Transcript Eye spy: Microbial Growth on contact lenses Theresa Edson

Eye Spy: Microbial Growth on Contact Lenses
Theresa Edson and Kyle Hilsabeck
Introduction:
Biofilms are “organized microbial systems consisting
of layers of microbial cells associated with surfaces
(Prescott, Harley, & Klein, 2002).” These microbial
communities play a major role in disease by providing
pathogens with nutrients and protection from host immune
responses. Objects such as contact lenses have shown to be
prime real estate for microbial growth, as they provide a
warm, moist environment for bacteria, the causative agents
in several serious ocular diseases. Bacterial contamination is
often associated with ocular infection and inflammation
during extended wear of contact lenses (Willcox MDP,
Harmis NY, Holden BA, 2002), and can occur through
improper handling techniques, including improper or lack of
hand sanitation, extended use beyond the recommended lens
lifetime, incorrect cleaning of lenses, and the use of human
saliva to remoisten dried-out lenses. The most commonly
found microbe found on contact lenses is the coagulasenegative staphylococci, especially Staphylococcus
epidermidis (Willcox, et al., 2002). This study was
conducted in order examine microbial growth on contact
lenses under different conditions, and we expect to find
extensive biofilm growth on contact lenses that have been
used incorrectly according to user instructions.
A.
B.
C.
D.
Figure 1. DAPI stain of eight well slide biofilms at 200X
magnification. Duplicate wells from top to bottom: (A) New lens.
(B) Daily cleaning. (C) Continuous use. (D) Moistened with Saliva.
References:
•Willcox MDP, Harmis NY, Holden BA. (2002). “Bacterial populations on
high-Dk silicone hydrogel contact lenses: the effect of length of wear in
asymptomatic patients.” Clinical and Experimental Optometry, 85: 172-175.
•Prescott, Harley, & Klein. (2002). Microbiology, 5th ed. McGraw Hill: Boston.
620-621.
•Difco Laboratories. (1998). Difco Manual, 11th ed. Sparks, MD: Becton
Dickinson and Co.
Materials and Methods:
Contact lenses were exposed to a total of four
different environments. Two lenses were worn as
recommended by the manufacturer with daily removal
and cleaning. One of these was then moistened with
saliva before culturing. A third lens was worn for nine
days without removal or cleaning, and the fourth lens
was a brand new, unused lens taken directly out of its
package to serve as a control.
All four lenses were placed in tubes filled with
TSB with 5% glucose and 5% sucrose media; all tubes
were incubated at 37° C for two days. From there a
series of dilutions and spread plates were mixed up and
plated according to standard procedure, with
concentrations ranging from 10-1 to 10-8. Plate counts
were taken, and isolated colonies were suspended in
TSB and transferred to EMB, MSA, PIA, and Blood
Agar plates to test for enterobacteria, Staphylococcus,
Pseudomonas, and hemolytic bacteria, respectively.
Culture from the lens that had been worn according to
daily use instructions was used to inoculate tubes filled
with lactose, sucrose, and dextrose solutions, TSI and
citrate slants, and SIMS media.
An eight-well slide was filled with 400 μL TSB
with 5% glucose and 5% sucrose, and 50 μL of culture
per well, using two wells per sample. The slide was
incubated at 37° C for one day, and the substrate was
suctioned off with a pipette. The cells were then stained
with DAPI according to provided instructions to
examine biofilm characteristics on the slide.
Results:
Substantial bacterial growth was observed on all
contact lenses with the exception of the brand new lens.
Spread plates from each culture appeared to contain a pure
culture of the same microbe, most likely Staphylococcus
epidermidis. This was confirmed when results from
biochemical tests and differential media showed no growth
on EMB or PIA media, the blood agar grew white colonies
with no clearing, and pinkish colonies grew on MSA media.
Isolated colonies tested positive for sugar fermentation
without producing gas, and tested negative for H2S
production, citrate utilization, and starch hydrolysis; these
tests also confirm the presence of S. epidermidis.
The eight well slide with DAPI stain showed the most
growth on contacts that had been worn continuously,
followed by lenses that had been moistened with saliva,
lenses worn according to daily recommendations, and brand
new lenses, which showed minimal growth (Figure 1). The
biofilm taken from daily use contacts showed a distinctive
honeycomb pattern.
Conclusions:
Staphylococcus epidermidis is able to grow on contact
lenses.
 Bacterial growth on contact lenses is difficult to avoid,
even when worn and cared for correctly.
 Proper technique can help reduce the number of
bacteria on contact lenses