Genevieve Flock, Shankumar Mooyottu and Kumar Venkitanarayanan

Efficacy of Octenidine Hydrochloride in Reducing Hyper-virulent
Clostridium difficile Spores on Stainless Steel Surface
Genevieve Flock, Shankumar Mooyottu and Kumar Venkitanarayanan*
Department of Animal Science, University of Connecticut, Storrs, CT 06269-4040, *Corresponding author : [email protected]
ABSTRACT
Clostridium difficile (CD) is a spore forming nosocomial pathogen
which causes a toxin-mediated enteric infection in humans. The spores of CD
can survive on abiotic surfaces for several months, and act as a source of new
and recurrent infections by feco-oral route. Octenidine hydrochloride (OH) is
a positively-charged bispyridinamine, exhibiting antimicrobial activity against
a variety of bacteria. This study investigated the sporicidal efficacy of OH
against CD spores on stainless steel surface.
Two hypervirulent, CD isolates were separately grown on brain heart
infusion agar for 10 days at 37oC in an anaerobic chamber. Spores were
purified using standard protocols. Suspensions containing 106 spores per 100 µl
were inoculated on stainless steel discs (16 mm diameter), which were airdried for 1 h, and treated with 0, 1, 2, 3, 4 and 5% of OH in 62% ethanol for 10
min. Untreated control (0% OH) and ethanol control (62% ethanol) were also
included. Following incubation, the discs were transferred to tubes containing
5 ml PBS and glass beads, vortexed and sonicated for 2 min to remove viable,
attached spores from the disc surface. Serial-dilutions were made and 1 ml of
the dilutions was pour-plated. Additionally, the discs were transferred onto
plates to determine the residual viable spores surviving on the discs.
Octenidine hydrochloride was significantly effective in killing CD spores
on steel discs (P < 0.05). In CD strain 1870, 1% OH reduced spores by 1 log
CFU/ml, whereas 2, 3, and 4% OH reduced by 2 log CFU/ml and 5% OH
decreased spores by ~ 3 log CFU/ml compared to controls. In CD strain 1805,
1, 2, 3 and 4% OH reduced spores by 1 log CFU/ml, whereas 5% OH
decreased spores by ~ 2 log CFU/ml compared to controls.
No residual spores were found on OH-treated discs after washing,
whereas a substantial amount of spores were present on control discs. The
results suggest that OH could potentially be used as a sanitizer to reduce CD
spores on stainless steel surfaces for controlling nosocomial infections of the
pathogen.
C. difficile Strains, Culture Conditions and Treatments
C. difficile ATCC strains 1805 and 1870 were separately grown in brain
heart infusion (BHI) agar for 10 days at 37°C in an anaerobic chamber for
spore cultivation. Each stainless steel disc was inoculated with 106 spores/100
µL, air dried and then subjected to treatments; control (only spore inoculum),
ethanol (62% ethanol) and OH treatments (1, 2, 3, 4 and 5% OH dissolved in
62% ethanol). The sporicidal efficacy of OH was determined by dilution and
plating of spores on BHI+T agar (brain heart infusion agar supplemented
with 0.1% sodium taurocholate). Moreover, residual spores on discs were
visualized.
Figure 1: Effect of Octenidine Hydrochloride on
C. difficile ATCC 1870 Spores
C. difficile strain ATCC 1870
SUMMARY
 Octenidine hydrochloride significantly reduced spores of C. difficile
strain ATCC 1870 on stainless steel discs. 1% OH reduced spores by 1
log CFU/ml; 2, 3 and 4% OH reduced by 2 log CFU/ml, and 5% OH
decreased spores by ~ 3 log CFU/ml compared to controls.
Figure 2: Effect of Octenidine Hydrochloride on
Strain2ATCC 1805 Spores
Strain3
Strain
3
C. difficile
 1, 2, 3 and 4% OH reduced spores of C. difficile ATCC 1805 by 1 log
CFU/ml, whereas 5% OH decreased spores by ~ 2 log CFU/ml
compared to controls.
RQ
 No residual spores of C. difficile could be detected on discs after OH
treatment.
C. difficile strain ATCC 1805
Clostridium difficile (CD) spores are shed in the feces of patients
CONCLUSION
Sporicidal Efficacy of Octenidine Hydrochloride on Stainless Steel
Octenidine hydrochloride could potentially be used as a disinfectant on
hospital surfaces to control nosocomial infections of C. difficile.
Strain2
StrainStrain3
3
OBJECTIVE
To determine the sporicidal efficacy of octenidine hydrochloride against
Clostridium difficile strains ATCC 1805 and 1870.
Residual C. difficile Spores on Steel Discs
C. difficile Spore Preparation
INTRODUCTION
infected with bacterium. The spores are resistant to environmental stresses,
and can survive on hospital surfaces for long periods of time. Commonly
affected surfaces in hospitals include bedpans, blood pressure cuffs, and
floors, which can lead to the transmission of CD in humans. Transmission of
CD from healthcare workers to patients though the feco-oral route or
environment to patients though shared instruments, furniture or contaminated
hospital surfaces has been reported.
Spores of CD are highly resistant to typical cleaning procedures. A safe
and effective sporicidal disinfectant against C. difficile spores is currently not
available. During severe CD outbreaks, 10% sodium hypochlorite is used in
hospitals, however, this high concentration of chlorine can corrode hospital
equipment, and trigger respiratory difficulties in healthcare workers.
Octenidine hydrochloride (OH) is a bispyridinamine compound that is
safe for use on skin, wounds, and as a mouth rinse. Recent studies have shown
that OH is effective against Staphylococcus aureus and Listeria
monocytogenes by way of disruption of the prokaryotic cell membrane.
RESULTS
MATERIALS & METHODS
* Treatment significantly different from control (p<0.05)
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