The Effectiveness of antiseptics on Pseudomonas aeruginosa, Staphylococcus aureus, and E.coli.
By: Abigail L. Stowell (Animal Science), Amy Poss (Biology), and Amanda Steffens (Biochemistry)
Abstract
Antiseptics are essential in controlling bacterial populations on the skin surface. The effectiveness of antiseptics is dependent on several properties: fast acting with a prolonged duration of activity, activity against all likely contaminants and pathogens, nontoxic, noncarcinogenic and non-teratogenic (does not cause malformation of embryo or fetus) to host cells,
and having minimal systemic absorption. The effectiveness of these antiseptics on gram-positive and gram-negative bacterium differ depending on the antimicrobial action of the drug. The antimicrobial activity of a drug on a bacteria can be inferred by disk diffusion, Minimum inhibitory concentration (MIC), and Minimal bactericidal concentration (MBC)
tests. These tests were used to determine the level of activity and the effectiveness of nitrofurazone, iodine tincture and hydrogen peroxide against Pseudomonas aeruginosa (PA), E. coli (EC), and Staphylococcus aereus (SA). The results of these tests demonstrated that iodine was most effective as a bactericidal agent in all three of the bacteria SA, PA, and EC.
Methods:
Background
Iodine tincture (2.4% Sodium Iodide and 47% alcohol) is believed to kill bacteria,
by precipitating or congealing the bacteria’s proteins through halogenation with free
iodine. This activity can be increased by lowering the pH. Iodine tincture is broad
spectrum, meaning it is effective against a wide variety of microorganisms. It has
Unfortunately its activity is limited to only 4-6 hrs and its absorption into damaged
skin and mucous membranes can increase serum levels 104 times normal levels. If
pregnant, this can penetrate the placenta and eventually cause hypothyroidism or
goiters in the fetus.
(Fig.1)
Chemical Formula:
K+[I-]
Hydrogen Peroxides bactericidal activity is attributed to its oxidizing ability. It is
relatively unstable and when in warm conditions tends to degrade into water and
oxygen. It is a relatively weak antiseptic and therefore is used primarily for
cleansing wounds only. Because of its instability, also has a very short half-life.
(Fig.2)
Chemical Formula:
Nitrofurazone is a bactericidal agent, with a broad spectrum of activity. It is primarily
effective against most gram-positive bacteria and facultative gram-negative bacilli.
Topical nitrofurazone is not absorbed into the blood to any substantial degree even
after extensive use. Sensitive bacterium contain a flavoprotein reductase that causes
the transformation of nitrofurazone into a chemical that reacts with proteins. (6).
Discussion:
Results:
Test I. Disk Diffusion
Using an inoculation loop and aseptic procedures, a culture of SA, EC, and PA was
prepared. The cultures contained thee loops of bacteria and 10 ml of Nutrient broth.
After preparing cultures they were incubated at 37°C for 24 hrs. Mueller Hinton afar
was prepared using 38g of mix per liter of distilled water. 39 sterile Petri dishes were
filled with 20ml of agar per plate. The plates were left to cool and were stored in the
refrigerator till used. After the 24hr. incubation period, 13 plates per bacteria were
inoculated using a sterile swab and swabbing in a continuous pattern in three
directions to form a bacterial lawn. Nine absorption disks for each antiseptic
(Neosporin, nitrofurazone, iodine, and hydrogen peroxide) were prepared by soaking
them in an antiseptic. Excess antiseptic was either left to drip off or was scraped off
using a sterile forceps. The disks were separated out into sterile Petri dishes and
allowed to dry in the 37°C incubator for 10 minutes. After drying absorbance disks
were placed onto the plate cultures. For each bacteria three absorbance disks of each
antiseptic was tested (1 disk/plate). A control using a blank disk was prepared for each
bacteria. The plates were incubated again at 37°C and clearance values were taken
from each at 24 and 48 hrs (As in Fig. 7). Neosporin was eliminated from further
study because of its inability to diffuse on the plates.
P. aeruginosa
E. coli
S. aureus
Iodine
10.00 mm
12.67 mm
9.67 mm
Hydrogen Peroxide
17.37 mm
18.67 mm
17.33 mm
Nitrofurazone
15.53 mm
10.67 mm
25.00 mm
Test I. Disk Diffusion discussion (Table 1 and Graph 1):
The disk diffusion test provided evidence that iodine and hydrogen peroxide
maintained consistent results among all three species bacteria. Nitrofurazone
demonstrated the greatest degree of effectiveness on SA (gram-positive). Our results
showed that Nitrofurazone was less effective against PA and EC (gram-negative
bacteria). Hydrogen peroxide had the greater clearance on gram-negative bacteria, than
did iodine and nitrofurazone. Iodine and nitrofurazone illustrated variable diffusibility
on Mueller-Hinton agar. The nitrofurazone had an extremely high standard deviation,
while iodine and hydrogen peroxide’s clearance values were more consistent. This
could be due to nitrofurazone’s relatively gel-like physical state at room temperature,
and could possibly affect its diffusibility and hinder the experimenter’s ability to
maintain equal concentrations of antiseptic on the disks.
Average
Table 1. Average Zone of inhibition (mm) after 24 hour
incubation
Test II. MBC Discussion:
The MBC results were disregarded because the bacterial growth in the broth was too
large of an amount to affected by the antiseptics used. An actual human wound would
most likely never contain the large amount of bacteria we had in our test tubes. Under
realistic conditions, one would place antiseptics on the wound before it would ever
reach such large quantities of bacteria within 24 hours.
35.00
30.00
25.00
(Fig. 3)
Chemical Formula:
O
NO2
CH=N-NHCONH2
The bacteria used in the experiments were Pseudomonas aeruginosa, Staphylococcus
aureus, and E.coli.
Pseudomonas aeruginosa (Fig.4) is a Gram-negative, aerobic rod. They are
common inhabitants of soil and water, but can also be found on the surface of plants and
animals. This bacteria is an opportunistic bacteria. If given the chance it can cause
urinary tract infections, respiratory system infections, dermatitis, soft tissue infections,
and a variety of systemic infections, particularly in patients with severe burns, cancer
and AIDS patients who are immunosuppressed. The bacterium is respiratory and never
fermentative, it can grow in the absence of O2 if NO3 is available as a respiratory
electron acceptor. Pseudomonas aeruginosa is often observed growing in "distilled
water" which is evidence of its minimal nutritional requirements. Its optimum
temperature for growth is 37° . This bacteria is also know for it’s resistance to
antibiotics. It’s resistant to so many because of it’s permeable barrier provided by its
outer membrane lipopolysaccharide. It also maintains antibiotic resistant plasmids (rfactors) which can be passed on to later generation.
Staphylococcus arueus (Fig.5) is in the Bacterial family Micrococcaceae, and are
Gram-positive spherical bacteria that form clusters that look like grapes. Each spherical
cell is about 1 micrometer in diameter. Staphylococcus arueus form large yellow
colonies in rich mediums. It can grow at a temperature range of 15-45 degrees calculus,
however the most rapid growth is at temperatures near that of body temperature. S.
aureus is a nonmotile, nonsporeforming facultative anaerobe, which produces lactic acid
upon fermentation of glucose. S. aureus is considered a potential pathogen. It can be
found in humans in nasal passages and on skin and mucous membranes. S. aureus is a
leading cause of soft tissue infections, and is a major cause of hospital acquired
infections of surgical wounds and needle sticks. S. aureus also causes toxic shock
syndrome (TSS), food poisoning, pneumonia, meningitis, and boils.
Escherichia coli (Fig.6) is a common gram-negative, rod-shaped intestinal
bacterium. It grows at about 37 degrees Celsius and is very viable in many environmental
conditions such as changes in pH, chemicals, and temperature. It is a facultative
anaerobe which can ferment acid and lactose. E. coli lives in a mutualistic relationship
with humans and other warm blooded animals where it survives in the GI tract. It is the
predominant facultative organism in the human GI tract. It is a useful bacterium for the
analysis of water for fecal contamination. E. coli can cause gastroenteritis or urinary
tract infections, and is a common cause of food poisoning. E. coli can be contracted
through undercooked contaminated beef, drinking contaminated water or unpasteurized
milk, and by person to person through hand to mouth contact.
Bacterial Lawn Growth
Zone of Inhibition(mm)
Zone Of Inhibition
20.00
Iodine
Hydrogen Peroxide
15.00
Nitrofurazone
10.00
5.00
Diameter in mm (clearance value)
Fig.7. Bacterial Plates for Disk Diffusion
EC plate with Hydrogen Peroxide
0.00
P. aeruginosa
E. coli
S. aureus
Bacteria Used
Test II. MBC test on pregrown bacteria cultures.
Ten cultures of each bacteria SA, EC, and PA was prepared using 5ml of nutrient broth
and three loops of bacteria. The cultures were then incubated at 37°C for 24hrs. Eight
sterile Mueller Hinton Agar dishes were prepared using 20ml of agar per dish and
allowed to cool. Three 24hr nutrient broth cultures of each bacteria tested was used
per antiseptic. In the first culture 1ml of an antiseptic was used, in the second .5ml of
the same antiseptic was used, and in the third .25ml of the antiseptic was used. This
was repeated for the other two antiseptics being tested. One culture per bacteria was
left as a control group. The samples were then reincubated for 24hrs at 37°C. After
incubation of the test tubes the plates were split into quadrants and streaks of each test
tube was done. The plates were then incubated for 24hrs at 37°C and the presence of
growth was observed and noted.
Graph 1. Average Zone of Inhibition (mm) after 24 hour
incubation
1ml/5ml NA
Cidal
Growth
Turbidity
Pseudomonas aeruginosa
Iodine
E. coli
Conclusion:
Staphylococcus aureus
Pseudomonas aeruginosa
Hydrogen Peroxide
E. coli
Staphylococcus aureus
Pseudomonas aeruginosa
Test III. MIC and MBC tests
Nitrofurazone
For each bacteria, ten test tubes with 5ml of nutrient broth were inoculated with three
loops of 24 hr. nutrient broth culture. Three of these inoculated tubes were used per
antiseptic. In test tube 1, 1ml of antiseptic was added. In test tube 2, .5ml of
antiseptic was added, and in test tube number 3, .25ml of antiseptic was added. This
was repeated for each of the antiseptics. One sample of each bacteria was kept as a
control. The antiseptic cultures were then incubated at 37°C for 24 hrs. After
incubation samples were observed for turbidity. The samples that did not demonstrate
turbidity were used for the MBC test.
.05 ml/5ml NA
Cidal
E. coli
Staphylococcus aureus
Growth
Turbidity
Pseudomonas aeruginosa
Iodine
E. coli
Staphylococcus aureus
Pseudomonas aeruginosa
Hydrogen Peroxide
A streak plate was taken for each of the non-turbid bacterial samples. The streak
plates were incubated for 24hrs at 37°C. After the 24hr incubation period the presence
or absence of growth was noted and the data was analyzed.
E. coli
Staphylococcus aureus
S. aureus
Overall, the results support the hypothesis that iodine is the most effective
bactericidal agent in the presence of moisture. This suggests that iodine is the best
choice to use as an antiseptic for wounds. Hydrogen peroxide diffuses considerably
and works fairly well in limiting the number of bacteria present, but does not have a
high degree of bactericidal activity when water is present. Previous research
suggests that while hydrogen peroxide makes a good cleaning agent, its bactericidal
action is very weak. Nitrofurazone controls S. aureus (gram positive) quite well,
but its effectiveness is diminished considerably in the gram negative bacteria tested.
When testing the various antiseptics using the disk diffusion methods diffusibility
issues were faced. Neosporan, which originally was an antimicrobial agent tested
was completely unable to diffuse on the agar. Iodine also demonstrated less ability
to diffuse in the Mueller-Hinton agar. Further experimentation using Nutrient Agar
and possible several others would allow for analysis of the effects of Agar on the
disk diffusion results.
Pseudomonas aeruginosa
Nitrofurazone
E. coli
Staphylococcus aureus
P. aeruginosa
Test III. MIC and MBC discussion (Table 2):
In some respect the MIC and MBC test better simulated a cutaneous infection that an
individual would experience after injury. The cells were allowed to cluster together,
had free motion in a fluid medium, and concentrations of the antiseptic were diluted.
The antiseptics were installed in a moisture rich environment that would imitate the
interior of a wound with blood or pus. In PA and SA hydrogen peroxide demonstrated
the least turbidity. When plating these cultures for the MBC test, growth developed in
all three concentrations. This could be due to the lack of the ability to penetrate all cells
in the medium, for example, cells that may “clump” together. This clumping process
would decrease the surface area of the bacteria cell that is in contact with the antiseptic,
therefore decreasing the antiseptics effectiveness. It could also be due to the antiseptics
degradation in the presence of a high degree of water activity or in high moisture
environments. Nitrofurazone’s MIC for SA was somewhere between 1ml and .5 ml.
For PA and EC, nitrofurazone demonstrated very little effectiveness at any of the tested
concentrations. Prior research demonstrated that nitrofurazone, although broadspectrum is effective primarily in gram-positive and facultative gram-negative bacteria
(6). When testing the MBC for the 1ml concentration of nitrofurazone there was
substantial growth present, this showed that although the concentration was high
enough to substantially inhibit growth, it was not strong enough to demonstrate a
complete kill of the sample. This could also be due to the “clumping” phenomena
discussed previously. Iodine was the most effective bactericidal agent. It killed all of
the bacteria (gram positive and gram negative alike) at concentrations of .5ml and 1ml.
Growth was observed at .25ml of antiseptic. This demonstrates that the MBC for this
agent is between .25ml and .5ml.
E.coli
0.25 ml/5ml NA
Cidal
Growth
Turbidity
References
1.
Pseudomonas aeruginosa
Iodine
Fig. 4. Scanning electron micrograph
of the common soil bacteria
Pseudomonas aeruginosa. Image ©
James A. Sullivan, CELLS alive!
Fig.5. Scanning electron micrograph of
Staphylococcus aureus
http://www.bact.wisc.edu/Bact330/lecturestaph
Iodine
E. coli
2.
Staphylococcus aureus
Fig.6. Electron micrograph of E. coli cells
http://www.slic2.wsu.edu:82/hurlbert/micro101/pag
es/Chap2.html#two_bact_groups
Staphylococcus aureus
Hydrogen Peroxide
Pseudomonas aeruginosa
3.
E. coli
4.
5.
Hydrogen
Peroxide
Pseudomonas aeruginosa
Nitrofurazone
Hypothesis:
Iodine is used extensively as a antiseptic for surgeries, wound care, and sanitizing
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(broad-spectrum). Previous research suggests that iodine tincture is the most effective
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Nitrofurazone, although it has a longer half-life it does not have as large of an
antimicrobial spectrum.
E. coli
6.
Staphylococcus aureus
7.
8.
Table 2. MIC and MBC test results
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