Abstract:
Bacillus cereus can cause irreversible eye damage in humans as early as nine hours
after initial infection through production of exotoxins, including hemolysin BL (Hbl)
and nonhemolytic entertoxin (Nhe) during endophthalmitis. Carvacrol is an extract
from oregano oil that may be able to treat B. cereus endophthalmitis, due to wellknown
antimicrobial and anti-inflammatory qualities. However, sublethal levels of
carvacrol may increase B. cereus virulence. The goal of this study was to measure
relative levels of regulator and virulence gene expression in subinhibitory
concentration (SIC) of carvacrol-stressed Bacillus cereus earlier shown to elevate
virulence when sublehtally-injured. Subinhibitory levels of carvacrol increase
relative expression of hblc, nheA, and plcR to 16s expression in B. cereus infected
human retinal pigment epithelium, ARPE-19 cells. The cytotoxic levels of carvacrol
in ARPE-19 cells were determined using a lactate dehydrogenase (LDH) cytotoxicity
assay. Methods ARPE-19 cells were cultured in DMEM/F12 supplemented with
10% fetal bovine serum (FBS). ARPE-19 cells were treated with 105 CFU/ml. B. cereus alone, or B. cereus + SIC (1 mM) of carvacrol. Relative differences in
expression of plcR, hblC, and nheA regulator and virulence gene expression in B.
cereus treated with SIC of carvacrol, and untreated B. cereus was quantified by mean
cycle threshold (CT) value comparisons using real-time PCR, and statistically
analyzed using one-way ANOVA in Minitab17, . DAPI staining was performed to
visualize B.cereus internalization in ARPE-19 cells 0, 24 and 48 hours post infection.
Results By RT-PCR, relative plcR, hblC, and nheA mRNA expression were not
different between SIC carvacrol-treated B. cereus levels compared to untreated
cultures at 0, 24 or 48 h post infection. DAPI staining revealed no detectable
invasion by B. cereus at 0, 24 and 48 h post infection. Cytotoxicity in ARPE-19 cells
was detected in response to carvacrol compared to the negative control. Percentage
of cytotoxicity in ARPE-19 cells infected with B. cereus and treated with SIC levels of
carvacrol was not significantly different. Conclusion These data indicate that
sublethal chemical stress by carvacrol did not change the potential virulence of this
pathogen via an up/down regulation in the global regulator (plcR) production and
virulence factors such as HblC, NheA and/or the global effector PlcR. Also, DAPI
staining findings showed that B. cereus does not detectably attach or invade ARPE-
19 cells through 48 hours after infection. However, carvacrol is cytotoxic to ARPE-
19 cells. Further investigation is needed to fully understand mechanism of effects of
carvacrol on ARPE-19 layer as an in vitro model for the blood retinal barrier.
