Previous studies from this laboratory have determined that rabbit
tears kill
S. aureus in vitro, and spermidine protects the
bacteria from this killing action.
10 Therefore,
experiments were conducted to determine the ability of spermidine (50
mM) to weaken host defenses and protect
S. aureus 8325-4 on
contact lenses applied to scarified rabbit corneas. Application of
lenses without the use of spermidine resulted in no significant change
in the number of bacteria on the lens from the time of inoculation
(10
4 CFU per lens) through 24 hours
(
P = 0.9355). However, similar inoculations augmented
by the use of spermidine resulted in more than a 2-log increase in CFUs
per lens by 24 hours PI (
P = 0.0027;
Table 1 ). More important, spermidine treatment resulted in an increase
in the number of bacteria in the cornea, an increase that was
significantly greater than the count in the inoculum (
P = 0.0002). Corneas of inoculated eyes not treated with spermidine did
not show a significant increase in CFUs over the inoculum
(
P = 0.4129). The bacteria on spermidine-treated lenses
caused an infection that increased the SLE score significantly over
that produced by bacteria on lenses not treated with spermidine
(
P ≤ 0.0001;
Table 1 ).
Infection of the rabbit cornea by
S. aureus 8325-4 adherent
to contact lenses treated with spermidine was confirmed by
histopathologic analysis
(Figs. 1A 1B) . The corneas at 24 hours after inoculation showed destruction of
the corneal epithelium and bacterial penetration into the stroma. An
association of bacteria and neutrophils was seen within the corneal
fissures created by scarification
(Fig. 1C) . Thus, rabbit corneas
topically inoculated with
S. aureus adherent to contact
lenses and treated with spermidine resulted in keratitis, as measured
by bacterial replication, inflammation, and pathologic changes.
To determine the role of α-toxin in the topical model of infection,
the replication and virulence of the parent 8325-4 strain was compared
with that of the α-toxin–deficient mutant, DU1090, and its rescued
strain, DU1090/pDU1212. The CFUs in the cornea of the parent,α
-toxin–deficient, and rescued strains were not significantly
different at 24 hours PI (6.59 ± 0.13, 6.65 ± 0.16,
6.12 ± 0.25 CFU/ml, respectively;
P ≥ 0.1959).
Each colony recovered from eyes infected with the parent or rescue
strain retained the hemolytic activity of an α-toxin–producing
strain, whereas those colonies recovered from eyes inoculated with the
mutant strain did not have the hemolytic activity characteristic ofα
-toxin. The SLE scores in eyes infected with the rescued strain were
comparable to those of eyes infected with the parent strain
(
P ≥ 0.1123) and scores with both strains were higher
than those of the α-toxin–deficient mutant at all times tested from
6 to 24 hours PI (
P ≤ 0.0081;
Fig. 2 ). Eyes inoculated with the parent or the rescued strains showed severe
ocular inflammation, including conjunctival injection, chemosis,
corneal infiltration, corneal edema, hypopyon formation, fibrin
accumulation in the anterior chamber, and iritis. The changes in the
eyes infected with the α-toxin–deficient mutant were trace to mild
and were limited to conjunctival injection, chemosis, corneal
infiltrate, and iritis. Corneas infected with the parent or rescued
strain had severe epithelial erosions, whereas each cornea infected
with the α-toxin–negative mutant had an intact corneal epithelium
(parent, 9.56 ± 0.87 mm; rescue, 8.40 ± 0.87 mm; mutant,
0.0 ± 0.0 mm;
P ≤ 0.0001). Inflammatory
changes in eyes infected with each strain, as described earlier, were
visible grossly
(Fig. 3) .
Histopathologic analysis of eyes infected with either of theα
-toxin–producing strains demonstrated the loss of the corneal
epithelium and the accumulation of PMN in the tear film within the
fissures created by scarification
(Figs. 4A 4C) . In contrast, eyes infected with the α-toxin–negative mutant
retained the corneal epithelium and demonstrated few PMNs in the tear
film associated with the cornea
(Fig. 4B) .