (chromosomal gamma-toxin-deficient mutant rescued by a plasmid encoding gamma-toxin), and Newman Delta(hla) (alpha-toxin-deficient) were intrastromally injected into rabbit corneas. Eyes were scored by slit lamp examination (SLE), and bacterial colony-forming units (CFU) per cornea were determined at 15, 20, and 25 hours after infection. Histologic examination of corneas was performed. Rabbits were immunized against alpha-toxin and subsequently challenged with S. aureus strain Newman. Western blot analyses of culture supernatants were performed to detect alpha-toxin production. RESULTS: All strains grew equivalently, producing approximately 7 log CFU per cornea at 25 hours after infection. SLE scores at 20 and 25 hours after infection revealed that strains Newman Delta(hlg) and Newman Delta(hla), although virulent, caused significantly less ocular damage and inflammation than their parent or the gamma-toxin genetically rescued strain (P