Transcript Association with disease severity in contact lens related keratitis

Biofilm bacterial diversity: Association with disease severity in
contact lens related keratitis
L. Wiley1, M. Mcallister1, L.A. Wiley2, T. Elliott2, D. Bridge2, J. Odom1, J. Olson2,
West Virginia University Eye Institute1, Department of Microbiology, West Virginia University2
Purpose: Biofilm formation in contact lens cases may
predispose to the development of contact lens related
keratitis. To better understand the composition of
contact lens case biofilm, we used 16S ribosomal RNA
(rRNA) gene sequencing to examine cases from
patients with mild keratitis, keratitis with focal infiltrate,
and contact lens related ulcers, as well as cases from
asymptomatic controls.
Heat map: number of bacterial isolates per patient (columns)
grouped by disease severity. Seven controls are not included
because no bacteria were isolated.
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Scanning Electron Micrograph Demonstrating Biofilm on a Contact Lens
from a patient with keratitis and a focal infiltrate
Mean visual acuity
Methods: Contact lens cases were obtained from 5
patients with mild diffuse keratitis, 8 with keratitis and
focal infiltrates, and 4 with contact lens related ulcers.
Eight cases from asymptomatic soft contact lens
wearers were processed as controls. Biofilms were
removed from lens cases by scraping and sonication,
and DNA was extracted using the Mo Bio Microbial DNA
Isolation Kit. Universal primers were used to amplify the
bacterial 16S rRNA gene, PCR products were purified,
cloned into the pCR 4-TOPO vector (Invitrogen), then
re-amplified and sequenced. Sequences were classified
by BLAST analysis against GenBank. Each sequence
was matched with at least one database entry at the
genus level (identity >95%).
Results: The number of bacterial types isolated from
the case correlated with increasing severity of disease
(Spearman rank order correlation <0.000001). There
was a statistically significant difference between the
number of bacterial types identified and the four clinical
groups: normal, mild keratitis, keratitis with focal
infiltrate and corneal ulcer, p=0.0006. All the affected
groups exhibited more bacterial types than the controls
(Mann-Whitney U test, p=0.0013). Presenting visual
acuity was correlated with number of bacteria identified
(p=0.011). Achromobacter and Stenotrophomonas were
predominant isolates.
Discussion: Biofilm formation in contact lens cases can be a potential
source of toxin production, and also may harbor organisms which cause
contact lens related keratitis.1,2
This involves a developmental cycle in which planktonic organisms
aggregate on a surface, and then irreversibly adhere to that substrate.
Microcolony formation occurs through cell to cell, quorum-sensing
signaling leading to increased bacterial diversity and eventual
elaboration of planktonic organisms.
While the early biofilm may produce toxins, the mature biofilm
evolves to a stage in which it begins to disperse metabolically active
planktonic organism, and these bacteria may promote the formation of
microabscess or frank corneal ulceration. Microcolonies of the mature
biofilm may be released from the main structure, facilitating rapid
development of contact lens biofilm.
We observed a predominance of gram negative organisms, such as
Achromobacter and Stenotrophomonas in early biofilms, whereas
advanced biofilms have more gram positive species and a more diverse
gram negative population. Our data suggests that Achromobacter and
Stenotrophomonas organisms may play an important role in establishing
the contact lens case biofilm.
Conclusion: Bacterial diversity from contact lens cases was correlated
with severity of disease and presenting visual acuity, and was greater
than asymptomatic controls. Achromobacter and Stenotrophomonas are
prominent residents of contact case biofilms.
References
1. McLaughlin-BorLace L, Stapleton F, Matheson M, Dart JK: Bacterial biofilm on contact lenses and lens storage cases in wearers
with microbial keratitis. J AppI Microbiol. 1998 May; 84(5):827-38.
2. Galentine PG, Cohen EJ, Laibson PR, Adams CD, Michaud R, Arentsen JJ: Corneal ulcers associated with contact lens wear.
Arch Ophthalmol. 1984; 102(6):891-94.
Biofilm bacterial diversity: Association with disease severity in contact lens
related keratitis
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This study provides additional support for the premise that contact lens case biofilm development and
progression are associated with the presence and severity of contact lens related anterior segment disease.
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The practice of testing contact lens solution effectiveness against the planktonic organisms may provide a
false sense of security in their ability to prevent infection in actual practice.
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This process of biofilm evolution is likely to occur in contact lens wearers who are noncompliant with proper
cleaning regimen.
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The practice of “topping” off case solution is an example of a practice certain to invite biofilm formation.
– Further study to determine the rate of biofilm formation and composition will be forthcoming
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Although fungus and protozoa were not evaluated in our study, Stenotrophomonas is a favored nutrient
species for Acanthamoeba and may be a precursor to such infections.