- Email: [email protected]
Bacillus cereus keratitis associated with contact lens wear1
Bacillus cereus Keratitis Associated with Contact Lens Wear Antonio Pinna, MD,1 Leonardo A. Sechi, PhD,2 Stefania Zanetti, PhD,2 Donatella Usai, PhD,2 Giovanni Delogu, PhD,2 Piero Cappuccinelli, MD,2 Francesco Carta, MD1 Objective: We report the first case of contact lens-related Bacillus cereus keratitis and ulcer associated with B. cereus contamination of the contact lens case. This is also the first study to investigate and establish the genetic identity of an organism isolated from the cornea and contact lens case in a patient with contact lens-associated keratitis. Design: Case report. Intervention and Testing: Conjunctival swabs and corneal scrapings from the left eye were inoculated for culture. The contact lens case was also cultured. Antibiotic susceptibility testing was determined by agar disk diffusion method. Initial treatment with topical ciprofloxacin and fortified tobramycin was given. Genetic analysis of the bacterial isolates was performed using polymerase chain reaction (PCR) with enterobacterial repetitive intergenic consensus primers (ERIC; ERIC-PCR). Susceptibility of B. cereus to heat and contact lens disinfecting solutions containing hydrogen peroxide, hydrogen peroxide-catalase, polyquaternium-1, and polyaminopropyl biguanide (PAPB) was tested. Main Outcome Measures: Clinical features, culture results, and antibiotic susceptibility testing were analyzed. The ERIC-PCR amplification products were visualized in ethidium bromide-stained agarose gel. Bacterial growth after exposure to heat and contact lens disinfecting solutions was assessed on blood agar plates. Results: B. cereus was grown from the conjunctiva, corneal ulcer, and contact lens case. All isolates were sensitive to gentamicin, tobramycin, ciprofloxacin, clindamycin, and vancomycin. The corneal ulcer gradually healed over the next 6 days. Results of ERIC-PCR showed that the isolates from the cornea and contact lens case were indistinguishable, thus demonstrating the source of infecting organism to be the contaminated contact lens case. Exposure to a temperature of 80°C for 20 minutes and incubation with hydrogen peroxide-catalase, polyquaternium-1, and PAPB for the minimum recommended time failed to kill B. cereus. Only exposure to hydrogen peroxide for 4 hours eradicated the organism. Conclusions: B. cereus should be considered a possible etiologic agent of contact lens-associated keratitis. Heat and many types of contact lens disinfecting solutions may be ineffective in eradicating B. cereus from contaminated contact lens cases. Only prolonged exposure to hydrogen peroxide appeared to be sporicidal to B. cereus in this study. Ophthalmology 2001;108:1830 –1834 © 2001 by the American Academy of Ophthalmology. The genus Bacillus consists of gram-positive endosporeforming rod-shaped bacteria that may be aerobic or facultatively anaerobic. It contains 50 species, of which Bacillus subtilis, Bacillus anthracis, Bacillus cereus, Bacillus licheniformis, and Bacillus thuringiensis are the best known. Some strains have a tendency to stain variably, and portions of the bacterial rod may appear gram negative. The spores are able to resist heat, radiation, disinfectants, and desiccation. Most species are saprophytes widely distributed in the natural environment, but some species are opportunistic or Originally received: December 18, 2000. Accepted: April 30, 2001. Manuscript no. 200895. 1 Institute of Ophthalmology, University of Sassari, Sassari, Italy. 2 Department of Biomedical Sciences, Section of Experimental and Clinical Microbiology, University of Sassari, Sassari, Italy. The authors have no financial interest in any material used in this study. Reprint requests to Antonio Pinna, MD, Institute of Ophthalmology, University of Sassari, Viale San Pietro 43 A, 07100 Sassari, Italy. E-mail: [email protected].
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© 2001 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
obligate pathogens of animals, including humans. The main habitats are soils of all kinds and the water columns and bed deposits of fresh and marine water.1,2 The pathogenicity of Bacillus species for the eye has been recognized since the late nineteenth century.3 B. cereus has emerged as the most important ocular pathogen in this genus and possibly one of the most destructive organisms to affect the eye.4,5 This organism has been associated with severe posttraumatic keratitis and endophthalmitis after penetrating injury with vegetable or metallic matter. Specific enzymes and toxins including hemolysins, enterotoxins, and emetic toxin are produced by B. cereus. These virulence factors may be responsible for the devastating nature of the inflammatory reaction. Other Bacillus species that have been implicated as causative agents of keratitis include Bacillus brevis,6 Bacillus coagulans,6 Bacillus subtilis,7 Bacillus licheniformis,8,9 Bacillus laterosporus,10 and Bacillus thuringiensis.11 Bacillus organisms may produce infections of the cornea when host resistance is lowered.6 ISSN 0161-6420/01/$–see front matter PII S0161-6420(01)00723-0
Pinna et al 䡠 Contact Lens-associated B. cereus Keratitis To our knowledge, there have been no reports of cultureproven B. cereus keratitis associated with contact lens use with isolation of the organism from both the cornea and contact lens case. Other authors have described a corneal ulcer12 and multiple corneal epithelial opacities7 in wearers whose contact lens cases were contaminated with B. cereus, but they were not able to detect the infecting organism on corneal samples. We report herein a case of contact lensrelated B. cereus corneal ulcer associated with B. cereus contamination of the patient’s contact lens case. Genetic analysis of the bacterial isolates from the cornea and the contact lens case was performed to ascertain whether the strains were identical. Susceptibility of B. cereus to heat and to four commercial contact lens disinfecting solutions containing hydrogen peroxide, hydrogen peroxide-catalase, polyquaternium-1, and polyaminopropyl biguanide (PAPB) was tested.
Case Report A 46-year-old man had used daily wear soft contact lenses for 10 years without experiencing any problem. Each night he removed his lenses and rinsed them with thimerosal-preserved saline solution. He then placed the lenses in his contact lens case. He disinfected his lenses with heat three to four times each week. He replaced the saline in his case whenever he disinfected the lenses and never rinsed or air-dried his case. Two days before examination, he experienced a red, painful left eye. On examination, best-corrected visual acuity was 20/20 in the right eye and 20/80 in the left eye. Results of slit-lamp examination of his right eye were normal. His left eye showed conjunctival hyperemia and edema. The left cornea had a central ulcer approximately 4 mm in diameter, with a surrounding ring of anterior stromal infiltration. The anterior chamber showed 3⫹ cells and flare. A few keratic precipitates were also present. A diagnosis of microbial keratitis was made. Specimens were collected from the inferior fornix of the palpebral conjunctiva and the corneal ulcer using sterile cotton swabs and a sterile platinum spatula, respectively. Conjunctival swabs and corneal scrapings were then used to inoculate directly fresh blood, chocolate, McConkey, and Sabouraud’s agar plates and thioglycolate broth. Specimen collection and cultures for the detection of bacteria and fungi were performed according to a previously recommended protocol.13 In addition, slides of the corneal scrapings were examined by Gram’s stain. His contact lens case was also cultured. The contact lens case was opened without touching the interior of the case, and a sterile cotton swab was used to culture the solution inside the case. Cultures were performed on blood, chocolate, McConkey, and Sabouraud’s agar plates and thioglycolate broth. Gram’s stain of the corneal scrapings revealed gram-positive rods. Cultures from the conjunctiva, corneal ulcer, and left compartment of his contact lens case yielded a heavy growth of Bacillus organisms, which were later identified morphologically and biochemically as B. cereus (Fig 1 and Table 1).1,2 Antibiotic susceptibility testing determined by the agar disk diffusion method (Kirby-Bauer method) revealed that all isolates were susceptible to gentamicin, tobramycin, ciprofloxacin, clindamycin, and vancomycin and were resistant to penicillin, piperacillin, amoxicillin– clavulamic acid, ceftriaxone, oxacillin, rifampin, and cotrimoxazole. The right compartment of the patient’s lens case showed no bacterial growth. Soon after specimen collection, a regimen of topical fortified
Figure 1. Scanning electron micrographs showing Bacillus cereus recovered from corneal scrapings (original magnification, ⫻5000 [A], ⫻10,000 [B]).
tobramycin (1.5%) hourly, ciprofloxacin (0.3%) six times daily, and atropine (1%) twice daily was begun, and the corneal ulcer gradually healed over the next 6 days. Three weeks after initial examination, best-corrected visual acuity in the left eye was 20/20. A superficial anterior stromal scar was present in the area of the previous ulcer. A conjunctival swab taken on this occasion showed growth of Staphylococcus epidermidis, but no Bacillus organisms.
Genetic Analysis of Bacterial Isolates Enterobacterial repetitive intergenic consensus (ERIC) sequences are repetitive elements of 126 bp restricted to transcribed regions of the bacterial chromosome. Their position in the genome is different in different species.14 Polymerase chain reaction (PCR) with ERIC primers (ERIC-PCR) has recently been used to establish clonal relationship between different strains of B. cereus.15 Genetic analysis of the B. cereus isolates from the cornea and contact lens case was performed by ERIC-PCR. The chromosomal DNA of the two isolates of B. cereus was extracted as previously reported.16 The primers used for amplification were ERIC1R (5⬘ATGTAAGCTCCTGGGGATTCAC-3⬘) and ERIC2 (5⬘-AAGTAAGTGACTGGGGGCGTGA-3⬘), each at a concentration of 0.5 M. Polymerase chain reaction amplifications were carried out in a final volume of 50 l with a reaction mixture containing 20 mM
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Ophthalmology Volume 108, Number 10, October 2001 Table 1. Properties of Isolates from Conjunctiva, Corneal Ulcer, and Contact Lens Case versus Properties of Bacillus cereus1,2 Properties
Isolates
Bacillus cereus
Gram’s stain Cell diameter Spore shape and location
Gram-positive rod ⬎1.0 m Ellipsoidal, subterminal ⫹ ⫹ ⫹ Resistant
Gram-positive rod ⬎1.0 m Ellipsoidal, subterminal ⫹ ⫹ ⫹ Resistant
⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺
⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫾ ⫾ ⫺ ⫾ ⫹ ⫺ ⫹ ⫹ ⫹
Motility Anaerobic growth Hemolysis of sheep cells Susceptibility to penicillin Acid from D-glucose D-arabinose D-xylose D-trehalose D-mannitol Fructose Sorbitol Glycerol Glycogen Inulin Salicin Gelatin hydrolysis Indole production Nitrate reduction Utilization of citrate Voges-Proskauer test
Tris hydrogen chloride (pH 8.3), 50 mM potassium chloride, 1.5 mM magnesium chloride, 200 M (each) deoxynucleoside triphosphate (dATP, dGTP, dTTP, and dCTP; Pharmacia LKBBiotechnology, Uppsala, Sweden), 1 U of Taq polymerase (GibcoBRL, Gaithersburg, MD), and 5 l of sample containing 100 ng of chromosomal DNA. The reaction mixtures were overlaid with one drop of paraffin oil and then incubated at 94°C for 2 minutes, followed by 35 cycles at 94°C for 45 seconds, at 52°C for 1 minute, and at 70°C for 40 seconds, and a final extension at 70°C for 20 minutes. Ten microliters of reaction products were analyzed by electrophoresis in 1.8% Metaphor agarose gel (FMC Bioproducts, Rockland, ME) in 0.04 M Tris-acetate-0.01 M edetic acid (pH 8.0) at 90 V for 90 minutes. After electrophoresis, the gel was stained with ethidium bromide and amplification products were visualized under ultraviolet light. The ERIC-PCR was performed in triplicate by a masked individual (LAS). The results of B. cereus ERIC-PCR testing are presented in Figure 2. The PCR amplification revealed that the bacterial isolates from the cornea and the contact lens case had identical patterns.
Susceptibility of Bacillus cereus to Heat and Contact Lens Disinfecting Solutions A pure subculture of B. cereus isolated from the patient’s contact lens case was obtained on agar blood plates. A suspension of the organism was made in sterile phosphate-buffered saline solution (PBSS) at a concentration of 1 ⫻ 106 colony-forming units (cfu)/ ml. One milliliter of suspension was then placed into three separate sterile test tubes. The first test tube was incubated at 60°C for 20 minutes, the second at 80°C for 20 minutes, and the third at 100°C for 20 minutes. After incubation, 0.5 l of suspension was removed from the test tubes and seeded on blood agar plates, which were then incubated at 37°C for 24 hours. The test tubes incubated at 60°C and 80°C showed growth of B. cereus. Conversely, the test tube incubated at 100°C showed no growth.
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Figure 2. Agarose gel electrophoresis (1.8% Metaphor agarose) of DNA amplification of Bacillus cereus isolates obtained by polymerase chain reaction with enterobacterial repetitive intergenic consensus (ERIC) primers (ERIC-PCR). Lanes: M, 100 bp molecular marker (Gibco-BRL, Gaithersburg, MD); A, corneal isolate; B, contact lens case isolate. Isolates showed identical ERIC-PCR patterns.
Suspensions of B. cereus were also made in the following contact lens disinfecting solutions: hydrogen peroxide (3%; minimum recommended time, 6 hours), polyquaternium-1 (0.0011%; minimum recommended time, 4 hours), and PAPB (0.0001%; minimum recommended time, 4 hours). Ten microliters of overnight culture of B. cereus at a concentration of 1 ⫻ 108 cfu/ml were inoculated into 990 l of disinfecting solution so as to reach a final concentration of 1 ⫻ 106 cfu/ml. The suspensions were distributed in 24-well plates (1 ml per well). Control wells received 1 ml of B. cereus suspended in sterile PBSS at a concentration of 1 ⫻ 106 cfu/ml. The hydrogen peroxide (3%)-catalase (0.1 mg/ tablet) system (minimum recommended time, 6 hours) was also tested. B. cereus suspended in hydrogen peroxide at a concentration of 1 ⫻ 106 cfu/ml was placed into the container provided by the manufacturer, which was filled up to the recommended level. The enzyme catalyst was present during the incubation as instructed by the manufacturer. At different times (4, 6, 8, and 24 hours), 0.5 l of suspension was removed from the wells and the container and seeded on blood agar plates, which were then incubated at 37°C for 24 hours. Susceptibility of B. cereus to the disinfecting solutions for contact lenses is summarized in Table 2.
Discussion In this report, we describe the first case of contact lensrelated B. cereus keratitis associated with B. cereus contamination of the patient’s contact lens case. Genetic analysis showed that the bacterial isolate from the cornea was indistinguishable from the contact lens case isolate, thus demonstrating the source of the infecting organism to be the contaminated contact lens case. The lens case contained heat-resistant spores that survived exposure to a temperature of 80°C for 20 minutes. Commercial contact lens disinfecting solutions containing hydrogen peroxide-catalase, polyquaternium-1, and PAPB used for the minimum recommended time failed to kill B. cereus. Only exposure to
Pinna et al 䡠 Contact Lens-associated B. cereus Keratitis Table 2. Bacillus cereus Growth at Different Times after Exposure to Disinfecting Solutions for Contact Lenses
Disinfecting Solution Hydrogen peroxide (3%) (minimum recommended time, 6 hours)
Polyquaternium 1 (0.0011%) (minimum recommended time, 4 hours)
Polyaminopropyl biguanide (0.0001%) (minimum recommended time, 4 hours)
Phosphate-buffered saline solution
Hydrogen peroxide (3%) catalase (0.5 mg/ tablet) (minimum recommended time, 6 hours)
Exposure Time (hours)
Bacillus cereus Growth
4
No growth
6 8 24 4
No growth No growth No growth Growth
6 8 24 4
Growth Growth Growth Growth
6 8 24 4 6 8 24 4
Growth Growth Growth Growth Growth Growth Growth Growth
6 8 24
Growth Growth Growth
hydrogen peroxide (3%) for 4 hours eradicated the organism. Bacillus organisms are a rare but serious cause of endophthalmitis and corneal ulcers. B. cereus, in particular, has emerged as one of the most virulent organisms to affect the eye. This organism produces enzymes and toxins including phospholipases, proteases, hemolysins, enterotoxins, and emetic toxin, which may contribute to its pathogenicity.1,2 Although clinical and experimental evidence indicates that the biochemical environment of the vitreous is optimal for a rapid multiplication of B. cereus,5 little is known about the clinical importance of this organism in ocular surface pathologic conditions. Although corneal ulcers caused by Bacillus organisms in soft contact lens wearers have been described in the literature,7,9,17 to our knowledge there have been no reports of culture-proven B. cereus keratitis associated with daily wear soft contact lenses with isolation of the organism from both the cornea and lens case. Other authors have described corneal ulcers12 and multiple corneal epithelial opacities that stained with fluorescein7 in patients whose contact lens care systems were contaminated with B. cereus, but they were not able to detect any infecting organism on corneal samples. Donzis et al7 suggested that the multiple corneal epithelial lesions seen in their patient may represent a hypersensitivity reaction to B. cereus antigens or a reaction to the toxins produced by the organism. Likewise, bacterial toxins and enzymes may have played a role in the development of B. cereus keratitis in our patient. Indeed, these virulence factors, along with corneal microtrauma resulting from contact lens manipulation, may cause corneal epithelial break-
down. These surface changes may act as avenues for the inoculation of the organism and allow for its proliferation within the corneal tissue. Studies of patients with contact lens-associated corneal ulcers have shown contamination of the lens care systems.18,19 Microbial contamination of the lens care system may represent the source of the infecting organisms in corneal ulcers associated with contact lens wear.20,21 Mayo et al22 cultured strains of Pseudomonas aeruginosa with the same physiologic properties, antibiograms, serotypes, and plasmid profiles from both the cornea and the saline solutions of five patients with infectious corneal ulcers. However, only a study establishing genetic identity between the bacterial isolates from the corneal ulcer and the contact lens care system can really demonstrate that the contaminated contact lens care system is the source of the infecting organism. To our knowledge, this report is the first to investigate the genetic relatedness of bacterial isolates obtained from a contact lens-associated corneal ulcer and the patient’s contact lens case. The ERIC-PCR analysis has recently been shown to be a simple, rapid, and high-resolution method to establish clonal relationship between different strains of B. cereus.15 In our study, DNA fingerprinting with ERIC-PCR revealed that the strains of B. cereus isolated from the patient’s corneal ulcer and contact lens case had identical patterns, thus demonstrating the source of the infecting organism to be the contaminated contact lens case. This report suggests that genetic analysis, apart from being mainly a research tool with epidemiologic applications, may also represent a clinically useful tool in individual patients. Bacillus organisms have been found in the contact lens care systems of 7% of asymptomatic patients.23 These organisms form spores that are resistant to heat and many types of chemical disinfectants. Thus, after a care system is contaminated, the organism may be difficult or even impossible to eradicate from the system using recommended lens care techniques. The lens case of our patient contained heat-resistant spores that survived exposure to a temperature of 80°C for 20 minutes. Furthermore, incubation for the minimum recommended time with hydrogen peroxide-catalase, polyquaternium-1, and PAPB failed to kill B. cereus. Only exposure to hydrogen peroxide (3%) for 4 hours eradicated the organism. Our results are consistent with those of Donzis et al,7 who reported that B. cereus was killed after 5 hours of incubation in hydrogen peroxide (3%), whereas PAPB and thimerosal-chlorhexidine were not effective in eradicating the organism after 5 and 24 hours of incubation, respectively. Apart from being sporicidal to Bacillus organisms, prolonged exposure to hydrogen peroxide (3%) has also been reported to be effective against other organisms involved in contact lens-associated corneal ulcers, including Staphylococcus aureus, P. aeruginosa, Serratia species, Klebsiella species, Candida albicans, and Acanthamoeba castellanii.24,25 Hydrogen peroxide is a respectable disinfectant, but it is toxic to human tissue as well. For this reason, it is necessary to neutralize fully any hydrogen peroxide adherent to the lens before the lens is reapplied to the eye. This can be done by enzymatic means (catalase). The manufacturer recommends adding a
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Ophthalmology Volume 108, Number 10, October 2001 catalase tablet to the lens case at the beginning of disinfection. This system proved to be ineffective at killing B. cereus. Because the enzyme catalyst is present from the very beginning of the disinfection step, the hydrogen peroxide is neutralized long before any disinfection can occur, and the lens is essentially soaking in saline solution. Therefore, the need for an adequate exposure time before neutralization must be stressed. B. cereus produces a broad-spectrum lactamase and is thus resistant to penicillin, ampicillin, and cephalosporins. It is also resistant to trimethoprim, but it is almost always susceptible to clindamycin, erythromycin, chloramphenicol, vancomycin, and the aminoglycosides.2 In the case described here, treatment with topical fortified tobramycin (1.5%) and ciprofloxacin (0.3%) resulted in healing of the corneal ulcer. Other authors have reported topical gentamicin alone or in combination with other antibiotics to be successful in the treatment of Bacillus species keratitis.7–10,17 Overall, topical aminoglycoside antibiotics, given early, appear to be the best treatment for corneal ulcers caused by Bacillus organisms. The case reported here indicates that B. cereus may be of pathologic significance in contact lens wearers. Further studies are necessary for a better understanding of the mechanism by which this potentially pathogenic organism may cause contact lens-associated keratitis. Early treatment with topical fortified aminoglycoside antibiotics may yield a cure. Prolonged exposure of the contact lens to hydrogen peroxide (3%) appears to be effective in preventing B. cereus keratitis in soft contact lens wearers.
8. 9. 10. 11. 12.
13. 14.
15.
16.
17. 18.
References 1. Sneath PHA. Endospore-forming gram-positive rods and cocci. In: Sneath PHA, Mair NS, Sharpe ME, Holt JG, eds. Bergey’s Manual of Systematic Bacteriology. Baltimore: William & Wilkins, 1986; v. 2, sect. 13. 2. Logan NA, Turnbull PCB. Bacillus and recently derived genera. In: Murray PR, editor-in-chief. Manual of Clinical Microbiology, 7th ed. Washington, DC: American Society for Microbiology, 1999; chap. 23. 3. Poplawska S. Zur Aetiologie der Entzu¨ndung des Auges nach Verletzung durch Fremdko¨rper. Arch Augenheilkd 1891;22: 337–54. 4. Davenport R, Smith C. Panophthalmitis due to an organism of the Bacillus subtilis group. Br J Ophthalmol 1952;36:389 –92. 5. O’Day DM, Smith RS, Gregg CR, et al. The problem of Bacillus species infection with special emphasis on the virulence of Bacillus cereus. Ophthalmology 1981;88:833– 8. 6. van Bijsterveld OP, Richards RD. Bacillus infections of the cornea [case report]. Arch Ophthalmol 1965;74:91–5. 7. Donzis PB, Mondino BJ, Weissman BA. Bacillus keratitis
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19. 20. 21. 22.
23. 24. 25.
associated with contaminated contact lens care systems [case report]. Am J Ophthalmol 1988;105:195–7. Tabbara KF, Tarabay N. Bacillus licheniformis corneal ulcer [letter]. Am J Ophthalmol 1979;87:717–9. Groden LR, Brinser JH. Outpatient treatment of microbial corneal ulcers. Arch Ophthalmol 1986;104:84 – 6. Tabbara KF, Juffali F, Matossian RM. Bacillus laterosporus endophthalmitis [case report]. Arch Ophthalmol 1977;95: 2187–9. Samples JR, Buettner H. Corneal ulcer caused by a biologic insecticide (Bacillus thuringiensis) [letter]. Am J Ophthalmol 1983;95:258 – 60. van Setten GB, Tervo T, Tarkkanen A. Akute Keratitis und die Kontamination von Kontaktlinsenpflegesystemen mit Bacillus cereus [Engl abstract]. Klin Monatsbl Augenheilkd 1989;195: 28 –31. Wilhelmus KR, Liesegang TJ, Osato MS, Jones DB. Laboratory Diagnosis of Ocular Infections. Washington, DC: American Society for Microbiology, 1994; Cumitech Series 13A. Sechi LA, Zanetti S, Dupre´ I, et al. Enterobacterial repetitive intergenic consensus sequences as molecular targets for typing of Mycobacterium tuberculosis strains. J Clin Microbiol 1998; 36:128 –32. Shangkuan YH, Yang JF, Lin HC, Shaio MF. Comparison of PCR-RFLP, ribotyping and ERIC-PCR for typing Bacillus anthracis and Bacillus cereus strains. J Appl Microbiol 2000; 89:452– 62. Sechi LA, Pinna A, Pusceddu C, et al. Molecular characterization and antibiotic susceptibilities of ocular isolates of Staphylococcus epidermidis. J Clin Microbiol 1999;37: 3031–3. Ormerod LD, Smith RE. Contact lens-associated microbial keratitis. Arch Ophthalmol 1986;104:79 – 83. Weissman BA, Mondino BJ, Pettit TH, Hofbauer JD. Corneal ulcers associated with extended-wear soft contact lenses. Am J Ophthalmol 1984;97:476 – 81. Mondino BJ, Weissman BA, Farb MD, Pettit TH. Corneal ulcers associated with daily-wear and extended-wear contact lenses. Am J Ophthalmol 1986;102:58 – 65. Stenson S. Soft contact lenses and corneal infection [editorial]. Arch Ophthalmol 1986;104:1287–9. Pitts RE, Krachmer JH. Evaluation of soft contact lens disinfection in the home environment. Arch Ophthalmol 1979;97: 470 –2. Mayo MS, Cook WL, Schlitzer RL, et al. Antibiograms, serotypes, and plasmid profiles of Pseudomonas aeruginosa associated with corneal ulcers and contact lens wear. J Clin Microbiol 1986;24:372– 6. Donzis PB, Mondino BJ, Weissman BA, Bruckner DA. Microbial contamination of contact lens care systems. Am J Ophthalmol 1987;104:325–33. Zanetti S, Pinna A, Juliano C, et al. Effect of contact lens disinfecting solutions on microorganisms isolated from contact lens cases. Eur Bull Drug Res 1994;3:67–70. Zanetti S, Fiori PL, Pinna A, et al. Susceptibility of Acanthamoeba castellanii to contact lens disinfecting solutions. Antimicrob Agents Chemother 1995;39:1596 – 8.
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© 2001 by the American Academy of Ophthalmology Published by Elsevier Science Inc.
obligate pathogens of animals, including humans. The main habitats are soils of all kinds and the water columns and bed deposits of fresh and marine water.1,2 The pathogenicity of Bacillus species for the eye has been recognized since the late nineteenth century.3 B. cereus has emerged as the most important ocular pathogen in this genus and possibly one of the most destructive organisms to affect the eye.4,5 This organism has been associated with severe posttraumatic keratitis and endophthalmitis after penetrating injury with vegetable or metallic matter. Specific enzymes and toxins including hemolysins, enterotoxins, and emetic toxin are produced by B. cereus. These virulence factors may be responsible for the devastating nature of the inflammatory reaction. Other Bacillus species that have been implicated as causative agents of keratitis include Bacillus brevis,6 Bacillus coagulans,6 Bacillus subtilis,7 Bacillus licheniformis,8,9 Bacillus laterosporus,10 and Bacillus thuringiensis.11 Bacillus organisms may produce infections of the cornea when host resistance is lowered.6 ISSN 0161-6420/01/$–see front matter PII S0161-6420(01)00723-0
Pinna et al 䡠 Contact Lens-associated B. cereus Keratitis To our knowledge, there have been no reports of cultureproven B. cereus keratitis associated with contact lens use with isolation of the organism from both the cornea and contact lens case. Other authors have described a corneal ulcer12 and multiple corneal epithelial opacities7 in wearers whose contact lens cases were contaminated with B. cereus, but they were not able to detect the infecting organism on corneal samples. We report herein a case of contact lensrelated B. cereus corneal ulcer associated with B. cereus contamination of the patient’s contact lens case. Genetic analysis of the bacterial isolates from the cornea and the contact lens case was performed to ascertain whether the strains were identical. Susceptibility of B. cereus to heat and to four commercial contact lens disinfecting solutions containing hydrogen peroxide, hydrogen peroxide-catalase, polyquaternium-1, and polyaminopropyl biguanide (PAPB) was tested.
Case Report A 46-year-old man had used daily wear soft contact lenses for 10 years without experiencing any problem. Each night he removed his lenses and rinsed them with thimerosal-preserved saline solution. He then placed the lenses in his contact lens case. He disinfected his lenses with heat three to four times each week. He replaced the saline in his case whenever he disinfected the lenses and never rinsed or air-dried his case. Two days before examination, he experienced a red, painful left eye. On examination, best-corrected visual acuity was 20/20 in the right eye and 20/80 in the left eye. Results of slit-lamp examination of his right eye were normal. His left eye showed conjunctival hyperemia and edema. The left cornea had a central ulcer approximately 4 mm in diameter, with a surrounding ring of anterior stromal infiltration. The anterior chamber showed 3⫹ cells and flare. A few keratic precipitates were also present. A diagnosis of microbial keratitis was made. Specimens were collected from the inferior fornix of the palpebral conjunctiva and the corneal ulcer using sterile cotton swabs and a sterile platinum spatula, respectively. Conjunctival swabs and corneal scrapings were then used to inoculate directly fresh blood, chocolate, McConkey, and Sabouraud’s agar plates and thioglycolate broth. Specimen collection and cultures for the detection of bacteria and fungi were performed according to a previously recommended protocol.13 In addition, slides of the corneal scrapings were examined by Gram’s stain. His contact lens case was also cultured. The contact lens case was opened without touching the interior of the case, and a sterile cotton swab was used to culture the solution inside the case. Cultures were performed on blood, chocolate, McConkey, and Sabouraud’s agar plates and thioglycolate broth. Gram’s stain of the corneal scrapings revealed gram-positive rods. Cultures from the conjunctiva, corneal ulcer, and left compartment of his contact lens case yielded a heavy growth of Bacillus organisms, which were later identified morphologically and biochemically as B. cereus (Fig 1 and Table 1).1,2 Antibiotic susceptibility testing determined by the agar disk diffusion method (Kirby-Bauer method) revealed that all isolates were susceptible to gentamicin, tobramycin, ciprofloxacin, clindamycin, and vancomycin and were resistant to penicillin, piperacillin, amoxicillin– clavulamic acid, ceftriaxone, oxacillin, rifampin, and cotrimoxazole. The right compartment of the patient’s lens case showed no bacterial growth. Soon after specimen collection, a regimen of topical fortified
Figure 1. Scanning electron micrographs showing Bacillus cereus recovered from corneal scrapings (original magnification, ⫻5000 [A], ⫻10,000 [B]).
tobramycin (1.5%) hourly, ciprofloxacin (0.3%) six times daily, and atropine (1%) twice daily was begun, and the corneal ulcer gradually healed over the next 6 days. Three weeks after initial examination, best-corrected visual acuity in the left eye was 20/20. A superficial anterior stromal scar was present in the area of the previous ulcer. A conjunctival swab taken on this occasion showed growth of Staphylococcus epidermidis, but no Bacillus organisms.
Genetic Analysis of Bacterial Isolates Enterobacterial repetitive intergenic consensus (ERIC) sequences are repetitive elements of 126 bp restricted to transcribed regions of the bacterial chromosome. Their position in the genome is different in different species.14 Polymerase chain reaction (PCR) with ERIC primers (ERIC-PCR) has recently been used to establish clonal relationship between different strains of B. cereus.15 Genetic analysis of the B. cereus isolates from the cornea and contact lens case was performed by ERIC-PCR. The chromosomal DNA of the two isolates of B. cereus was extracted as previously reported.16 The primers used for amplification were ERIC1R (5⬘ATGTAAGCTCCTGGGGATTCAC-3⬘) and ERIC2 (5⬘-AAGTAAGTGACTGGGGGCGTGA-3⬘), each at a concentration of 0.5 M. Polymerase chain reaction amplifications were carried out in a final volume of 50 l with a reaction mixture containing 20 mM
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Ophthalmology Volume 108, Number 10, October 2001 Table 1. Properties of Isolates from Conjunctiva, Corneal Ulcer, and Contact Lens Case versus Properties of Bacillus cereus1,2 Properties
Isolates
Bacillus cereus
Gram’s stain Cell diameter Spore shape and location
Gram-positive rod ⬎1.0 m Ellipsoidal, subterminal ⫹ ⫹ ⫹ Resistant
Gram-positive rod ⬎1.0 m Ellipsoidal, subterminal ⫹ ⫹ ⫹ Resistant
⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺
⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫾ ⫾ ⫺ ⫾ ⫹ ⫺ ⫹ ⫹ ⫹
Motility Anaerobic growth Hemolysis of sheep cells Susceptibility to penicillin Acid from D-glucose D-arabinose D-xylose D-trehalose D-mannitol Fructose Sorbitol Glycerol Glycogen Inulin Salicin Gelatin hydrolysis Indole production Nitrate reduction Utilization of citrate Voges-Proskauer test
Tris hydrogen chloride (pH 8.3), 50 mM potassium chloride, 1.5 mM magnesium chloride, 200 M (each) deoxynucleoside triphosphate (dATP, dGTP, dTTP, and dCTP; Pharmacia LKBBiotechnology, Uppsala, Sweden), 1 U of Taq polymerase (GibcoBRL, Gaithersburg, MD), and 5 l of sample containing 100 ng of chromosomal DNA. The reaction mixtures were overlaid with one drop of paraffin oil and then incubated at 94°C for 2 minutes, followed by 35 cycles at 94°C for 45 seconds, at 52°C for 1 minute, and at 70°C for 40 seconds, and a final extension at 70°C for 20 minutes. Ten microliters of reaction products were analyzed by electrophoresis in 1.8% Metaphor agarose gel (FMC Bioproducts, Rockland, ME) in 0.04 M Tris-acetate-0.01 M edetic acid (pH 8.0) at 90 V for 90 minutes. After electrophoresis, the gel was stained with ethidium bromide and amplification products were visualized under ultraviolet light. The ERIC-PCR was performed in triplicate by a masked individual (LAS). The results of B. cereus ERIC-PCR testing are presented in Figure 2. The PCR amplification revealed that the bacterial isolates from the cornea and the contact lens case had identical patterns.
Susceptibility of Bacillus cereus to Heat and Contact Lens Disinfecting Solutions A pure subculture of B. cereus isolated from the patient’s contact lens case was obtained on agar blood plates. A suspension of the organism was made in sterile phosphate-buffered saline solution (PBSS) at a concentration of 1 ⫻ 106 colony-forming units (cfu)/ ml. One milliliter of suspension was then placed into three separate sterile test tubes. The first test tube was incubated at 60°C for 20 minutes, the second at 80°C for 20 minutes, and the third at 100°C for 20 minutes. After incubation, 0.5 l of suspension was removed from the test tubes and seeded on blood agar plates, which were then incubated at 37°C for 24 hours. The test tubes incubated at 60°C and 80°C showed growth of B. cereus. Conversely, the test tube incubated at 100°C showed no growth.
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Figure 2. Agarose gel electrophoresis (1.8% Metaphor agarose) of DNA amplification of Bacillus cereus isolates obtained by polymerase chain reaction with enterobacterial repetitive intergenic consensus (ERIC) primers (ERIC-PCR). Lanes: M, 100 bp molecular marker (Gibco-BRL, Gaithersburg, MD); A, corneal isolate; B, contact lens case isolate. Isolates showed identical ERIC-PCR patterns.
Suspensions of B. cereus were also made in the following contact lens disinfecting solutions: hydrogen peroxide (3%; minimum recommended time, 6 hours), polyquaternium-1 (0.0011%; minimum recommended time, 4 hours), and PAPB (0.0001%; minimum recommended time, 4 hours). Ten microliters of overnight culture of B. cereus at a concentration of 1 ⫻ 108 cfu/ml were inoculated into 990 l of disinfecting solution so as to reach a final concentration of 1 ⫻ 106 cfu/ml. The suspensions were distributed in 24-well plates (1 ml per well). Control wells received 1 ml of B. cereus suspended in sterile PBSS at a concentration of 1 ⫻ 106 cfu/ml. The hydrogen peroxide (3%)-catalase (0.1 mg/ tablet) system (minimum recommended time, 6 hours) was also tested. B. cereus suspended in hydrogen peroxide at a concentration of 1 ⫻ 106 cfu/ml was placed into the container provided by the manufacturer, which was filled up to the recommended level. The enzyme catalyst was present during the incubation as instructed by the manufacturer. At different times (4, 6, 8, and 24 hours), 0.5 l of suspension was removed from the wells and the container and seeded on blood agar plates, which were then incubated at 37°C for 24 hours. Susceptibility of B. cereus to the disinfecting solutions for contact lenses is summarized in Table 2.
Discussion In this report, we describe the first case of contact lensrelated B. cereus keratitis associated with B. cereus contamination of the patient’s contact lens case. Genetic analysis showed that the bacterial isolate from the cornea was indistinguishable from the contact lens case isolate, thus demonstrating the source of the infecting organism to be the contaminated contact lens case. The lens case contained heat-resistant spores that survived exposure to a temperature of 80°C for 20 minutes. Commercial contact lens disinfecting solutions containing hydrogen peroxide-catalase, polyquaternium-1, and PAPB used for the minimum recommended time failed to kill B. cereus. Only exposure to
Pinna et al 䡠 Contact Lens-associated B. cereus Keratitis Table 2. Bacillus cereus Growth at Different Times after Exposure to Disinfecting Solutions for Contact Lenses
Disinfecting Solution Hydrogen peroxide (3%) (minimum recommended time, 6 hours)
Polyquaternium 1 (0.0011%) (minimum recommended time, 4 hours)
Polyaminopropyl biguanide (0.0001%) (minimum recommended time, 4 hours)
Phosphate-buffered saline solution
Hydrogen peroxide (3%) catalase (0.5 mg/ tablet) (minimum recommended time, 6 hours)
Exposure Time (hours)
Bacillus cereus Growth
4
No growth
6 8 24 4
No growth No growth No growth Growth
6 8 24 4
Growth Growth Growth Growth
6 8 24 4 6 8 24 4
Growth Growth Growth Growth Growth Growth Growth Growth
6 8 24
Growth Growth Growth
hydrogen peroxide (3%) for 4 hours eradicated the organism. Bacillus organisms are a rare but serious cause of endophthalmitis and corneal ulcers. B. cereus, in particular, has emerged as one of the most virulent organisms to affect the eye. This organism produces enzymes and toxins including phospholipases, proteases, hemolysins, enterotoxins, and emetic toxin, which may contribute to its pathogenicity.1,2 Although clinical and experimental evidence indicates that the biochemical environment of the vitreous is optimal for a rapid multiplication of B. cereus,5 little is known about the clinical importance of this organism in ocular surface pathologic conditions. Although corneal ulcers caused by Bacillus organisms in soft contact lens wearers have been described in the literature,7,9,17 to our knowledge there have been no reports of culture-proven B. cereus keratitis associated with daily wear soft contact lenses with isolation of the organism from both the cornea and lens case. Other authors have described corneal ulcers12 and multiple corneal epithelial opacities that stained with fluorescein7 in patients whose contact lens care systems were contaminated with B. cereus, but they were not able to detect any infecting organism on corneal samples. Donzis et al7 suggested that the multiple corneal epithelial lesions seen in their patient may represent a hypersensitivity reaction to B. cereus antigens or a reaction to the toxins produced by the organism. Likewise, bacterial toxins and enzymes may have played a role in the development of B. cereus keratitis in our patient. Indeed, these virulence factors, along with corneal microtrauma resulting from contact lens manipulation, may cause corneal epithelial break-
down. These surface changes may act as avenues for the inoculation of the organism and allow for its proliferation within the corneal tissue. Studies of patients with contact lens-associated corneal ulcers have shown contamination of the lens care systems.18,19 Microbial contamination of the lens care system may represent the source of the infecting organisms in corneal ulcers associated with contact lens wear.20,21 Mayo et al22 cultured strains of Pseudomonas aeruginosa with the same physiologic properties, antibiograms, serotypes, and plasmid profiles from both the cornea and the saline solutions of five patients with infectious corneal ulcers. However, only a study establishing genetic identity between the bacterial isolates from the corneal ulcer and the contact lens care system can really demonstrate that the contaminated contact lens care system is the source of the infecting organism. To our knowledge, this report is the first to investigate the genetic relatedness of bacterial isolates obtained from a contact lens-associated corneal ulcer and the patient’s contact lens case. The ERIC-PCR analysis has recently been shown to be a simple, rapid, and high-resolution method to establish clonal relationship between different strains of B. cereus.15 In our study, DNA fingerprinting with ERIC-PCR revealed that the strains of B. cereus isolated from the patient’s corneal ulcer and contact lens case had identical patterns, thus demonstrating the source of the infecting organism to be the contaminated contact lens case. This report suggests that genetic analysis, apart from being mainly a research tool with epidemiologic applications, may also represent a clinically useful tool in individual patients. Bacillus organisms have been found in the contact lens care systems of 7% of asymptomatic patients.23 These organisms form spores that are resistant to heat and many types of chemical disinfectants. Thus, after a care system is contaminated, the organism may be difficult or even impossible to eradicate from the system using recommended lens care techniques. The lens case of our patient contained heat-resistant spores that survived exposure to a temperature of 80°C for 20 minutes. Furthermore, incubation for the minimum recommended time with hydrogen peroxide-catalase, polyquaternium-1, and PAPB failed to kill B. cereus. Only exposure to hydrogen peroxide (3%) for 4 hours eradicated the organism. Our results are consistent with those of Donzis et al,7 who reported that B. cereus was killed after 5 hours of incubation in hydrogen peroxide (3%), whereas PAPB and thimerosal-chlorhexidine were not effective in eradicating the organism after 5 and 24 hours of incubation, respectively. Apart from being sporicidal to Bacillus organisms, prolonged exposure to hydrogen peroxide (3%) has also been reported to be effective against other organisms involved in contact lens-associated corneal ulcers, including Staphylococcus aureus, P. aeruginosa, Serratia species, Klebsiella species, Candida albicans, and Acanthamoeba castellanii.24,25 Hydrogen peroxide is a respectable disinfectant, but it is toxic to human tissue as well. For this reason, it is necessary to neutralize fully any hydrogen peroxide adherent to the lens before the lens is reapplied to the eye. This can be done by enzymatic means (catalase). The manufacturer recommends adding a
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Ophthalmology Volume 108, Number 10, October 2001 catalase tablet to the lens case at the beginning of disinfection. This system proved to be ineffective at killing B. cereus. Because the enzyme catalyst is present from the very beginning of the disinfection step, the hydrogen peroxide is neutralized long before any disinfection can occur, and the lens is essentially soaking in saline solution. Therefore, the need for an adequate exposure time before neutralization must be stressed. B. cereus produces a broad-spectrum lactamase and is thus resistant to penicillin, ampicillin, and cephalosporins. It is also resistant to trimethoprim, but it is almost always susceptible to clindamycin, erythromycin, chloramphenicol, vancomycin, and the aminoglycosides.2 In the case described here, treatment with topical fortified tobramycin (1.5%) and ciprofloxacin (0.3%) resulted in healing of the corneal ulcer. Other authors have reported topical gentamicin alone or in combination with other antibiotics to be successful in the treatment of Bacillus species keratitis.7–10,17 Overall, topical aminoglycoside antibiotics, given early, appear to be the best treatment for corneal ulcers caused by Bacillus organisms. The case reported here indicates that B. cereus may be of pathologic significance in contact lens wearers. Further studies are necessary for a better understanding of the mechanism by which this potentially pathogenic organism may cause contact lens-associated keratitis. Early treatment with topical fortified aminoglycoside antibiotics may yield a cure. Prolonged exposure of the contact lens to hydrogen peroxide (3%) appears to be effective in preventing B. cereus keratitis in soft contact lens wearers.
8. 9. 10. 11. 12.
13. 14.
15.
16.
17. 18.
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