Download 44-The role of materials and surfaces in the transmission of bacteria

PROJECT N°44
The role of materials and surfaces in the transmission of bacteria in public places:
A case study of a school
Camilla Hurst
European School Luxembourg I
23, Boulevard Konrad Adenauer, 1115 - Luxembourg, Luxembourg
S6 EN
Abstract
This research project is a study into the bacteria found in schools and measures that can be
taken to reduce the transmission from person to person.
This project has been carried out in
collaboration with the Luxembourg Institute of Science and Technology (LIST).
The first stage of the project identified the bacteria in the school with DNA sequencing. While
the majority of the bacteria were harmless, Staphylococcus saprophyticus (a cause of urinary tract
infections) was identified. We also found Propionibacterium acnes, and a bacteria belonging to the
genus Neisseria (which might provoke meningitis, a sexually-transmitted disease, and other diseases).
The project continued with tests as to the survival of bacteria on a range of surfaces and in
particular on various soft and hard woods, copper and plastic. The results show a very quick death rate
for the bacteria on pine (nearly all gone after 15 minutes). Plastic, was the worst performer, with
bacteria still present on the plastic surface after more than 10 hrs. The project attempted to identify
the compounds in wood that might contribute to its antibacterial properties. Solutions of wood
extracts were prepared by soaking sawdust in physiological serum and in ethanol. The water-based
solutions had little effect, while the ethanol based solutions did reduce bacterial present in the test
samples by approximately 10-fold over a 2 day period.
The chemical composition of these extracts
was analysed with High Performance Liquid Chromatography. This showed that there were more
compounds present in the ethanol extractions, of which the predominant compound found is
associated with plant defensive systems.
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The mechanisms by which pine disinfects are complex and remain unclear. There is likely to
be a physical effect where bacteria are absorbed in the wood. Pine is also antibacterial as we are using
the natural defensive mechanisms of a tree when it is subject to bacterial invasion – namely resins that
protect the tree from intrusions. This is a complex reaction that is appears to depend upon the
combination of the chemicals found in the resins.
Materials can be used to reduce transmission risks, for example by using untreated pine for
work surfaces. However, door handles pose a particular problem due to the frequency with which they
are touched.
The only way to quickly remove bacteria from a door handles is through repeated
cleaning. A prototype door handle was built that dispenses a small quantity of disinfectant each time
it is used.
This was well-received by a sample of users.
This work has been derived from a school environment, but is relevant for other public
buildings such as hospitals, and care homes.
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1. Introduction
Though many infections can be controlled by the body’s defensive mechanisms, antibiotics may be
necessary to help the body kill bacterial intrusions. Unfortunately, there is growing resistance to
antibiotics. Some bacteria such as Staphylococcus aureus have shown an ability to adapt to each new
antimicrobial agent that is developed. This bacterium is present of the skin of 30% of population, but
if it enters the body through cuts and scrapes it can lead to boils and abscesses. In extreme case it can
cause general sepsis and death (harvardmagazine.com/2014/05/superbug; Accessed, 1/2/2017).
In the US it has been estimated that 2 million people each year are infected by resistant bacteria, of
which
some
23,000
die
(www.huffingtonpost.com/entry/infection-all-antibiotic-
resistant_us_587960f4e4b0b3c7a7b16e29; Accessed, 1/2/2017). Alternative estimates are that as
many as 50,000 people die each year in the US and Europe due to the inability to control infections
caused by resistant bacteria. On the world scale, the number has been put at 700,000 deaths per year
(www.amr-review.org; Accessed, 1/2/2017).
The problem is becoming much more serious. Estimates by the Review on Antimicrobial Resistance
(commissioned in July 2014 by the UK Prime Minister; www.amr-review.org; Accessed, 1/2/2017)
place the possible worldwide annual deaths as many as 10 million by 2050.
Many policies are needed to battle this issue. A particular problem is excessive use of antibiotics with
animals (giving a small quantity of antibiotics leads to weight gain) exposing bacteria to low levels of
antibiotics in the environment and allowing them to adapt through natural selection. As much as 80%
of antibiotics in the US are used in animals. Antibacterial resistance can then pass to humans through
the food supply. Antibiotics are also spread through animal urine and faeces allowing further
resistance to develop in the natural environment (www.ncbi.nlm.nih.gov/pmc/articles/PMC4378521/;
Accessed, 1/2/2017).
Bacteria are also able to share genetic resistance horizontally from one to the other and not just
through reproduction (www.theatlantic.com/health/archive/2017/01/colistin-resistance-spread/;
Accessed, 1/2/2017). For example, in November 2015 a new gene that was resistant to the antibiotic
of last resort, colistin, was found in China. This gene, known as mcr-1, is not found in the bacteria’s
chromosomes, but on a free-floating piece of bacteria DNA called a plasmid. Bacteria carrying this
plasmid can share copies of it with other bacteria that they come into contact with, allowing the gene
to spread rapidly. In fact, already in 2016 a woman in the United States was found to have a urinary
tract infection caused by Escherichia coli bacteria with mcr-1. This bacterium had a total of 15 genes
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for resistance to other antibiotics (www.scientificamerican.com/article/dangerous-new-antibioticresistant-bacteria-reach-u-s/; Accessed, 1/2/2017). As colistin is used extensively for animals in China,
but not in people, the mcr-1 gene is likely to have developed from excessive use of antibiotics in animal
feedstocks.
In another case, a recent news story has told of the case of an American woman who contracted
Klebsiella bacterium during an operation in India. This bacterium was resistant to all 26 antibiotics
available in the US, leading to the eventual death of the woman in September 2016 from sepsis
(www.newscientist.com/article/2118046-woman-dies-from-infection-resistant-to-all-availableantibiotics/; Accessed, 1/2/2017).
These examples show the possibility of new resistant species developing in one country and being
spread over long distances. Poor management of antibiotics in distant locations can have very
widespread consequences.
However, some 80 per cent of infections are spread through person-to-person contact. Therefore,
one important measure is to improve hygiene and to reduce hand-to-hand transmission to start with.
This research project focuses on these issues with the case study of a school.
This project has been
carried out in collaboration with the Luxembourg Institute of Science and Technology (LIST).
This year’s project builds upon earlier work I undertook to identify bacteria in the school. The problem
will be tackled using an integrated approach from a variety of angles to reduce risk. In this context,
one theme is to look at the role of surfaces in transmission. It was found that pine effective is in
naturally reducing bacteria levels. Tests were performed both on pine’s effectiveness and on the
reasons why. The goal was to understand how surfaces at risk of contamination can be adapted to
reduce transmission. The second theme has been concerned with the surface of door handles. They
pose particular challenges due to their frequent use. The prototype for an automatically disinfecting
door handle was developed.
The initial work on the identification of bacteria in the school is summarised briefly in Section 2.
Section 3 examines the antibacterial properties of pine. Section 4 looks at the disinfection of door
handles. Section 5 concludes.
This work has been derived from a school environment, but is relevant for other public buildings such
as hospitals, and care homes.
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2.
Earlier research: Identification of the bacteria in the school
Earlier research involved an examination of the bacteria found in the school. Test samples were taken
from a range of surfaces around the school and cultivated on agar jelly. The highest level of bacterial
contamination was found on surfaces such as the library table, banisters, door handles. Conversely,
places that are thought to be “dirty” are properly cleaned (toilets, water fountain) and had low levels
of bacteria present. Most likely surfaces such as tables and the banister only get very superficial
cleaning as they are not thought to be “dirty”.
Samples from the more contaminated surfaces (the library table, the banister, a canteen chair, and
the entrance door handle) were also incubated at 37°C, or body temperature, for 3 days under 3
different atmospheric conditions using chocolate agar. This is a very rich medium and it resembles the
conditions bacteria would face inside a body.
The bacteria cultivated by this process were identified using DNA testing with a PCR or polymerase
chain reaction (see en.wikipedia.org/wiki/Polymerase_chain_reaction en; accessed 2/2/2017, and
en.wikipedia.org/wiki/Sanger_sequencing; accessed 2/2/2017). This was done with equipment and
laboratory support from LIST. This analysis found that the majority of the bacteria were harmless and
associated to the human skin “microflora” (which protect us from intrusion of harmful bacteria). The
entrance door handle had a bacterium that is associated to the human mouth microbiota, and is
typically associated to the aerosols created when sneezing.
More importantly, Staphylococcus
saprophyticus was found on the bannister. S. saprophyticus causes 10-20% of urinary tract infections
(UTIs). In females 17–27 years old, it is the second most common cause of community-acquired UTI,
after Escherichia coli.
The library table produced the broadest range of results. On this table we found a bacterium from the
Dermacoccaceae family. This sample only had a sequence identity of 90%-94% with the best-matching
database entries, meaning that we have discovered an unprecedented bacterium that has not been
described so far. The family of bacteria to which it is closest is usually found on dry surfaces such as
tables and desks. They are also found in dust, skin, water and plants.
In terms of health, it also had the following bacteria present:

Propionibacterium acnes. These bacteria are responsible for acne, the skin condition well
known for teenagers.
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
Bacteria belonging to the genus Neisseria. This bacterium bears resistant pathogens which
might provoke meningitis, a sexually-transmitted disease (gonorrhea), inflammation and other
diseases
The results show a number of bacteria present in the school that could cause the spread of disease –
from acne to urinary infections.
On the school banister we also found a variant of Kaistella. This bacterium originates from South Korea
and while it has been found in many other locations this is the first time it has been discovered in a
European environment. While this bacteria is not harmful, this result shows the potential for bacteria
to travel long distances, and to mutate to its environment. As mentioned in the introduction, this
raises issues regarding the distribution of harmful diseases on a world-scale.
Full details are given in the paper: What do we bring home from school? A study on the transmission
of bacteria on school premises, Camilla Hurst, presented to the ESSS in 2015.
3. Tests of various materials
3.1. Background: Comparison of woods, plastic and copper
The next question in this research project was to explore the natural anti-bacterial properties of
materials. The goal is to find surfaces that would naturally reduce bacteria. This would help prevent
the creation of resistant bacteria.
Internet research showed that metals such as copper reduce the growth of bacteria. For example,
surfaces made with copper alloys reduce the transmission of disease-causing organisms and can
reduce
patient
infections
in
hospital
intensive
care
units
by
(https://en.wikipedia.org/wiki/Antimicrobial_copper-alloy_touch_surfaces;
as
much
accessed
as
58%
2/02/2017).
This is known as the oligodynamic effect.
In earlier research (What do we bring home from school? A study on the transmission of bacteria on
school premises, Camilla Hurst, presented to the ESSS in 2015), I studied the impact of copper by
copper-plating one side of a steel door handle using the electrolysis of copper sulphate. A group of
students used both the steel handle and the copper handle to enter a room. Sample were taken from
the handles and cultivated in Petri dishes of chocolate agar at 37°C in an oxygen-reduced atmosphere
to represent the conditions in the body. While this is only one test, the copper plated handle reduced
the amount of bacteria by 1.8 times, or almost by 50%. This was an encouraging result, which
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suggested that the materials used could indeed be important. If materials could eventually kill
bacteria, this means that surfaces made from these materials would be constantly self-cleaning.
In order to continue this work I designed an experiment to test the extent to which bacteria could
survive on a range organic and in-organic of surfaces. The surfaces tested were four woods (Pine;
Paulownia a very light softwood; Beech and Oak), as well as Copper, and Plastic.
The outline of this experiment was to place the same amount of bacteria on each surface and see on
which surface the bacteria would die the fastest. Of course, each surface was exposed to the same
environment and over the same amount of time. For this phase of the experiment, unharmful E.coli
supplied by the LIST was cultivated and used.
Firstly, bacteria were placed in a solution of physiological serum. Serial dilutions were performed to
know the amount of bacteria present, which was found to be 11205 bacteria per millilitre. On each
material several 7cm by 2cm columns were measured. A total of 1ml of the bacteria solution was
spread on each of these columns. Each material was swabbed at regular intervals, each time using a
different column. Threefold dilutions of the swabbed bacteria were then prepared (the original
concentration, 10 times diluted and 100 times diluted). A total of 50 microliters of the swabbed
bacteria was placed in the agar petri dishes. The petri dishes were then incubated at 32°C (ambient
temperature) for 2 days and then colony counted. This temperature was chosen to find out what
bacteria would be present in the environment.
The results are shown in the table below.
Amount of bacteria on each surface
300
250
200
150
100
50
0
Paulownia
Oak
Pine
Copper
Beech
Plastic
Note: the 1st column is after 15 minutes; the 2nd after 2 hours 35 minutes; the 3rd after 7 hours 20
minutes; and, the 4th after 10 hours 40 minutes. These times were chosen as the experiment was
carried out during school days and the tests were constrained by the class schedule.
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Pine and copper showed a very rapid reduction in bacteria levels with only small trace levels detected
after the first time interval (15 minutes). By the second time period (155 minutes), no bacteria were
found on the Paulownia and Oak surfaces. The worst surface of all was the plastic, which still had traces
of the bacteria after almost 11 hours. This was surprising as plastic is so widely proposed as a clean
and hygienic material when in fact it keeps bacteria alive for the longest.
It is likely that a wood’s physical property is one of the reasons for its antibacterial
effect. It is able to absorb the bacteria and hence remove it from the surface.
However, in the earlier experiment mentioned above, the Paulownia wood was by far
the most absorbent (as shown in the photo). In fact, it was so absorbent that some of
the bacteria solution spread even outside of the designated area. Nonetheless, pine
performed better than Paulownia.
Further experiments were carried out to consider this issue further. The same test was done with
copper for comparison. In this test, a bacterial solution was prepared much like in the previous
experiment, and placed into sterile tubes. The same volume of grounded copper and pine particles
(1ml) was submerged in 5ml of bacterial serum. The tubes were regularly shaken and some 50
microliters were removed from the solution every 20 minutes and dilutions were done (for three times
in total). The extracted solution was placed in Petri dishes and incubated for 2 days at 32°C. With this
test, the bacteria were fully eliminated within an hour by both materials, confirming their antibacterial
properties.
Since the sawdust was dry it will have absorbed some of the solution in that sample and with it some
bacteria. However, this should not have influenced the density of bacteria that remained in the
solution, as there is no reason to suppose that bacteria would be preferentially absorbed by the wood.
Any effect of the sawdust in this test should be due to its chemical content rather than physical
absorption.
A few other studies have found similar results. For example, one study examined the survival of
bacteria on various woods (pine, spruce, larch, beech, maple, poplar and oak) and polythene. It found
that bacterial levels declined most rapidly on pine and slowest on plastic. Oak was the next best
performing wood with intermediate results for the other woods. The conclusion was that both pine
and oak have good hygienic properties. (www.wilms.com/Hygiene/presse/wilmshf5920057281.pdf).
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If the way in which pine destroys bacteria can be understood, the optimal way of include these
properties in work surfaces could be developed.
That was the objective of the next stage of the
project.
3.2. Tests of the components of pine
The resins in wood act as a natural defensive mechanism to invasion by bacteria and insects. Resins
are found throughout a tree, but are more concentrated in heartwood (the closed dead inner part of
the tree which in the case of pine is filled with resin). However, these resins flow to areas where the
surface/bark of the tree is damaged.
The compounds in these resins that may have anti-bacterial properties include:

Turpenes.

Resin acids

Phenolic compounds.
(see,
for
example,
biotuli-hanke.fi/files/download/BIOTULI-raportti_YangJaakkola2012.pdf
www.chemeng.ntnu.no/research/paper/Online-articles/Heum_Organic_extractives.ppt, and
chemistry.umeche.maine.edu/Green/Afternoon/Cole.pdf; accessed 1/2/2017)
A test was constructed for tannin, a phenol, as one study has already successfully tested the
antimicrobial effect of tannin. These phenols can bind and precipitate proteins, thus amino acids and
other organic compounds. This happens when the tannins form hydrogen bonds with proteins. If it
can form bonds with bacterial protein then they may become denatured impairing metabolism.
(www.academia.edu/5617816/Test_of_Antimicrobial_Activity_of_Tannins_Extract_from_Guava_Lea
ves_to_Pathogens_Microbial_By_Meigy_Nelce_Mailoa; Accessed, 1/2/2017)
Tannins have insoluble tannin-ferric complexes, meaning that there can be metal ion deprivation. This
is important because ferric solution reacts with tannins by changing colour. By mixing tannin with iron
sulfate, and iron chloride, a water-soluble ferrous tannate complex is formed. This is a dark blue
pigment (using iron and reacting it with tannin was actually a medieval way of making ink) making this
a very rapid test of tannin concentration. In my experiment I used both iron(II)sulphate and
iron(III)chloride to see whether the difference between Fe2+ and Fe3+ would affect the dissociation of
the tannin.
The oak wood instantly dyed to a dark blue with both iron salts, as shown in the picture. The soft
woods didn’t react with the iron salts, showing that tannin is more abundant in hard wood.
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Pine with iron sulphate
Oak with iron sulphate
Tannin is evidently not what makes pine antibacterial.
In order to explore this further, pine sawdust was soaked in water in order to extract soluble
components of the wood resins. For the first experiment a solution of pine extract was formed by
soaking 15 g of pine sawdust in 30 ml of physiological solution for 48 hours. The sawdust was then
filtered out to leave only soluble components in the solution. The bacteria used was S. epidermidis a
harmless skin bacteria. Although this not usually pathogenic, it is the case that patients with
compromised immune systems may be at risk of developing infection notably in hospitals
(en.wikipedia.org/wiki/Staphylococcus_epidermidis; accessed 15/3/2107).
Bacteria were added to a sample of 10 ml of this solution. A similar quantity of bacteria was added to
pure physiological serum. After 1 hour, 50µl of the various dilutions (in total five 10-fold dilutions) of
the sample were distributed on petri dishes of agar jelly. After being cultivated at 37 centigrade for
48 hours, the bacterial colonies found on each dish were counted.
The results were:
Physiological serum with bacteria
Sample 1: 247 @ dilution of 10-4
Sample 2: 214 @ dilution of 10-4
Pine extract with bacteria
Sample 1: 183 @ dilution of 10-4
Sample 2: 177 @ dilution of 10-4
While pine extract has reduced the level of bacteria, this was not by a significant amount.
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To test this further, the experiment was repeated with two variations. In the above experiment, the
concentration of solutes in the extract solution may not have been particularly high due to the limited
contact period between pine and water. Therefore, in this experiment, 15g of sawdust was added to
100ml of water. This mixture was boiled and kept at this temperature of 30 minutes. The solution
was left for five days, boiled for a further 30 minutes at this time, then left for another 5 days. As the
pine was left in contact with water for a much longer period than before, this should have increased
the concentration of extract. Internet sites for the home production of pine oils suggest soaking pine
needles for a 2 week period (e.g. www.articles.mercola.com/herbal-oils/pine-oil.aspx; accessed
1/02/2017). Boiling could have opposite effects. The higher temperature should increase the
dissolving of extracts in the wood, but this may have been compensated by the evaporation of these
chemicals. However, the boiling point of many pine extracts is reported to be well above 100 degrees
(en.wikipedia.org/wiki/Pine_oil; accessed 1/2/2017) so the result should be to increase
concentrations.
Secondly, as wood may contain compounds that are not soluble in water a small quantity of sawdust
was added to a sample of physiological serum. The sawdust that was used was from the sample that
had been soaked for 10 days to remove water-soluble components. It was microwaved beforehand
to kill of the possible bacteria on the sawdust.
As before, bacteria were added to each of these samples and they were left for a period of 175
minutes. Evidently, a control sample with no bacteria was kept. After the dilutions (again a total of
five 10-fold dilutions) these various solutions were cultivated on agar dishes for 48 hours at a
temperature of 37 degrees.
The colony counts for each of these samples were as follows:
Physiological serum with bacteria: 67 @ dilution of 10-5
Pine extract with bacteria: 53 @ dilution of 10 -5
Physiological serum with sawdust and with bacteria: 19 @ dilution of 10 -5
As before, the extract of soluble chemicals from pine has reduced the quantity of bacteria in this test
though by an insignificant amount. The presence of sawdust with the bacteria has had a slightly more
significant reduction in the bacteria present, perhaps indicating the presence of hydrophobic
compounds remaining within the sawdust.
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Since many of the extracts mentioned above, such as turpenes do not dissolve in water and would
have remained in the sawdust, extracts were prepared with ethanol. In particular 20g of sawdust was
left in 100ml of ethanol for 12 days. For comparison, a similar solution was made with water based
serum as before.
These samples were shaken at regular intervals to ensure good contact between
the pine and the solvent.
The ethanol was evaporated by vaporisation in a gas chamber. This was necessary as the alcohol
solvent might kill the bacteria. As ethanol is extremely volatile, the alcohol naturally evaporates from
the solution into the air of the gas chamber, which is continually changed allowing the reaction to
continue. After 20 minutes, all the ethanol was evaporated from the solution, and only the solvents
from the pine remained. The extracts produced were placed in suspension of 480µl of serum with
20µl of the extracted solution. A similar amount (500µl) of the pine extracts in water, and water were
also placed in Eppendorf tubes.
As the previous experiments showed that the concentration of the pine extracts might not be
sufficiently concentrated, commercial pine oil was purchased as another comparator
(en.wikipedia.org/wiki/Pine_oil), and in particular pure pine oil produced by Naissance
(www.enaissance.co.uk/pine-needle,-scotch-organic-essential-oil); accessed 2/2/2017). This extract is
100% Pine Scotch Oil. It is produced by steam distilling the needle, twigs and cones of pine trees. In
this case the species of pine was Pinus sylvestris, which is commonly found in Europe. As with the
extracts from pin in ethanol, 20µl of this oil was added to 480µl of serum.
As before S. epidermidis was used. In this experiment the bacteria were cultivated in nutrient broth,
which allowed them to reproduce at a much greater rate than on Petri dishes. In addition, the nutrient
broth allowed the bacteria to continue to grow during the experiment. This was done so that the
bacteria would not die naturally die, but would only be reduced in the presence of an antibacterial
molecule. Some 1 ml of these bacterial solutions were added to each of the Eppendorf tubes.
The solution was analysed after 5 time intervals, to see the evolution of the number of bacteria. The
first time interval was directly after the bacterial solution was added to the extracts. The second was
60 minutes later. In these first two intervals, a total of 4 dilutions of the extracted solution were
performed (from 10-2 times to 10-5 times). As per usual, these dilutions were done by adding 50µl of
the bacterial solution to 450µl of serum.
12
The third test was done after 105 minutes, followed by a day later and two days later. These last three
intervals saw a total of 5 dilutions of the extracted bacterial solution (from 10-2 times to 10-5 times).
Some 50µl of the dilutions were placed on petri dishes, and cultivated at 37°C.
The results are as follows:
Time
interval
zero
Water
Commercia
l pine oil
Pine extract with
ethanol
Pine extract with
water
Dilution
50
71
69
70
10-5
1 hour
98
104
72
109
10-5
1h45min
78
111
74
122
10-5
1 day
193
153
35
313
10-5
123
13
2 days
2 days
10-5
23
10-6
45
The extract with ethanol has reduced the bacteria present by more than ten-fold with respect to the
control. The commercial pine oil, which presumably contains similar compounds, separated from the
serum limiting the contact between the bacteria and the oil.
This shows that there are chemicals in the extraction in ethanol that are not present in the extraction
in water. However, the reduction of bacteria in this test is not as significant the reduction on pine
wood itself. This may be due to the concentration of pine extraction in the experiment which is less
than the levels found in wood. However, a reasonable conclusion is that the hydrophobic elements
in resins are a source of wood’s anti-bacterial properties.
3.3. Test of the chemical composition of pine
The next step was to study and compare the compounds in the pine extraction from both water and
ethanol. The chemicals were separated into their constituent compounds using High Performance
Liquid
Chromatography
with
equipment
at
LIST
(this
technology
is
described
in
www.chemguide.co.uk/analysis/chromatography/hplc.html; accessed 1/2/2017).
Chromatography has a mobile and a stationary phase. The mobile phase consists of the pine
extractions along with liquid solvents flowing through the stationary phase in a column, composed of
silica gel. This gel is made so that it is non-polar in comparison to the polar solvents. The polarity of
the solvent creates new intermolecular forces with the molecules of the pine extraction. Depending
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on the degree of the polarity of the molecule in the extraction, these forces can range from being
relatively strong hydrogen bonds to relatively weak Van der Waal forces. The more polar molecules
will be attracted more strongly to the solvent and they will tend to move more with the solvent. The
less polar molecules will be attracted less to the solvent, as the only intermolecular force present are
the Van der Waals forces. Consequently, the non-polar molecules in the extraction will travel more
slowly.
Once they have been separated by the column, the molecules reach a detector. As organic molecules
absorb different wavelengths of ultra-violet light, a UV beam of light is shone at the passing molecules.
A UV detector is positioned on the opposite side of the stream allowing an instant reading. When the
UV detector distinguishes a peak indicating the presence of a molecule, this molecule is diverted to a
mass spectrometer.
The masses distinguished can then be researched in databases for more
information on that molecule.
The databased used for these samples was MELTIN
(en.wikipedia.org/wiki/METLIN; accessed 1/2/2017).
The equipment at LIST used for this experiment is shown in the photos below:
The chromatogram of the extracts used in the last experiment (after 12 days soaking) is shown below.
In black are the molecules present in the ethanol extraction, and in red are those present in the water
extraction. Each peak represents a molecule varying in UV light absorption. As discussed above, polar
molecules in the extractions travelled faster, so the closer the peak is to the y-axis, the more polar the
molecule is.
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We can observe that the number of molecules in the ethanol extract is greater than that with water.
The increase in number of peaks suggests that there are more compounds present in the ethanol
extraction.
Peak at 22.62 minutes
minsmin
This graph shows the same results as the graph above, but over a reduced time interval.
In this graph it is possible to see that the base line for the extractions with water (in red) is greater
than that of the extractions in ethanol (in black). This is due to the fact that the solvents used in the
mobile phase can also absorb UV light. Therefore only peaks above the base line can be taken into
account.
As explained, each peak represents a compound passing through the detector. The area underneath
the peak represents the amount of that compound present. Looking at the results, one can see that
there are more compounds present in the ethanol extraction than there are in the water extraction.
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There is one peak which is much greater than the others, with an absolute maxima at 22.6 minutes.
This is compound is present in both extractions, but it is possible to see that there is considerably more
of the compound in the extraction with ethanol.
The next graph includes the results from the mass spectrometry. It shows the UV light intensity
absorption over the mass detected for each compound.
When inserting the UV wavelength absorption and the mass on international databases, a series of
molecules were candidates. The molecule that best matched all the values found during the
experiment was Hydroxy-pinoresinol. This molecule is shown in the picture below.
This molecule is a lignin derivative. Lignin is found in the cell wall of wood as the arrangement of these
organic polymers is very rigid, and important for the structure of the cell wall. Pinoresinols are
associated as a secondary metabolite. These are molecules, made in the vacuole of the plant cell, and
stored in the cell wall where the lignin is found. Secondary metabolites, unlike primary metabolites
are not necessary for the plants survival. However, among other things, they play a role in protecting
the plant. They could be described as the plant’s “immune system”. For example, they are important
during biotic stress: when a fungi or bacteria enters wood. This compound is phenolic, the same group
mentioned earlier as being antibacterial (experiment with tannin). However, it is interesting that this
particular compound does not seem to be identified in other studies of the antibacterial properties of
wood. It has been found as a potent anti-oxidant in olive oil. Details on how pinoresinolic compounds
may have benefical effeczs on human health can be found in :
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http://www.academia.edu/26656738/Pinoresinol_and_1acetoxypinoresinol_two_new_phenolic_compounds_identified_in_olive_oil.
It maybe that this is molecule, or secondary metabolites in general, have antibacterial properties to
protect the wood. As secondary metabolites of plants can vary in amount and presence, this could
explain the advantagious antibacterial properties of some woods. However, to analyse whether this
molecule is antibacterial, and has this effect in several species of pinewood, that would mean
extracting all the molecules in pinewood and testing each one separately. The sheer number of all the
molecules present means that this would be very time consuming.
Given the range of molecules, and the complexity of identifying them, it is much more efficient to use
the pinewood surface itself, as whatever these antibacterial chemicals are, they will be present in the
material.
3.4. Conclusions on pine
It is important to know why pine has antibacterial properties so we can better understand how to use
it or similar materials in public spaces. However, the mechanisms by which pine disinfects are complex
and remain unclear. There is likely to be a physical effect where bacteria are absorbed in the wood.
Pine is also antibacterial as we are using the natural defensive mechanisms of a tree when it is subject
to bacterial invasion – namely resins that protect the tree from intrusions. This may be a complex
reaction that depends upon the combination of the very many chemicals found in the resins. The
complexity of the natural world makes it difficult to arrive at simple theories of what this is.
We can still conclude that untreated pine should be considered for work surfaces in public places and
particularly in higher-risk environments such as schools, hospitals and care homes.
4. Automated disinfection of surfaces
4.1. Student hygiene
Since the application of pine surfaces has its limitations as a health measure, it is also important to
understand peoples’ behaviour and to explore associated measures. Indeed, the introduction of
bacteria to the school is due to a lack of hygiene by students (not washing hands during the day, and
particularly after going to the toilet, sneezing on their hands, etc.) who leave these bacteria on surfaces
in the first place. In fact, a study in 2014 showed that 26% of people in the UK had faecal matter on
their hands. Only 13% of the people polled said they would avoid eating unless they had washed their
hands(www.huffingtonpost.co.uk/2014/09/16/hands-are-dirtier-than-publicsurfaces_n_5827782.html; accessed 1.02.17).
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At an earlier stage in the project, I gave a questionnaire to 50 students at the school in order to have
more information on hygiene practices. Its purpose was to understand the regularity with which
students wash their hands, and whether they do this in key moments during the day (after using public
transport, after going to the toilet, after laboratory experiments, before eating, etc).
The average frequency of hand washing in school was 2.1 times per day, though one third of these
usually did not involve soap and were rather hand-rinsing. While some 90% often wash their hands
after going to the toilet, only just over 50% always do this. Hand washing is much less common before
eating (48% never or rarely wash their hands before eating) or upon arrival at school after using public
transport (60% never or rarely wash their hands). This increases the risk of transmission of bacteria
from outside the school and consequent infection during eating.
The main reasons given for not washing hands more frequently is lack of time and critical views on the
facilities available (some two-thirds of students consider the school to be dirty due to the number of
people and a lack of general hygiene).
An alternative to physically removing bacteria with soap and water is to use disinfectant to kill bacteria.
Over two-thirds of students said they would use disinfectant dispensers often and as many as 90%
would use disinfectant at least sometimes.
To test this further, a hand disinfectant machine was borrowed
from a local business.
This was placed for one week in a
prominent position by the entry door of the school.
The
machine was battery powered and dispensed a standard
quantity of disinfectant when triggered by a photo sensor. In
one week, it is estimated that the machine was used 600 times,
or an average of 120 times per day. While this is only 6 per cent of the school population, this result
is from only one dispenser which was placed without any information or awareness-raising.
It appears that dispensers of disinfect could be particularly useful to prevent the transmission of
bacteria around the school. This point is used in the next sections.
4.2. The problem of door handles
Desk surfaces, chairs, banisters must be regularly cleaned to reduce bacteria – the need for disinfecting
of these “clean” surfaces is over-looked by cleaning staff. As we have discussed, the use of materials
that passively reduce bacteria such as pine may also be desirable.
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A particular concern is door handles since they are touched very frequently during the day. The aim
was to investigate whether the design of the door handle affects the transmission of bacteria.
Consequently, research for this project examined the transmission risk of various door handles. The
different handles considered were:
1) A standard handle (i.e. a horizontal lever)
2) A long vertical bar
3) A flat plat used to push a door
4) A round door knob
A picture of the test rig for these handles is shown here.
The experiment measured both the surface contact area for each door handle as well as the area
touched on the door. This was done with UV-sensitive paint that is invisible to the naked eye. A group
of volunteers were asked to use each door handle using gloves, and after each area the contact area
was measured under UV light.
A simple model of the risk of transmission was developed. In particular, it was assumed that the
probably of transmission to the fomite is proportional to the area of contact per person, a. This is
spread with equal probability across the total area of the door handle, A, that is touched by people.
Therefore, the resulting density of bacteria on the door handle is reduced by a/A. Finally, the
transmission to another person from a contaminated handle is again proportion to the area of contact,
a.
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With these assumptions, the probability of transmission is proportional to:
a (initial contact) * a/A (spread or dilution) * a (second contact)
which is equivalent to (a3/A).
The results of this experiment generated the transmission risk factors given below. The coefficient
(a3/A) has been scaled to 100 for the worst performing door handle.
Standard
Round
lever
Knob
a, contact area per person (cm3)
53
A, total effective area touched (cm3)
Index of transmission risk
Door type
Plate
Pole
34
79
68
75
65
400
395
100
31
62
39
Both the plate and pole have a high contact area with the hand – surprisingly high for the plate where
a large part of the palm is used to push the door. However, a larger area is used to touch the door,
thus diluting the transmission risk with respect to a standard lever. The best is the round doorknob
because this is only touched by part of the fingers. While the possible area to touch is also smaller
there remains several alternative ways to hold the handle.
What we see with these results is that the greater risk of transmission is with a standard lever door
handle. This is because of the high contact area when the door handle is gripped together with the
small area available to hold – everybody is touching the same area. Unfortunately, most of the door
handles in the school, indeed in most public places, are exactly of the traditional lever type. As already
discussed, door handles are highly contaminated surfaces. For this reason they require more regular
cleaning than the once-per-day frequency of cleaning staff. The NHS gives some tips on the most
effective cleaning methods (see www.nhs.uk/Livewell/homehygiene/Pages/prevent-germs-fromspreading.aspx; accessed 3/02/2017).
4.3. Automated disinfection of door handles
The risk posed by people touching door handles would be eliminated if it were possible to have door
handles that automatically disinfect themselves after use.
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There are critical issues in designing such a door handle:

Even if limited to higher-risk doors where there is a frequent flow of people (such as toilets)
there are many doors that would benefit from this type of door handle. Low cost would be a
critical part of having widespread adoption.

Given that doors are replaced infrequently, the solution should be limited to the door handle
without changing the structure of the door itself.
These limitations rule out more complex electric or battery powered sprays or re-engineering of doors,
in favour of the distribution of a small quantity of disinfectant each time the door is used. This would
force people to use disinfectant. The large majority of students have said that they would readily use
hand disinfectants, but it is possible that some may find this intrusive. However, hand disinfectants
evaporate very rapidly so the inconvenience of having wet hands passes very quickly if only a small
quantity of disinfectant is used. The public value of a clean environment would seem to justify this.
The lever door handle is particularly suited for this approach:

As we have seen, the same area is always touched when a standard door lever is used so the
area to be cleaned is relatively small and focused.

There is considerable leverage/torque that can be used to power a mechanism.
The next stage of this project was to build a prototype to prove the principle.
I examined many commercial available products that include sprays as well as equipment used for
soap dispensers. The same pump mechanism is used in all sprays for cleaning material and cosmetics.
It operates as follows:
a) Lever is used to move a piston within a cylinder
b) One end of the cylinder is connected to a reservoir. The other end is connected to a nozzle.
c) At the intake end, a valve controls the direction of flow of the fluid as the piston moves up and
down. This is achieved with a ball bearing held in place by a small spring. As the piston moves
towards the intake this valve closes.
d) As the same time the movement of the piston opens a small hole that has an outlet linked to
the nozzle. The contents of the cylinder are pushed out of the nozzle.
e) The piston moving in the other direction closes the hole to the outlet, but releases the ball
bearing. Liquid is sucked into the cylinder.
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In this way, moving the lever generates a pump action (more explanation is give at:
www.oberk.com/packaging-crash-course/whats-inside-a-fine-mist-sprayer). The components are
shown in the photo below.
It is possible to build this mechanism into a door handle with an external reservoir of disinfectant and
a perforated tube distributing the product on the handle. Only a small quantity of disinfectant should
be dispensed each time – only sufficient to cover the handle.
The concept is illustrated below:
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Based upon this idea a prototype was built. It is shown below:
The prototype was tested with a sample of 29 students and staff at the school. A confidential
questionnaire was completed by these people after they had used the self-disinfecting door handle.
As background information, the sample group reported the following:

An average washing of hands in the school of 2.6 times per day, slightly higher than the survey
mentioned before. However, this remains a low number. The overwhelming complaints
related to lack of soap, inadequate hand drying facilities, and insufficient time. Some argued
that touching the door handle to exit the toilets would simply make their hands dirty again so
there was not any point in washing them.

An average use of 10 door handles per day in school. This number had a wide range from only
a few times to over 20.
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Each user of the door handle was asked to rate the experience in the following categories:
Very inconvenient
Somewhat bothersome
Convenient
Great contribution to school hygiene
As many as 90 percent of users rated the door handle as “convenient” or better. Some one-third gave
the highest level of support for the installation. This is a very encouraging result.
In terms of locations, respondents were asked to comment on where it might be either not-useful, or
useful for toilet doors, main doors, or classroom doors. Nearly all participants (93%) thought that toilet
doors should have disinfection, while over half (55%) also saw wider use with commonly used doors
in the school.
This is a small sample, but given the overwhelming support from users it was decided not to pursue
additional trials at this stage.
In a commercial model, the pump mechanism could be fitted on the door handle, or could be partially
included inside the lock mechanism. The disinfectant container needs to be on the outside of the door
so that it can be easily changed. Nonetheless, tubes from outside could be fed into the lock area via a
hollow spindle for the handle.
In any case, the pump is a relatively small component that could be fitted on the exterior next to the
door handle. A drawing of what this may look like is shown below:
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4.4. Conclusions on the automated disinfection of door handles
We have demonstrated that a low cost device could be used to have door handles that automatically
disinfect. To encourage adoption, these appliances must have relatively low maintenance. Ideally,
this should be incorporated in the normal cleaning schedule of the surrounding facilities.
The cost of disinfectant is approximately €20 per 1.2 litre container (data from amazon.co.uk, HandCleaners.co.uk, and others). If each use of the door handle discharged 0.5 ml of disinfectant
(approximated one-half the quantity distributed by commercial hand dispensers), this would be
sufficient for 2400 uses.
Assume on average 30 people per toilet and average use of 3 times per day (building standards vary
according to country – in the UK you would need one toilet per 20 students in a school, though some
will be used more than others: British Standard 6465-1:2006, Sanitary Installation, quoted by
www.washroomcubicles.co.uk/how-many-toilets-do-you-need/). Each toilet door would be opened
90 times per day (30 x 3) or 450 openings per week. This would require a change of disinfectant
container approximately once every five weeks. The weekly cost would be less €4 for an average of
30 people, or € 0.025 per person per day.
A classroom used by 20 students, 8 times per day, would have of the order of 800 door openings per
week. In this case, one container of disinfectant would be used every 3 weeks.
These figures show that replacing the container of disinfectant, which could be done by the regular
cleaning staff, should not pose a particular cost. Very frequent use of 450-500 times a day would be
required to consume one container in a week.
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This appliance could be fitted to any frequently used door, but it is more likely to be accepted in toilets
as people understand better the need for hygiene in these locations. It would need to be clearly
labelled so that people realise it is better to take any gloves off they may have before opening the
door. In fact, their use should be done with a general campaign regarding the need for cleanliness and
hygiene in public spaces.
5. Conclusions for public health
Bacteria are present around our school especially in locations that are least expected. Places that are
thought to be contaminated maybe cleaner than elsewhere – the toilet seat had the least amount of
bacteria. However, some surfaces such as the library and classroom tables, the banister, and sports
equipment probably only get very superficial cleaning. Indeed, our DNA sequencing showed that a
number of potentially disease causing bacteria were found on the library table and banister. In fact,
cleaning staff should be encouraged to disinfect these apparently “clean” surfaces. Cleaning materials
should also be disinfected after use to prevent them acting as a fomite.
Materials such as plastics or plastic coated materials are often chosen because they are easy to clean.
However, if they are not continually disinfected, plastic is the worst possible material.
Having surfaces that naturally reduce bacteria can complement cleaning.
This comes from the
appropriate choice of material. A variety of tests performed for this project have shown that pine has
antibacterial properties: pine should be used over plastic where possible.
Pine is antibacterial because it contains compounds that have a natural antibacterial effect to protect
the tree. As with many natural systems, a complex collection of compounds are likely to be responsible
for this. This was clearly shown in the results of the Liquid Chromatography of pine extracts. It is
therefore, appropriate to use pine itself rather than to try to duplicate the behaviour of pine with other
materials.
These results are relevant for other environments, such as the home. For example, untreated wooden
chopping boards are better in the kitchen than plastic ones.
Door handles pose a particular problem because of their frequency of use.
Analyses of the way in
which door handles are touched as well as students’ behaviour with respect to hygiene suggest the
possible use of self-disinfection door handles that dispense a small quantity of disinfectant with each
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operation. A prototype was designed that shows that these door-handles can be made at low cost.
Tests also show a high acceptance of this idea by users. Typical applications would be school toilets,
but could also include other high risk environments such doors in hospitals and care homes.
While awareness of hygiene has been raised in the past during risky flu epidemics it has been quickly
forgotten. With the threat of bacterial resistance to antibiotics and the huge costs to society that
would cause, it seems a good time to remind people of these facts. As Professor Michael Gilmore of
Harvard University (www.harvardmagazine.com/2014/05/superbug) has warned: “sometime soon an
epidemic will begin.” It is time to take measures to ensure the cleanliness of public places.
6. Acknowledgements
I would like to thank the Luxembourg Institute of Science and Technology and in particular Drs Christian
Penny and Christelle André for their help in this project.
Particular thanks are due to Dr Penny who has supported this project from the very beginning. He was
able to explain complex subjects in an understandable way and responded almost immediately to any
queries. This project wouldn’t have been possible without him. A big thank you!
I would also like to thank Mr Bennett, my mentor, for his support.
I would finally like to thank everyone else who helped me along the way, especially my family for
putting up with the drilling and sawing noises!
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