(Investigative Ophthalmology and Visual Science. 2001;42:1243-1246.)
© 2001
by The Association for Research in Vision and Ophthalmology, Inc.
In Vitro Model of Infectious Crystalline Keratopathy: Tissue Architecture Determines Pattern of Microbial Spread
Thomas K. H. Butler1,
Harminder S. Dua1,
Richard Edwards2 and
James S. Lowe3
1 From the Larry A. Donoso Laboratory for Eye Research, Division of Ophthalmology and Vision Sciences, the
2 Division of Microbiology and the
3 Division of Pathology, University of Nottingham, United Kingdom.
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Abstract
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PURPOSE. To develop an in vitro model of infectious crystalline keratopathy
using human corneal buttons and to test the hypothesis that the
compactness of the corneal stroma determines the pattern of microbial
spread.
METHODS. Twenty human corneal buttons obtained after penetrating keratoplasty
for keratoconus (KC) and eight human corneal buttons obtained from eye
bank (EB) donor eyes were maintained in organ culture. Fourteen buttons
(10 KC and 4 EB donors) were maintained in a turgid state (swollen,
edematous) and 14 in a nonturgid state (compact, normal state of
deturgescence) by the omission or addition of 5% dextran to the
culture medium. Eight KC and four EB nonturgid buttons and eight KC and
four EB turgid buttons were inoculated with Streptococcus
viridans (Lancefield group G, gram-positive) organisms. Two KC
nonturgid and two KC turgid buttons were inoculated with
Klebsiella oxytoca (gram-negative) organisms. Bacterial
migration and spread in the tissue were observed by light and electron
microscopy.
RESULTS. Of the nonturgid buttons, six KC buttons and all four EB buttons
inoculated with S. viridans and both KC buttons
inoculated with K. oxytoca demonstrated an arborizing,
crystallike pattern of bacterial spread. In the turgid buttons, five KC
and all four EB buttons inoculated with S. viridans and
both KC buttons inoculated with K. oxytoca demonstrated
globular, amorphous colonies. This was in complete contrast to the
needlelike branching appearance seen in nonturgid corneal buttons.
Electron microscopy confirmed an interlamellar spread of the bacterial
colonies.
CONCLUSIONS. This is the first in vitro model of bacterial keratitis. It
demonstrates that the pattern of spread of bacteria within corneal
tissue is largely determined by the compactness of the corneal stroma.
Altering tissue architecture changed the pattern of bacterial migration
and spread. This model has considerable potential in further
understanding host–microbe interactions and microbial spread that
occurs during infection.
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Introduction
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As a model of infection in human tissue, the transparency
of the cornea provides a unique opportunity to directly visualize the
pattern of spread of an infective process. However, the host
inflammatory response often obscures details of spread of the
infection. In certain clinical situations, such as prolonged topical
steroid usage (after corneal grafting), herpetic keratitis, and topical
anesthetic abuse,1
2
3
the host response is deficient or
absent. In such situations the pattern assumed by the invading and
colonizing pathogens is easily visible. In vivo, bacteria and fungi
have been seen to migrate and spread in an arborizing pattern of
sharply demarcated creamy-white opacities, which form a branching,
arborescent network within the corneal stroma, giving the lesion a
crystalline appearance.4
Therefore, the term infectious
crystalline keratopathy (ICK) is used to describe the condition.
The pathogenesis of ICK remains obscure. Previous studies have
suggested that intrinsic properties of the infecting organism determine
this unique pattern of growth,5
6
7
in combination with
some predisposing condition in the cornea, as mentioned. However, the
multitude of organisms identified from these lesions, including
different species of Streptococcus,5
6
7
Staphylococcus spp., Haemophilus spp.,
Enterococcus spp. (and other gram-negative
bacteria),8
and Candida spp.9
(and
other species of fungus), led us to hypothesize that it was the
characteristics of the host tissue, rather than that of the invading
organism, that determined the pattern of microbial spread and
migration. We tested this hypothesis in vitro by establishing a model
of human corneal infection.
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Methods
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Corneal Buttons
The protocol adhered to the tenets of the Declaration of
Helsinki, and informed consent was obtained from all
participants. Twenty corneal buttons (7.5–8 mm in diameter),
obtained at the time of penetrating keratoplasty from patients with
keratoconus (KC) were used in the study. The patients had a clinical
and topographic diagnosis of KC. There were 14 men and 6 women aged
between 23 and 42 years. Sixteen patients were wearing contact lenses,
but none had corneal infections or ulcers at any time. The reasons for
corneal transplant were intolerance to contact lens (n = 5), inability to achieve adequate contact lens fit (n = 11), and unwillingness to consider contact lens wear
(n = 4). Patients were randomly selected, but
individuals with excessive scarring or a history of acute hydrops were
excluded. Host trephination diameter was 7.5 mm in 14 patients and 8 mm
in 6 patients. In addition, 8 corneal buttons (8 mm diameter) were
obtained from eye bank (EB) donor eyes. The corneal buttons were left
to equilibrate for 48 hours in Eagle’s minimum essential medium (MEM;
Gibco–Life Technologies, Paisley, Scotland, UK) with 2% fetal calf
serum, 10 mM HEPES buffer, and 5% dextran (10 KC and 4 EB buttons) and
in the same medium without dextran (10 KC and 4 EB buttons), at 25°C.
Medium with dextran maintained the corneal buttons in a compact,
nonturgid state, whereas medium without dextran allowed the corneal
stroma to imbibe water and become swollen or turgid.
Corneal-Thickness Measurement
An ultrasound pachymeter (Bausch and Lomb, Rochester, NY) was
used to measure thickness of corneal buttons after equilibration in the
respective organ culture media for 48 hours. The mean thickness of the
nonturgid buttons was 601 ± 26 µm (range, 546–655). The mean
thickness of the turgid buttons was more than 1000 µm. The ultrasound
pachymeter used has a maximum range of 1000 µm. All turgid buttons
measured 1000 µm (or more); therefore, it was not possible to
determine the SD for this group. In the patients with KC, preoperative
corneal thickness measurements were made at the apex and midperiphery
(site of inoculation). The mean was 461 ± 31.5 µm (range,
410–510) at the apex and 545 ± 33.1 µm (range, 495–592) at
the midperiphery.
Bacterial Viability in Medium
Streptoccus viridans (Lancefield group G,
gram-positive) and Klebsiella oxytoca (gram-negative)
organisms were suspended in phosphate-buffered saline (PBS; 1 x
107 organisms per milliliter). Bacterial
viability was checked in the organ culture medium in which the corneal
buttons were to be maintained, to ensure that the medium, with and
without dextran, supported the growth of both species of organism in
the experimental conditions. For this purpose, 100 µl of the
suspension of each of the organisms was inoculated into separate
culture tubes containing 10 ml of medium each, and maintained at
25°C, 32°C, and 37°C. Bacterial growth of both species was
confirmed at all temperatures, with optimal growth occurring at 25°C.
This was therefore the temperature at which the experiments with
corneal buttons were conducted.
Inoculation of Corneal Buttons and Organ Culture
Fifty microliters (1 x 107 organisms
per milliliter) of bacterial suspension was injected into the corneal
stroma at the midperiphery of the button. A tuberculin syringe with a
25-gauge needle was used. The needle was introduced from the
endothelial side, under a laboratory microscope. The inoculation was
undertaken in sterile conditions. Eight KC and four EB nonturgid
buttons and eight KC and four EB turgid buttons were each inoculated
with S. viridans (Lancefield group G) organisms. Two
nonturgid KC and two turgid KC buttons were each inoculated with
K. oxytoca organisms. The buttons were placed in sterile
tubes containing 10 ml of the respective medium and maintained at
25°C. The medium was changed every 24 hours and the buttons examined
daily, by light microscopy, to progressively document growth of the
bacterial colonies. Photographs were taken at x40 magnification.
Electron Microscopy
Before fixation, a sample of the stromal bacterial colonies was
taken for bacterial culture, to confirm that the identity of the
bacteria was the same as the one inoculated. Between 3 and 14 days, the
buttons were fixed in 2% glutaraldehyde and processed for light and
electron microscopy (transmission and scanning). Sections for light
microscopy were stained with toluidine blue. All samples conformed in
identity to the inoculated organisms.
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Results
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Nonturgid Buttons
A classic arborescent appearance was observed in six of the eight
nonturgid KC and all four EB buttons inoculated with S.
viridans (Lancefield group G). The other two did not show any
growth. Both the nonturgid KC buttons inoculated with K.
oxytoca also demonstrated the arborescent pattern. A striking,
well-demarcated, branching, crystallike network of bacterial colonies
was easily visible with standard light microscopy at low magnification
of wholemounts (Fig. 1A
) and sections (Fig. 1C)
. The main trunks had a segmented or beaded
pattern with finely tapered, needlelike terminal ends. The secondary
branches also demonstrated fine tapering terminal ends, but the
segmentation was less apparent. The pattern became visible within days
3 through 7 after inoculation and persisted with further branching and
subbranching over the following period (7 days maximum) of observation. \.
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Figure 1. (A) Light microscopy of an inoculated nonturgid corneal
button. The arborescent pattern of streptococci is clearly visible,
with fine, needlelike terminal ends appearing at the leading edge. The
larger main trunks of the colony have a beaded appearance (wholemount;
magnification, x40). (B) Light micrograph of an inoculated
turgid corneal button. The proliferating colonies of bacteria have no
definite structured pattern of growth. There are multiple globular
aggregates with no definable leading edge. This is in complete contrast
to the branching network seen in compact corneal buttons (wholemount;
magnification, x40). (C) Light micrograph of a section of
nonturgid cornea (toluidine blue). The compact regular arrangement of
the lamellae with interlamellar spread of the bacteria is clearly
visible. (D) Light micrograph of a section of turgid cornea
(toluidine blue). The lamellae are widely spaced with globular rounded
areas of bacterial colonization. This is in contrast to the needlelike
branching pattern seen in nonturgid corneas.
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Scanning electron micrograph views of the horizontally split corneal
buttons showed the tapering ends of bacterial colonies forming spikes
that were aligned in the direction of the underlying collagen lamellae
(Fig. 2A
). With light microscopy and transmission electron microscopy, the
lesions were seen to be composed purely of bacterial colonies
proliferating between the compact stromal lamellae. Several sections of
all specimens of nonturgid corneas were studied, and no translamellar
spread was noted in any section. This is demonstrated in the example
shown in Figures 1C
and 3A .
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Figure 2. (A) Scanning electron micrograph of a bacterial colony in a
nonturgid corneal button. The leading edge of the bacterial colony is
aligned with the underlying stromal lamella. The sharply tapering tip
gives the condition its characteristic clinical pattern. (B)
Scanning electron micrograph of an inoculated turgid corneal button.
Islands of colonies of bacteria can be seen in spaces between the
stromal lamellae. The stromal lamellae are widely separated, and the
colonies of bacteria show a diffuse growth pattern.
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Figure 3. (A) Transmission electron micrograph of bacterial colonies
in a nonturgid corneal button. The bacterial colonies are located
exclusively between the tightly packed stromal lamellae. There is no
translamellar spread. (B) Transmission electron micrograph
of an inoculated turgid corneal button (same specimen as in Fig. 2B
). A
large colony of bacteria can be seen within the stroma. The growth
pattern is diffuse within the widely spaced stromal lamellae.
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Turgid Buttons
In turgid corneae, light microscopy revealed a completely
different growth pattern with globular, amorphous colonies within the
stroma (five of eight KC buttons and all four EB buttons inoculated
with S. viridans and both KC buttons inoculated with
K. oxytoca; Figs. 1B
1D
). The remaining three buttons did
not show any growth.
With scanning electron microscopy, the bacterial colonies were visible
in large lacunae among the more widely spaced stromal lamellae, without
a definite leading point (Fig. 2B)
. Light microscopy (Fig. 1D)
and
transmission electron microscopy (Fig. 3B) , showed that the collagen
lamellae were widely displaced, and the bacterial colonies did not
demonstrate the typical arrayed structure. Instead, a mass of
proliferating colonies of bacteria was observed.
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Discussion
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The corneal stroma has a lamellar structure composed of
approximately 200 to 250 flattened bundles of collagen fibrils. Within
any given layer or lamella, the bundles run parallel to each other and
extend the entire width of the cornea. However, the direction of the
bundles, in alternate layers or lamellae, is at right angles to each
other (orthogonal) in the posterior part of the corneal stroma and
oblique to each other in the anterior part of the corneal
stroma.10
This highly organized architecture of the stroma
contributes to the natural transparency of the cornea. The transparency
of the cornea provides a unique opportunity to directly visualize the
pattern of spread of an infective process. Clinically, bacterial
infection of the cornea evokes a rapid host response with inflammatory
cells infiltrating the stroma and resulting in the formation of an
abscess or an ulcer. However, in several clinical conditions, notably
herpetic viral disease, topical anesthetic abuse, and prolonged steroid
usage, the host response is dampened, and the infective process can be
visualized.1
2
3
The model of bacterial infection of the cornea reported herein, is the
first in vitro model of this type of human corneal infection. With this
model, we were able to successfully replicate the special microbial
growth patterns seen in vivo. We were able to demonstrate this
distinctive type of growth with gram-positive and gram-negative
organisms, in compact nonturgid corneas. Loss of the compact structure
of the cornea in the hydrated, turgid corneal buttons, prevented the
formation of this pattern of growth. Although it cannot be directly
concluded that the lamellar structure of the cornea is the cause of
this kind of keratopathy, we have demonstrated that the compactness of
the lamellar architecture and to some extent the lamellar structure
itself, contributes to the formation of the clinical pattern seen in
ICK. In nonturgid buttons, every needlelike branch of the arborescent
pattern was confined to one interlamellar plane of the stroma. Several
sections of each specimen were studied, and translamellar spread was
not observed in any section. However, we did not examine serial
sections, and therefore cannot confirm the absence of translamellar
spread. Nonturgid corneal buttons do not lose their lamellar
architecture, but the lamellae become separated from each other by
fluid. This loss of compactness of the lamellar arrangement allows
bacteria to grow as globular colonies in the wide spaces between
lamellae, rather than as a branching network.
These observations support our hypothesis that it is the lamellar
compact nature of the corneal architecture, rather than an inherent
property of the infecting organism, that determines this pattern of
growth.
This model also provides the opportunity to visualize directly in situ
microbial migration and interaction with host corneal tissue. This
should aid in the further understanding of the pathogenesis of corneal
infections in particular and the mechanisms of bacterial adhesion and
spread within tissues in general.
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Footnotes
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Submitted for publication August 3, 2000; revised December 14, 2000;
accepted January 8, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be marked
"advertisement" in accordance with 18 U.S.C. 
1734
solely to indicate this fact.
Corresponding author: Harminder S. Dua, Division of Ophthalmology
and Visual Sciences, Eye, Ear, Nose & Throat Centre, University
Hospital, Queens Medical Centre, Nottingham NG7 2UH, UK.
harminder.dua{at}nottingham.ac.uk
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Abstract
Introduction
