Download A comparative study of the effectiveness of

A comparative study of the effectiveness of triclosan containing antibacterial soaps and
regular soaps on Gram positive and Gram negative bacteria
J. Y. Greenfield, S. A Rand, N. M. Chikwem, D. S. McKnight, T. L. Coleman, J. Toto and J. O Chikwem*
Department of Biology, Lincoln University, PA 19352
Abstract
The anti bacterial effectiveness of four non-triclosan containing hand soaps was compared with that of six
triclosan containing hand soaps. Dilutions of the soaps were used in the preparation of pour plates which
were then streaked with cultures of Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium
and Bacillus cereus. After incubation at 37oC for 18 hours, none of the four non-triclosan containing soaps
inhibited the growth of the gram-negative bacteria, E. coli, S. typhimurium and Ps. aeruginosa. However,
the two Gram-positive bacteria, B. cereus and Staph. aureus, were inhibited by some concentrations of
soaps. B. cereus was inhibited by all four brands of soap at concentrations of 1.0% and above, while only
DG Body inhibited Staph. aureus at 2%. On the other hand, all six triclosan containing hand soaps
inhibited the growth of S. typhimurium and E. coli at concentrations of 0.5% and above. None of the
hand soaps in this study inhibited the growth of Ps. aeruginosa. We therefore conclude that triclosan
containing hand soaps are more effective in inhibiting the growth of both Gram-positive and Gramnegative bacteria; however, none of the hand soaps is effective against Pseudomonas aeruginosa.
*Corresponding author: Email: [email protected]
Introduction
Tricolsan
(2,4,4’trichloro-2’hydroxydiphenylether) is a broad spectrum antibiotic commonly
used in a wide variety of domestic products as an
antiseptic and anti-plaque agent. (1) Triclosan is
present in antibacterial soaps, toothpastes,
mouthwashes, cosmetics, deodorants, first-aid
products and many commonly used house-hold
items. In fact, 75% of liquid soaps and nearly
30% of bar soaps contain anti-microbial agents,
the most common of which is triclosan. (2) In
Sweden alone, 25% of toothpastes sold to
consumers contain triclosan. (3)
Triclosan
works by blocking the active site of the enol-acyl
carrier protein reductase enzyme (ENR) which is
essential in the synthesis of fatty acids in
bacteria. (4) Binding of triclosan to this enzyme
increases the affinity of the enzyme for NAD,
resulting in the formation of ENR-NAD+ triclosan complex which is unable to participate
in the synthesis of fatty acids. (4) Since humans
do not have this enzyme, tricolosan is believed to
be relatively safe for humans. (4) Although
ticlosan possesses mostly anti-bacterial
IHE: Lincoln University Journal of Science; Vol.2 (1) 2011
properties, it is also thought to have some antifungal
and
anti-viral
properties.
(5)
Consequently, triclosan is also used in hospitals
for preventing hospital acquired infections, (6)
and for decolonizing patients whose skins have
methicillin resistant Staphylococcus aureus. (7)
In spite of the effectiveness of triclosan in
inhibiting microbial growth in commonly used
domestic products and in controlling nosocomial
infections, there is a growing concern that the
overuse of triclosan may lead to potential
dangers to humans and the environment. For
example, the American Medical Association is
concerned that despite the proliferation of
triclosan in consumer products, its use has not
been extensively studied. (8) Studies have
shown
that
some
bacteria
including
Staphylococcus aureus and Escherichia coli can
develop low resistance to tricolosan due to Fab1
mutation. (9) Pseudomonas aeruginosa also
possesses multi-drug efflux pump that pumps
triclosan out of the cell, (10) and Bacillus species
have alternative Fab1 genes to which triclosan
does not bind. (11) These studies therefore
indicate that the use of triclosan as an antimicrobial agent may not be justified for a wide
variety of bacteria. Some studies even conclude
that triclosan-induced-resistant bacteria are also
resistant to other drugs including those used for
the treatment of tuberculosis. (12)
Some studies suggest that adding triclosan to
river water and shining ultraviolet light on the
water results in the formation of dioxin. (13, 14)
There is therefore concern that dioxin toxicity
could lead to endocrine disruption and possible
interference with the function of the thyroid
hormones and estrogen. (14, 15) Triclosan has
indeed been shown to block the metabolism of
thyroid hormones. (16)
Since 95% of the
triclosan used in consumer products is disposed
of in residential drains, and since wastewater
treatment is ineffective for removing triclosan, a
large amount of triclosan is expected to
contaminate waterways. (17) The discharge of
triclosan into waterways could also affect the
structure and function of algal communities in
stream ecosystems, thereby, leading to possible
destruction of the balance of aquatic ecosystems.
(18)
These concerns have led the American Food and
Drugs Administration to state that beyond the
use of triclosan in toothpastes to prevent
gingivitis, there is no evidence that triclosan
provides extra benefits to health in other
consumer products. (19) The Environmental
Working Group scientists also reviewed multiple
studies and concluded that the widespread use
of triclosan did not give consumers increased
germ-killing benefits. (20) Some studies even
suggest that triclosan was much less effective
than hand soap against surface bacteria. (21)
Other studies stated that chlorheximide and
povidone-iodine detergent preparations were
significantly more effective than non-medicated
soaps, but triclosan products were not. (22) In
spite of these concerns, leading soap brands
including Softsoap, Dial, and Bath and Body
works are vigorously promoting the efficacy of
triclosan as an anti-bacterial agent. It is
therefore necessary to determine the antibacterial quality of triclosan-containing soaps.
Our hypothesis is that the non-triclosan
containing hand soaps are equally effective in
killing bacteria as the tricolsan-containing soaps.
IHE: Lincoln University Journal of Science; Vol.2 (1) 2011
Methods and materials
Ten types of hand soaps were used in this study:
four of the ten [Dial, DG Body, Careone, Soft
soap] did not contain triclosan. Six of the ten
hand soaps [Soft soap, Careone, DG body, Dial,
Safeguard, Equaline] contained triclosan.
Careone and DG body had the lowest
concentration of triclosan at 0.115%; Soft soap,
Dial and Equaline had triclosan concentration of
0.15%, and Safeguard had the highest
concentration of triclosan at 0.25%. All types
and brands of hand soaps were purchased from
local grocery stores.
Veal Infusion Agar was prepared by mixing 40
grams of Veal Infusion Agar powder with one
liter of distilled water, and heating the mixture
over a Bunsen burner until completely dissolved.
19 ml of the dissolved agar was pipetted into
each of 50 screw-cap tubes which were then
sterilized by autoclaving at 121oC for 15 minutes.
The tubes were removed from the autoclave and
placed into a 45oC water bath to prevent the
solidification of the media. Dilutions of soap
solutions were prepared in sterile distilled water
to give stock solutions of 40%, 20%, 10%, 5%
and 2.5%, and then, 1.0 ml of each soap dilution
was added to the 19 ml of molten and cooled
agar. The soap dilution and molten agar were
gently mixed together to give a final soap
concentration of 2, 1, 0.5, 0.25 and 0.125
percent.
The mixtures of agar and soap
solutions were then carefully poured into sterile
Petri dishes and allowed to solidify. Control
agar plates were also prepared with 1.0 ml of
distilled water in place of the soap solution.
All the bacterial cultures used in this study were
purchased from Wards Natural Science,
Rochester, New York. Cultures of Escherichia
coli (85 W 1660), Pseudomonas aeruginosa (85
W 1903), Salmonella typhimurium (85 W 1956),
Bacillus cereus (85 W 1615), and Staphylococcus
aureus (85 W 1941) were inoculated into tubes
of Veal Infusion Broth and incubated at 37°C
overnight. The broth culture of each bacterium
was diluted in saline to McFarland 0.5. The soap
infused agar and control agar were then
inoculated with a loop-full of each culture. The
five bacteria samples were streaked on each agar
plate in parallel rows. Plates were incubated
overnight at 37⁰C. Microbial growth was
observed the following day and results were
recorded. Heavy growth of the test bacteria on
the soap containing media was indicated as
three pluses; moderate growth was indicated as
two pluses; light growth was indicated as one
plus while no growth was recorded as NG.
soaps were effective against Bacillus cereus at
concentrations of 1% and 2% and DG Body only
killed Staphylococcus aureus at a concentration
of 2% (Table 1).
Table 2 shows the summary of the results with
triclosan-containing hand soaps. The results
show that irrespective of the concentration of the
hand soap, none of the hand soaps under test
killed Pseudomonas aeruginosa. In fact there
was profuse bacterial growth in all the plates.
However, all six triclosan containing hand soaps
were effective at all concentrations against
Salmonella typhimurium, Escherichia coli and
Staphylococcus aureus. Bacillus cereus was also
killed at all concentrations by Soft soap and
Safeguard; however, Careone, DG Body and Dial
were only effective against Bacillus cereus if the
soap concentration was at least 0.5%. Based on
the results, we can state that the triclosancontaining hand soaps were more effective
against the test bacteria than the non-triclosancontaining hand soaps.
Results
The results of this study are shown in Table 1
(non-triclosan containing hand soaps) and Table
2 (triclosan containing hand soaps). The results
show that all the non-triclosan containing hand
soaps were not effective against the three Gramnegative bacteria (Pseudomonas aeruginosa,
Escherichia coli and Salmonella typhimurium.
Dial, Careone and Soft soap were also not
effective against one of the Gram-positive
bacteria, Staphylococcus aureus. All four hand
Table 1: Antibacterial activity of non-triclosan containing hand soaps
Hand
soap
Dial
Concentration
Percent
2.0
1.0
0.5
0.25
0.125
Psudomonas
aeruginosa
+++
+++
+++
+++
+++
Salmonella
typhimurium
+++
+++
+++
+++
+++
Escherichia
coli
+++
+++
+++
+++
+++
Bacillus
cereus
NG
NG
NG
+
+++
Staph
aureus
+++
+++
+++
+++
+++
DG Body
2.0
1.0
0.5
0.25
0.125
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
NG
NG
NG
+
+++
NG
+
+
+++
+++
Careone
2.0
1.0
0.5
0.25
0.125
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
NG
NG
+
+++
+++
+
++
++
+++
+++
2.0
+++
+++
+++
1.0
+++
+++
+++
0.5
+++
+++
+++
0.25
+++
+++
+++
0.125
+++
+++
+++
+++ [Heavy growth]; ++ [moderate growth]; + [light growth]; NG [No growth]
NG
NG
NG
++
+++
++
++
+++
+++
+++
Soft soap
IHE: Lincoln University Journal of Science; Vol.2 (1) 2011
Table 2: Antibacterial activity of triclosan containing hand soaps
Hand soap
Concentration
Percent
2.0
1.0
0.5
0.25
0.125
Psudomonas
aeruginosa
+++
+++
+++
+++
+++
Salmonella
typhimurium
NG
NG
NG
NG
NG
Escherichia
coli
NG
NG
NG
NG
NG
Careone
2.0
1.0
0.5
0.25
0.125
+++
+++
+++
+++
+++
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
+
+
NG
NG
NG
NG
NG
DG Body
2.0
1.0
0.5
0.25
0.125
+++
+++
+++
+++
+++
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
+
+++
NG
NG
NG
NG
NG
Dial
2.0
1.0
0.5
0.25
0.125
+++
+++
+++
+++
+++
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
++
NG
NG
NG
NG
NG
Safeguard
2.0
1.0
0.5
0.25
0.125
+++
+++
+++
+++
+++
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
NG
2.0
+++
NG
NG
1.0
+++
NG
NG
0.5
+++
NG
NG
0.25
+++
NG
NG
0.125
+++
NG
NG
+++ [Heavy growth]; ++ [moderate growth]; + [light growth]; NG [No growth]
NG
NG
NG
NG
+
NG
NG
NG
NG
NG
Soft soap
Equaline
Discussion
The results of this study show that triclosancontaining hand-soaps are more effective
antibacterial
agents
than
non-triclosan
containing hand soaps. The only exception
among the test bacteria is Pseudomonas
aeruginosa that was not killed by any of the
hand soaps.
Studies have shown that
Pseudomonas aeruginosa possesses multi-drug
efflux pump that pumps triclosan out of the cell.
(10) It is therefore possible that the resistance of
this bacterium to triclosan results from this
mechanism. Overall, no hand soap is effective
against Pseudomonas irrespective of whether it
contains triclosan; therefore, other methods
must be used in its killing especially in hospital
environments. All triclosan containing hand
IHE: Lincoln University Journal of Science; Vol.2 (1) 2011
Bacillus
cereus
NG
NG
NG
NG
NG
Staph
aureus
NG
NG
NG
NG
NG
soaps are effective against Salmonella
typhimurium, Escherichia coli, Staphylococcus
aureus and Bacillus cereus as long as the
concentration of triclosan is above 0.5%. It is
therefore recommended that hand soaps should
be used above this concentration in order to kill
bacteria such as Bacillus cereus. This study also
showed that irrespective of the concentration of
triclosan in the hand soaps, the antibacterial
effect was identical. For example, there was no
significant difference between Safeguard with
0.25% triclosan and Careone and DG Body with
0.115% tricosan. Since there are concerns
regarding the overuse of triclosan in domestic
products, the results of this study show that the
concentration of triclosan can be reduced by half
without losing its anti-bacterial qualities. This
will reduce the amount of triclosan being
discharged into wastewater that eventually
pollutes waterways. (17) Finally, the results of
this study contradict the views of the American
Medical Association, (8) and the Food and Drugs
Administration (19) that triclosan does not
provide extra health benefits to consumers
outside of their use in toothpastes to control
gingivitis. The concern should be in controlling
the concentration of triclosan in domestic
products to avoid the problems of overuse and
abuse.
9.
10.
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IHE: Lincoln University Journal of Science; Vol.2 (1) 2011
IHE: Lincoln University Journal of Science; Vol.2 (1) 2011