Ocular infections caused by non-tuberculous mycobacteria: update on epidemiology and management.

BACKGROUND: To provide an update on the frequency, distribution, risk factors and in vitro susceptibility of ocular infections caused by non-tuberculous mycobacteria. DESIGN: Retrospective study of university clinic patients. PARTICIPANTS: One hundred thirty-nine patients with culture confirmed non-tuberculous mycobacteria infections seen at Bascom Palmer Eye Institute from January 1980 to July 2007. METHODS: Chart review of data collected included patients' demographics, risk factors, microbiological profiles and clinical outcomes. MAIN OUTCOME MEASURES: Frequency, distribution, risk factors and in vitro susceptibility of ocular infections caused by non-tuberculous mycobacteria. RESULTS: A total of 183 non-tuberculous mycobacteria isolates from 142 eyes were identified, with a fourfold increase in the number of eyes infected with non-tuberculous mycobacteria from 1980-1989 (13.4%) to 2000-2007 (56.3%). Eighty-three percent of non-tuberculous mycobacteria isolates were identified as M. abscessus/chelonae. The majority (91%) of isolates were recovered within 10 days. Common diagnoses included keratitis (36.6%), scleral buckle infections (14.8%) and socket/implant infections (14.8%). Identifiable risk factors were presence of biomaterials (63.1%), ocular surgery (24.1%) and steroid exposure (77%). The median time from diagnosis of culture positive non-tuberculous mycobacteria infection to resolution was 13 to 24 weeks. Combination therapy was used to treat 80% of infected eyes. In vitro susceptibility of non-tuberculous mycobacteria isolates were: amikacin, 81%; clarithromycin, 93%; and moxifloxacin, 21%. CONCLUSIONS: The incidence of ocular infections caused by non-tuberculous mycobacteria has increased within the last 8 years, with a high number of biomaterial associated infections among this group. Clinical diagnosis and microbiological confirmation of non-tuberculous mycobacteria infections remains challenging. Patient outcomes may be improved by early diagnosis, appropriate therapy and removal of biomaterials.

Comparison of azithromycin and moxifloxacin against bacterial isolates causing conjunctivitis.

OBJECTIVE: To examine in vitro resistance to azithromycin and moxifloxacin in bacterial conjunctivitis isolates. METHODS: MIC90s (Minimum Inhibitory Concentration) and resistance rates to azithromycin and moxifloxacin were determined based upon microtiter broth dilution and/or antimicrobial gradient test strips in a multicenter phase III study and confirmed externally. RESULTS: The most common isolates collected from bacterial conjunctivitis patients in the phase III study were Haemophilus influenzae (40.6%), followed by Staphylococcus epidermidis (19.3%), Propionibacterium acnes (17.3%), Streptococcus pneumoniae (16.8%), and Staphylococcus aureus (0.06%). MIC90s for all of these organisms were well below established resistance breakpoints for moxifloxacin, indicating no bacterial resistance. On the other hand, the MIC90 for H. influenzae was 3-fold higher than the resistance breakpoint for azithromycin, > or = 128-fold higher for S. epidermidis, 16-fold higher for S. pneumoniae and > or = 128-fold higher for S. aureus, indicating moderate to very high bacterial resistance to azithromycin. CONCLUSIONS: Resistance to azithromycin is more common than resistance to moxifloxacin in clinical isolates causing bacterial conjunctivitis.

Effects of toxin production in a murine model of Staphylococcus aureus keratitis.

PURPOSE: To investigate the corneal virulence of toxin-deficient mutants of Staphylococcus aureus in young and aged mice in a topical inoculation model of keratitis. METHODS: Corneas of young and aged A/J mice were scarified and topically inoculated with a log phase S. aureus parent strain (8325-4), an alpha-toxin-deficient mutant (DU1090), or an Agr-defective mutant (ISP546) deficient in production of multiple toxins or with purified alpha-toxin. Slit lamp examination (SLE) and histopathology were performed, and bacterial colony-forming units (CFU) and myeloperoxidase (MPO) activity were determined. RESULTS: The infection of young mice with the mutant strains demonstrated significantly lower SLE scores (P < or = 0.0001) and reduced histopathologic changes compared with infections with the parent bacterial strain. Either mutant strain of S. aureus produced SLE scores in aged mice through 9 days after infection (PI) that were significantly lower than those of aged mice similarly infected with the toxin-producing parent strain (P < or = 0.0001). Despite use of identical inocula, the CFU per eye were greater for the parent than the mutant strains from 1 to 5 days PI in the young mice (P < or = 0.0372) and from 1 to 3 days PI in the aged mice (P < or = 0.0018). MPO activities were at the maximum at day 1 PI and were similar overall for all infections. Administration of purified alpha-toxin caused greater gross and histopathologic changes in eyes of aged mice than in those of young mice. CONCLUSIONS: Bacterial toxins, and especially alpha-toxin, can mediate corneal disease in mice. Differences in severity of S. aureus keratitis in aged versus young mice correlates with their susceptibility to alpha-toxin.

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

Ocular virulence of capsule-deficient streptococcus pneumoniae in a rabbit keratitis model.

PURPOSE: Determine the ocular virulence of noncapsular Streptococcus pneumoniae in a rabbit keratitis model. METHODS: Mice were infected intraperitoneally with 10(5) colony-forming units (CFUs) of Avery's strain (capsular type 2) or strain R6 (a noncapsular derivative of type 2), and mortality was monitored daily. In addition, 10(5) CFU of each strain was injected into rabbit corneas. Bacterial loads in rabbit corneas were determined at 20 or 48 hours after infection. Slit lamp examination (SLE) of rabbit eyes was performed at 24, 36, and 48 hours after infection. Controls included corneas inoculated with bacterial suspension medium and UV-killed bacteria. RESULTS: One hundred percent mortality was observed in mice infected intraperitoneally with the encapsulated strain at 2 days after infection, whereas all mice infected with the nonencapsulated strain survived for 21 days. The nonencapsulated strain caused the same pathologic effects in the rabbit cornea as the encapsulated strain at 24, 36, and 48 hours after infection (P > or = 0.080). Control corneas showed no pathologic effects and had significantly lower SLE scores than corneas infected with live bacteria (P < or = 0.001). Mean bacteria log CFU +/- SEM recovered at 20 hours after infection were 7.069 +/- 0.094 for the encapsulated and 6.533 +/- 0.116 for the nonencapsulated strain (P = 0.001). Bacteria recovered from the corneas at 48 hours after infection were 6.712 +/- 0.349 and 1.807 +/- 0.462 for the encapsulated and nonencapsulated strains, respectively (P < 0.001). CONCLUSIONS: The S. pneumoniae noncapsular strain was as virulent in the rabbit cornea as was the encapsulated strain, but unlike the encapsulated strain, was avirulent in the mouse peritoneum.

Susceptibility of aged mice to Staphylococcus aureus keratitis.

PURPOSE: To determine the effect of age on the extent of pathogenesis of Staphylococcus keratitis in the mouse. METHODS: Corneas of young and aged mice (BALB/c, A/J, and C57BL/6) were scarified and topically inoculated with S. aureus. Slit lamp examination (SLE) and histopathology were performed, and bacterial colony forming units and myeloperoxidase activity were determined. RESULTS: SLE scores of infected eyes of aged mice were significantly higher at days 1 and 3 postinfection (PI) as compared to infected young mice. Histopathological changes observed in all aged mice were more severe than those in young mice. Young BALB/c and A/J mice demonstrated minimal signs of keratitis by day 3 PI, whereas aged mice of both strains demonstrated severe keratitis by day 3. Young C57BL/6 mice showed no clinical signs of keratitis, whereas aged C57BL/6 mice demonstrated moderate keratitis. CONCLUSIONS: Aged mice with S. aureus keratitis demonstrated increased pathology as compared to young mice.

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.

A new topical model of Staphylococcus corneal infection in the mouse.

PURPOSE: To establish, in the scarified mouse eye, a new model of Staphylococcus aureus keratitis suitable for studies of pathogenesis and host defense mechanisms. METHODS: Corneas of three strains of mice (BALB/c, A/J, and C57BL/6) were scarified and inoculated with S. aureus strain 8325-4. Mice underwent slit lamp examination (SLE) at 1, 3, 5, 7, and 9 days after infection and were killed. Histopathologic analyses, determination of bacterial colony-forming units (CFU), and myeloperoxidase (MPO) activity assays were performed at each time point. RESULTS: S. aureus keratitis developed in both BALB/c and A/J strains of mice, but not in C57BL/6. The BALB/c and A/J strains demonstrated greater susceptibility to infection, as evidenced by significantly higher SLE scores and more viable bacteria per infected eye than in C57BL/6 mice at 5, 7, and 9 days after infection (P

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.