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Ocular surface, contact lens and bacterial interactions in a rabbit model
Ocular Surface, Contact Lens and Bacterial Interactions in a Rabbit Model John K Dart FRCS*, John Peacock FIMLSt, Ian Grierson PhD~', David V Seal M D t organisms in a standard 10~tl. inoculum for all experiments. Contact lens material, design and fit. The CL's were made of a 75% water content hydrogel. The single curve piano design was modified from lenses made to curvatures calculated from photokeratoscopy. The D k / L was 13.3 X 10-9 (cm/sec) (ml 02 mlxmmHg) chosen to give a robust lens expected to cause anoxic stress in both the closed and open eye state (Holden 1984). Lenses were fitted to alignment with the cornea and sclera and to be mobile with lid pressure.
Introduction Pseudomonas aeruginosa is a virulent corneal pathogen to which contact lens (CL) wearers are particularly predisposed (Dart 1987). The pathogenesis of pseudomonas keratitis in contact lens wearers is not understood. The well established association between contact lens care material contamination and keratitis (Mayo 1986) is not found in all patients (Dart 1987). P. aeruginosa, in common with other pathogenic bacteria, adheres to hydrogel and PMMA surfaces in vitro (Dart 1986, Duran 1987, Slusher 1987, Butrus 1987). Neither of these findings explains why pseudomonas causes keratitis more frequently in CL users or why soft contact lens (SCL) users are more at risk than hard lens (HCL) users (Dart 1986). This suggests that additional factors must be important in the pathogenesis of this disease (Dart 1987) of which the interactions between the CL, the ocular surface and bacterium have not been studied. We have investigated the kinetics of bacterial colonisation (adherence and/or invasion) in the normal and traumatised rabbit eye and the effect of contact lens wear on this in the absence of trauma. We have then used this model to investigate the persistence of bacteria on the surface of the CL and factors that may affect this. The results of these investigations are reported here.
Materials and Methods Preparation and inoculation of bacteria A strain of Pseudomonas aeruginosa, from a case of human keratitis, was freshly isolated on blood agar. This was purity plated and one colony subcultured in broth, resuspended in glycerol broth and divided into aliquots for storage at -20°C. These were inoculated into broth cultures 21 hours before use resulting in 2 - 4 × 106 From the *Dept. of Clinical Ophthalmology and tDept, of Pathology, Institute of Ophthalmology, University of London, United Kingdom.
TransactionsBCLAInternational
Conference
1988
Preparation of corneas and controls. Female New Zealand white rabbits were used for all experiments. The right eyes were used for the experiments and the left as controls. For the experiment to measure the kinetics of bacterial colonisation of the normat and traumatised cornea three standardised corneal wounds were made with a 4mm trephine under topical anaesthesia. In the experiments on CL wear lenses were worn continuously for the duration of each. The corneal response to lens wear was assessed by ultrasonic corneal pachymetry before and after 3 days of wear, with fluorescein photography at the end of the experiments, and by scanning electron microscopy in some cases. Quantification of bacteria, statistical analysis and iocalisation of bacteria in the cornea and on the lens. Bacterial quantification was carried out using the Miles and Misra technique for viable counts of a homogenate for both the corneas and lenses. The Kruskal-Wallis one way analysis of variance by ranks was used to compare differences between several groups and the Kolmogorov-Smirnov two sample test for pairs. The Chi-square test was used for the analysis of proportional data. The 5 % probability level was used to determine significant departure from the null hypothesis. Localisation of bacteria was by scanning and transmission electron microscopy (e-m) of both corneas and lenses, in some cases. Ruthenium red, which stains polysaccharides nonspecifically, was used to stain the surface of some of the lenses before transmission e-re.
Results Experiment to investigate the kinetics of bacterial coionisation in the normal and tranmatised cornea Bacterial colonisation was assessed at different time periods from 7.5min. to 6 hours. P. aeruginosa adhered in large numbers, between 103 and 108, to the injured cornea with a large variance between rabbits. Analysis of variance showed no difference between the numbers of bacteria at different time periods although the counts were lower at 2 hours, suggesting that some adherent bacteria were cleared from the eye, before the onset of clinical infection at 4 hours. Bacteria were cleared from
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the normal cornea and conjunctiva by 4 hours. This showed that quantification of bacterial colonisation in this model was a sensitive way of assessing the potential of a corneal insult, such as contact lens wear, to predispose the cornea to infection by this test organism. This would be easier to quantify and study than clinical infection in a contact lens wearing model. This study also established that the organism, despite its virulence, was cleared from a normal eye within 4 hours. This time was then chosen for the subsequent experiments, in the lens wearing model, to establish whether lens wear might modify the corneal surface resulting in an increase in bacterial coli~nisation. Experiment to investigate the effects of 2 weeks of continuous contact lens wear on bacterial colonisation. The lenses were worn continuously for 14 days before inoculation 4 hours before sacrifice. Pachymetry had shown that the lens wearing cornea thinned in most cases as a result of tens wear. This was probably the result of increased evaporation from the tear film (Cedarstaff 1983) and to a decreased blink rate (Sherrard 1974), in the presence of the lens. However there were no differences between the control and lens wearing corneas as assessed by either fluorescein photography or scanning electron microscopy. Btween 10: and 10~ bacteria were recovered from the lens wearing and some of the control corneas but the differences were not significant. However large numbers of bacteria, up to 108, were recovered from some lenses. Experiments to investigate the persistence of bacteria on. the surface of the lens. Lenses were inoculated on insertion and removed for bacterial quantification at periods between 4 hours and 1 week. Bacteria were recovered from two thirds of lenses. There was no difference in the proportions of lenses that were colonised by bacteria at each period but the numbers increased significantly on the lenses throughout this period increasing uniformly to 107 or more at t week -greater than the number in the initial inoculum. In another experiment the same protocol was followed but the lenses were removed at 3 days and divided for quantitative bacteriology and transmission e-m, after staining with ruthenium red, for the presence of a polysaccharide film on the lens surface. This was found on all lenses but was thicker on the anterior lens surface and more so in the presence of bacteria which were enveloped in the material.
Discussion Contact lens (CL) wear is now the most important potentially preventable cause of bacterial keratitis. Over 20% of all cases admitted for treatment to some centres have been related to the use of CL's for the correction of low refractive errors (Dart 1987). Despite this there has been little investigation of the pathogenesis of this important complication of CL wear. The CL is a biomaterial, like intraocular lenses, artificial hips and heart valves, it has physiological, anatomical, mechanical and
96
biological effects on the tissues withwhich it is in intimate contact. The CL has profound effects on corneal 02 supply. It disturbs the normal flow of tears over the cornea and interferes with the normal lid/tear resurfacing mechanisms. Mechanical and physiological stresses may lead to breaches in the corneal epithelium, common in CL users and without which bacterial keratitis does not occur. The lens surface is not inert following a short period of lens wear. Some studies (Butrus 1987), although not all (Dart 1986), have suggested that lens deposits may increase bacterial adherence. However our demonstration of the polysaccharide biofilm on the surface of the lens, and its increase in the presence of bacteria, have shown that adherence, although necessary in the initial stages of colonisation, might be of less importance once the bacteria has created its own microenvironment on the lens surface. That this bi0~m__ may be of bacterial origin has been shown in in vitro experiments (Slusher 1987); however our studies show that the host may contribute to this as well. Our studies have also shown that a normal rabbit eye can clear a large inoculum of bacteria within hours whereas a majority of CL wearing eyes, in our model, do not and indeed bacteria can successfully colonise the CL surface increasing in numbers until they represent a massive potential bacterial inoculum. The presence of such large numbers of bacteria on a lens was not associated with any inflammation except in the case of one animal that developed keratoconjunctivitis. This inoculum would not necessarily be expected to cause inflammation unless an epithelial breach presented a favoarable environment for bacterial invasion. These preliminary studies raise many questions about the interaction of the CL, ocular surface and bacteria. It is likely that these interactions are of fundamental importance to our understanding of CL related keratitis. In addition the concept of the lens as a biomaterial may clarify our understanding of many CL related disorders. If our present findings can be extrapolated to the human extended wear SCL user they have implications for lens hygiene and the safety of continuously worn hydroget lenses.
References Butrus S, Klotz SA, Misra RP (1987) The adherence of Pseudomonas aeruginosa to soft contact lenses. Ophthalmology 94:1311-14 Cedarstaff TH and Tomlinson A (1983) A comparative study of tear evaporation rates and water content of soft contac~ lenses. Am J Oprom Physiol Opt 60: 167-174. Dart JKG, Badenoch PR (1986) Bacterial adherence to contact lenzes. CLAO J 12: 220-4. Dart JKG (1987) Bacterial keratitis in contact lens users. Br MealJ295: 959-960. Duran JA, Refojo MF, Gipson IK, Kenyon KR (1987) Pseudomonas attachment to new hydroget contact lenses. Arch Ophthalmot 105: 106-9. Holden BA, Mertz GW (1984) Critical oxygen levels to avoid corneal edema for daily and extended wear contact lenses. Invest Ophthalmot Vis ScL 25: 161t-67. Mayo MS, Cook WL, Schlitzer RL, Ward MA, Wilson LA, Aheam DG (1986) Antibiograms serotypes, and plasmid profiles of Pseudomonas aeruginosa associated with corneal ulcers and contact lens wear. J Clin Microbio124: 372-6. Sherrard ES (1974) Full thickness keratoplasty in the rabbit: an unsatisfactory index of donor integrity. Exp Eye Res 18: 135-142.
Transactions BCLA latenaationa| Conference 1988
Slusher MM, Myrvik QN, Lewis JC, Gristina A G (1987) Extendedwear lenses, biofitm, and bacterial adhesion. Arch Ophthalmo1105: 110-5.
Acknowledgements M. Mathieson for the preparation of the bacterial cultures, the staff of Moorfields Eye Hospital Contact Lens
TransactionsBCLAInternationalConference1988
Production Dept. for manufacture of the lenses, Professor G Woodward PhD for advising on lens design, D. Minassian FRCS MSc (Epid) for advising on the statistical methods used and Mrs S. Lawrence MA for library facilities. This work was supported in part by Moorfields Eye Hospital Locally Organised Grants 86/5 and 87/1.
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Introduction Pseudomonas aeruginosa is a virulent corneal pathogen to which contact lens (CL) wearers are particularly predisposed (Dart 1987). The pathogenesis of pseudomonas keratitis in contact lens wearers is not understood. The well established association between contact lens care material contamination and keratitis (Mayo 1986) is not found in all patients (Dart 1987). P. aeruginosa, in common with other pathogenic bacteria, adheres to hydrogel and PMMA surfaces in vitro (Dart 1986, Duran 1987, Slusher 1987, Butrus 1987). Neither of these findings explains why pseudomonas causes keratitis more frequently in CL users or why soft contact lens (SCL) users are more at risk than hard lens (HCL) users (Dart 1986). This suggests that additional factors must be important in the pathogenesis of this disease (Dart 1987) of which the interactions between the CL, the ocular surface and bacterium have not been studied. We have investigated the kinetics of bacterial colonisation (adherence and/or invasion) in the normal and traumatised rabbit eye and the effect of contact lens wear on this in the absence of trauma. We have then used this model to investigate the persistence of bacteria on the surface of the CL and factors that may affect this. The results of these investigations are reported here.
Materials and Methods Preparation and inoculation of bacteria A strain of Pseudomonas aeruginosa, from a case of human keratitis, was freshly isolated on blood agar. This was purity plated and one colony subcultured in broth, resuspended in glycerol broth and divided into aliquots for storage at -20°C. These were inoculated into broth cultures 21 hours before use resulting in 2 - 4 × 106 From the *Dept. of Clinical Ophthalmology and tDept, of Pathology, Institute of Ophthalmology, University of London, United Kingdom.
TransactionsBCLAInternational
Conference
1988
Preparation of corneas and controls. Female New Zealand white rabbits were used for all experiments. The right eyes were used for the experiments and the left as controls. For the experiment to measure the kinetics of bacterial colonisation of the normat and traumatised cornea three standardised corneal wounds were made with a 4mm trephine under topical anaesthesia. In the experiments on CL wear lenses were worn continuously for the duration of each. The corneal response to lens wear was assessed by ultrasonic corneal pachymetry before and after 3 days of wear, with fluorescein photography at the end of the experiments, and by scanning electron microscopy in some cases. Quantification of bacteria, statistical analysis and iocalisation of bacteria in the cornea and on the lens. Bacterial quantification was carried out using the Miles and Misra technique for viable counts of a homogenate for both the corneas and lenses. The Kruskal-Wallis one way analysis of variance by ranks was used to compare differences between several groups and the Kolmogorov-Smirnov two sample test for pairs. The Chi-square test was used for the analysis of proportional data. The 5 % probability level was used to determine significant departure from the null hypothesis. Localisation of bacteria was by scanning and transmission electron microscopy (e-m) of both corneas and lenses, in some cases. Ruthenium red, which stains polysaccharides nonspecifically, was used to stain the surface of some of the lenses before transmission e-re.
Results Experiment to investigate the kinetics of bacterial coionisation in the normal and tranmatised cornea Bacterial colonisation was assessed at different time periods from 7.5min. to 6 hours. P. aeruginosa adhered in large numbers, between 103 and 108, to the injured cornea with a large variance between rabbits. Analysis of variance showed no difference between the numbers of bacteria at different time periods although the counts were lower at 2 hours, suggesting that some adherent bacteria were cleared from the eye, before the onset of clinical infection at 4 hours. Bacteria were cleared from
95
the normal cornea and conjunctiva by 4 hours. This showed that quantification of bacterial colonisation in this model was a sensitive way of assessing the potential of a corneal insult, such as contact lens wear, to predispose the cornea to infection by this test organism. This would be easier to quantify and study than clinical infection in a contact lens wearing model. This study also established that the organism, despite its virulence, was cleared from a normal eye within 4 hours. This time was then chosen for the subsequent experiments, in the lens wearing model, to establish whether lens wear might modify the corneal surface resulting in an increase in bacterial coli~nisation. Experiment to investigate the effects of 2 weeks of continuous contact lens wear on bacterial colonisation. The lenses were worn continuously for 14 days before inoculation 4 hours before sacrifice. Pachymetry had shown that the lens wearing cornea thinned in most cases as a result of tens wear. This was probably the result of increased evaporation from the tear film (Cedarstaff 1983) and to a decreased blink rate (Sherrard 1974), in the presence of the lens. However there were no differences between the control and lens wearing corneas as assessed by either fluorescein photography or scanning electron microscopy. Btween 10: and 10~ bacteria were recovered from the lens wearing and some of the control corneas but the differences were not significant. However large numbers of bacteria, up to 108, were recovered from some lenses. Experiments to investigate the persistence of bacteria on. the surface of the lens. Lenses were inoculated on insertion and removed for bacterial quantification at periods between 4 hours and 1 week. Bacteria were recovered from two thirds of lenses. There was no difference in the proportions of lenses that were colonised by bacteria at each period but the numbers increased significantly on the lenses throughout this period increasing uniformly to 107 or more at t week -greater than the number in the initial inoculum. In another experiment the same protocol was followed but the lenses were removed at 3 days and divided for quantitative bacteriology and transmission e-m, after staining with ruthenium red, for the presence of a polysaccharide film on the lens surface. This was found on all lenses but was thicker on the anterior lens surface and more so in the presence of bacteria which were enveloped in the material.
Discussion Contact lens (CL) wear is now the most important potentially preventable cause of bacterial keratitis. Over 20% of all cases admitted for treatment to some centres have been related to the use of CL's for the correction of low refractive errors (Dart 1987). Despite this there has been little investigation of the pathogenesis of this important complication of CL wear. The CL is a biomaterial, like intraocular lenses, artificial hips and heart valves, it has physiological, anatomical, mechanical and
96
biological effects on the tissues withwhich it is in intimate contact. The CL has profound effects on corneal 02 supply. It disturbs the normal flow of tears over the cornea and interferes with the normal lid/tear resurfacing mechanisms. Mechanical and physiological stresses may lead to breaches in the corneal epithelium, common in CL users and without which bacterial keratitis does not occur. The lens surface is not inert following a short period of lens wear. Some studies (Butrus 1987), although not all (Dart 1986), have suggested that lens deposits may increase bacterial adherence. However our demonstration of the polysaccharide biofilm on the surface of the lens, and its increase in the presence of bacteria, have shown that adherence, although necessary in the initial stages of colonisation, might be of less importance once the bacteria has created its own microenvironment on the lens surface. That this bi0~m__ may be of bacterial origin has been shown in in vitro experiments (Slusher 1987); however our studies show that the host may contribute to this as well. Our studies have also shown that a normal rabbit eye can clear a large inoculum of bacteria within hours whereas a majority of CL wearing eyes, in our model, do not and indeed bacteria can successfully colonise the CL surface increasing in numbers until they represent a massive potential bacterial inoculum. The presence of such large numbers of bacteria on a lens was not associated with any inflammation except in the case of one animal that developed keratoconjunctivitis. This inoculum would not necessarily be expected to cause inflammation unless an epithelial breach presented a favoarable environment for bacterial invasion. These preliminary studies raise many questions about the interaction of the CL, ocular surface and bacteria. It is likely that these interactions are of fundamental importance to our understanding of CL related keratitis. In addition the concept of the lens as a biomaterial may clarify our understanding of many CL related disorders. If our present findings can be extrapolated to the human extended wear SCL user they have implications for lens hygiene and the safety of continuously worn hydroget lenses.
References Butrus S, Klotz SA, Misra RP (1987) The adherence of Pseudomonas aeruginosa to soft contact lenses. Ophthalmology 94:1311-14 Cedarstaff TH and Tomlinson A (1983) A comparative study of tear evaporation rates and water content of soft contac~ lenses. Am J Oprom Physiol Opt 60: 167-174. Dart JKG, Badenoch PR (1986) Bacterial adherence to contact lenzes. CLAO J 12: 220-4. Dart JKG (1987) Bacterial keratitis in contact lens users. Br MealJ295: 959-960. Duran JA, Refojo MF, Gipson IK, Kenyon KR (1987) Pseudomonas attachment to new hydroget contact lenses. Arch Ophthalmot 105: 106-9. Holden BA, Mertz GW (1984) Critical oxygen levels to avoid corneal edema for daily and extended wear contact lenses. Invest Ophthalmot Vis ScL 25: 161t-67. Mayo MS, Cook WL, Schlitzer RL, Ward MA, Wilson LA, Aheam DG (1986) Antibiograms serotypes, and plasmid profiles of Pseudomonas aeruginosa associated with corneal ulcers and contact lens wear. J Clin Microbio124: 372-6. Sherrard ES (1974) Full thickness keratoplasty in the rabbit: an unsatisfactory index of donor integrity. Exp Eye Res 18: 135-142.
Transactions BCLA latenaationa| Conference 1988
Slusher MM, Myrvik QN, Lewis JC, Gristina A G (1987) Extendedwear lenses, biofitm, and bacterial adhesion. Arch Ophthalmo1105: 110-5.
Acknowledgements M. Mathieson for the preparation of the bacterial cultures, the staff of Moorfields Eye Hospital Contact Lens
TransactionsBCLAInternationalConference1988
Production Dept. for manufacture of the lenses, Professor G Woodward PhD for advising on lens design, D. Minassian FRCS MSc (Epid) for advising on the statistical methods used and Mrs S. Lawrence MA for library facilities. This work was supported in part by Moorfields Eye Hospital Locally Organised Grants 86/5 and 87/1.
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