Note: Descriptions are shown in the official language in which they were submitted.
- ~\
g7~
BACKGROUND OF THE INVENTION
This invention relates to the chemical binding of
antimicrobials onto a substrate bearing a hydroxyl group and
particularly to substances such as cellulose, starches and
leather.
The binding of antimicrobial substances to
substrates such as textiles has been of interest for many
years. Prior art efforts in this direction have not been
entirely satlsfactory because it has been difficult to
attach the antimicrobial to the substrate and still have
the antimicrobial retain biostatic or biocidal qualities.
Further problems exist in that the antimicrobials of the
! prior art could generally be readily washed away from the
treated substrate with water and/or with detergents.
It has been known to bond enzymes to a substrate
through the use of cyanuric chloride, and in such cases the
enzymes are immobilized. To our knowledge, however, anti-
microbials have not been successfully chemically bonded to
a substrate such as cellulose to resist repeated detergent
,~0 washing and still retain inhibitory effort on the growth of
microorganisms. Accordingly, it is an object of this
~ invention to provide methods and compositions for the chemical
`, bonding o~ antimicrobial compounds to substrates such as
cellulose, starches, and leather which cannot be easily
removed by washing with water and/or detergents.
The examples of some of the uses of antimicrobials
chemically bonded to cellulose would be in the treatment of
cloth used in hotels or hospitals for uniforms, bedding and
' the like, to prevent or minimize the presence of bacteria. ~ -
-- 1 -- .
~ "' '.
~ ~ .
` ' , . . . . .
1(~87g'`7~
The antimicrobials may also be bonded to bandages or bandage
pads, to be used direc~ly on superficial wounds, for example.
Besides the use o~ the bonded antimicrobials on clothing in
general, still other uses would be for diapers, and other
undergarments. Further, the bonded antimicrobials may be
used in industrial processes on filter media or on packaging
and the like to maintain sterility or to reduce the number
of undesirable microorganisms. The non-migrating, bonded
antimicrobials are not absorbed into the human body when
used on bedding, clothing, bandages or the like. When used
on ~ilter media or packaging surface~ active antimicrobials
will not contaminate the filtered or packaged substance.
On leather, such as in shoes, the bound antimicrobial can
provide built in resistance to fungi, molds and bacteria.
When bound to starch, the antimicrobials can be readily
; mixed into body powders and the like. Thus, bonded non-
migrating antimicrobials which could render surfaces sterile
have substantial economic value.
Accordingly, it is an object of the invention to
provide methods and compositions for chemically bonding ` `
antimicrobials to substrates.
It is a further object of the invention to provide
methods and compositions as set ~orth above wherein the
chemically bonded antimicrobial resists removal from the
substrate by detergent washing.
The invention accordingly comprises the several
steps and the relation of one or more of such steps with
respect to each of the others, and the composition possessing
the features, properties, and the relation of cohstituents,
which are examplified in the following detailed disclosure,
... - . .
L0~7~7
` .
and the scope of the invention will be indicated in the
` claims.
Other objects of the invention will in part be
- obvious and will in part appear hereinafter.
SUMMARY OF THE INVENTION
- The invention comprises the bonding of antimicrobials
onto hydroxyl containing substrates such as cellulose,
starches, or leather by reacting the hydroxyl groups of the
substrate with cyanuric chloride and then reacting of amine,
guanido or quaternary ammonium groups of the antimicrobials
with the attached cyanuric chloride. The general reactions
are believed to be as follows:
-:
Substrate -O ~ C ~C-Cl ,N
Substrate -O- C ~C-Cl
iN ~ ~N ¦l I
, C ~C ~ +HCl
i Cl
Cl
;20
!~1) Cyanuric Chloride
.
NH2-R Amine containing
Antimicrobial
: .
HCl ~ Substrate -O-C ~C-Cl
. Il I ~ . ,
N~ ~N
HN-R
~30
_ 3 _
~ ~1 0~5179~7
.:~
` The reaction ta~es place in an aqueous alkaline solution
such as a sodium hydroxide solution with a pH of about 9
to 10. The cyanuric chloride is preferably prepared in a ;:~
solvent such as aioxane or acetone. The process is ~:.
particularly useful in bonding antimicrobial compounds having
amino groups to cellulosic substrates, such as cotton,
rayon, cellulose acetate, etc.
: The invention is ef~ective in chemically bonding
antimicrobials to hydroxyl containing substrates and
particularly those antimicro~ials having amine, guanido or
quaternary ammonium hydroxyl groups. The cyanuric chloride
bonds to the substrate and to the antimicrobial to provide
.chemical bonds which resist removal by detergent washing
while maintaining inhibitory action against one or more
microorganisms.
SPECIFIC DESCRIPTION OF PREFERRED EMBODIMENTS
_ ... . . . _ .
,:' OF THE INVENTION
. The chemical binding of antimicrobial or antiseptic
.1 .compounds onto a hydroxyl containing substrate proceeds in
120 accordance with the above reactions to form the composltion:
. . .
~N ~ ..
:i~ N~C~N
.. R2
. .,
wherein X is the substrate which had contained the hydroxyl
group, one of Rl or ~i2 may be chlorlne and the other an
. amine, guanido or quaternary ammonium containing anti-
microbial compound or Rl and R2 may each be the same or
, ', ' '
' - ' ' ' '
~ 79'77
a dif~erent amine, guanido or quaternary ammonium containing
antimicrobial compound which is chemically bonded to the -
triazine ring through the nitrogen atom o~ the amine,
guanido or quaternary ammonium group.
A number of amino, guanido or quaternary ammonium
containing antimicrobials have been chemically bonded to
a ~ellulosic substrate, such as cotton, by cyanuric chloride.
These are chlorhexidine acetate, streptomycin, trimethyl
octyl decyl ammonium chloride, and mixtures o~ all three
of these antimicrobials. Other substances which have been
bound to a hydroxyl containing substrate are neomycin
sulfate, alkyldimethyl benzyl ammonium chloride and cetyl
dimethyl benzyl ammonium chloride. Further, of the
antimicrobials tested, the ones showing the strongest
inhibition against the growth of E. coli were chlorhexidine
acetate, streptomycin sulfate and neomycin sulfate as well
as ~uaternary ammonium compounds such as alkyldimethyl
benzyl ammonium chloride, trimethyl octyl decyl ammonium
chloride and cetyl dimethyl benzyl ammonium chloride.
In bacteriological testing the bonded antimicrobials
showed inhibitory action against E. coli, Pseudomonas
aeruginosa, Bacillus subtilis and Saccharomyces diastaticus.
,
It was also found that cyanuric chloride used alone has
some inhibitory effect for specific microorganisms.
The chemically bound antimicrobials showed
substantial resistance to being washed from cotton fabric
by several well-known laundry detergents. -
In the above reactions the cyanuric chloride is
bound to the hydroxyl bearing substrate through a carbon
atom with hydrochloric acid formed as a reaction by-product.
.~
~ ~ .. . . .. . .. . .
77
The amine, guanido or quaternary ammonium containing
antimicrobial compound is then bound to the triazine ring
` of the cyanuric chloride, replacing one or both of the other
chlorine atoms through the nitrogen of the antimicrobial,
again producing hydrochloric acid as a by-product.
- The cyanuric chloride or 2, 4, 6 tri-chloro-l,
3, 5, triazine is, for-example, prepared as a 2.5% solution
in dioxane or acetone as a solvent, and the pH of the solution
is adjusted to between 9 and 10 with lN NaOH. In the
following examples of the invention the procedures for pre-
paring the test samples shown in Tables 1 and 2 were as
follows. A number of 1 inch square cotton patches were
immersed in lN NaOH for about 15 minutes. The excess
., .
~ solution was poured off a~d the squares were washed with
; excess warm tap water (55C.) to wash out the alkali. Then
the washed squares were immersed in 2.5% solution of cyanuric
i chloride, prepared as above, again with the p~ of the
solution being adjusted to between 9 and 10 with lN NaOH.
The cotton squares were permitted to stand in the cyanuric
chloride solution for about 30 minutes. The excess cyanuric
chloride solution was then poured off and the squares were
washed twice with acetone containing 5% glacial acetic acid
to stop any further reaction and to neutralize the
alkalinity of the solution. The cotton patches were then
washed two more times with acetone in order to remove any
excess cyanuric chloride and were well rinsed with warm tap
water. Some of the cotton patches were then air dried and
analyzed for nitrogen by the Kjeldahl method.
; A number of the cyanuric chloride treated cotton
squares were then immersed in an aqueous solution of
- 6 -
'.:
., , , . - . . .
. , .
9~
:
chlorhexidine acetate (having 200 mg of chlorhexidine
diacetate per 100 ml of solution) and the pH was adjusted
to between 9 and 10 with lN NaOH. The cotton patches were
stirred with the solution and permitted to stand overnight
at room temperature, permitting reaction between the anti-
microbial and the cyanuric chloride bound to the cotton.
Other cotton patches were dipped into a streptomycin sulfate -~
solution containing 200 mg of streptomycin sulfate per 100 ml
and still other cotton patches were treated with a 0.5%
aqueous solution of trimethyl octyl decyl ammonium chloride.
The patches were all then removed from their respective
solution, rinsed with large amounts of warm tap water at
; about 55C. and air dried. A number of patches were then
taken for further nitrogen analysis to determine the amount
of antimicrobial compound which was bound to the cyanuric
chloride. Using the nitrogen values and weight of the
patches, the amounts of bound cyanuric chloride and bound
antlmicrobials were then calculated. These values are set
forth in Tables below.
The inhibitory activity of the cotton squares with
antimicrobial substances bound to them were tested against
Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa,
' and Saccharomyces diastaticus. The inhibitory effect as to
these microorganisms was determined in the following manner.
Cultures of each organism were grown in appropriate
~; broths and then 1 ml of each culture was diluted to 100 ml
with water. Two-tenths of a ml of this suspension was
pipetted onto a treated cotton square and allowed to stand
at room temperaturé for about 15 minutes. Each square was
~, 30 placed in a petri dish and 20 ml of water added and stirred
- 7 ;
. . . , .. , ~, .
.,
'' .. , ~. . ~ ~ ' . . . ...
-- ~Lq.)~'7S~77
to re-suspend organisms of inoculating culture. Then 1 ml
and 0.1 ml samples of that suspension were placed onto a
nutrient agar plate. After 24 hours of incubation at 30C.
the colonies on the plate were counted. An untreated -
cotton square or ones treated only with sodium hydroxide
or cyanuric chloride were similarly processed as controls.
Those cotton squares which showed less than 10% of the number
of colonies obtained with the controls are considered to
be inhibitoxy.
In khe following Tables the cotton patches to
which antimicrobials are bound through cyanuric chloride
were tested for retention of antimicrobial action after
, washing. For such testing the following procedure was
employed. After 18 hours of immersion in antimicrobial
solution the cotton patches were removed, well rinsed with
warm water, and placed in a detergent solution of about
1 gm of-detergent per liter of solution. The patches were
then stirred in the detergent for about 10 minutes, the
detergenk is poured off and the cotton was well washed
again with warm tap water at about 55C. Such washing
with detergent followed by a warm water rinse was repeated
two more times and finally the patches were air dried and
tested as described above for their microbial inhibitory
activity against the organisms shown in the followin~ Tables.
In the Tables the following abbreviations are used:
C = C~anuric Chloride (2, 4~ 6 trichloro 1, 3, 5
triazine)
M.W. = 184.5, N = 22.7%. Prepared as a 2.5%
; solution in either dioxane or acetone.
-- 8 --
.
,
,
1(38~
_ A - Chlorhexidine
Formula in Merc~ Inde~, ~ighth Edition, 1968, p. 23~.
M.W. = 505.5, N = 27.7%. Prepared in a concentration of
200 mg as chlorhexidine acetate per 100 ml of H20 -
pH = about 9Ø pH adjusted to 10 before use.
SPM = Streptomycln
Formula in Merck Index, Eighth Edition, 1968, p. 984-5
M.W. 736.58, N = 13.3%. Prepared in a concentration
of 200 mg as streptomycin sulfate per 100 ml of H2O ~
pH about 5Ø pH adjusted to 9 - 10 hefore use.
AQR =- Arquad 18 50
50% aqueous solution of trimethyl octyl decyl ammonium
chloride - ClgH39N tCH3)3Cl - M.W. -- 350.0, N - ~.0gO.
Prepared in a concentration of 2 m1 in 200 ml of H2O.
pH about 5.8. p~ adjusted to 9 - 10 with lN NaOII before use.
DM = A mixed solution (dilute) of CHA (133 my), SPM
(133 mg) and AQR (0.67 ml) in 200 ml of water adjusted
to pH 9.0 to 10Ø
CM = A mixed solution (concéntration of CHA (~00 mg)
SPM (400 mg), and AQR (2.0 ml) in 200 ml of water
adjusted to pH 9.0 to 10Ø
A =*All
A retail packaged detergent used ln clothes washing machines
-Label states that All contains surfactants, sodium carbonate,
sodium silicate, bleach, borax, and brighteners. It is
~: ,
; used in concentration of 1 g per liter (pH about 10). It is
bio-degradable, conta~ns no phosphorous and is manu~actured
by Lever Brothers Co., New York, New York.
.
, *Trade Mark
i 30
,`.1 ~ 9
`I A~
.. .. ~ . . .
. . . ~
`~, ....... - . . ~ .
t`. . ' - :
. '-" ' ,
~ ~(987~'~'7
T ~*Tide
rqtail~ ~ .ergent used in cloth~s washing machilles. Label
states that Ti~e contains anionl.c surfactants,.sodil~
caxbonate, sodium silicate, sodi~n sulfate, ~abric whit:ener,
and perfume. Used in concentration of 1 g per liter ~pll
about 10). It is biodegradable and is manufactured by
Proctcr & Gamble, Cinc~.nnati, Ohio~
J =*~oy
, _ ___
A retail wàshing liquid used for washing dishes and contains
anionic and nQnionic surfactants, ethyl alcohol as a
stabilizing agent, and perfume. Used in concentrati.on of
1 g per liter (p~ 7.3). It contains no phosphorous and
.is manufactured by Proctor & ~amble, Cincinnati, Ohio.
..
TNTC
Too numerous to count.
. Examples:
¦ C-CHA-T Indicates cotton treated with cyanuric chlo.~idc
(C), followed by reactioll with chlorhexidine
acetate ~CHA), followed by washing with.Tide (T).
C-A Indicates cotton treated with ~yanuric chloride
(C), followed by washing with A].l (A).
NaOH Indicates cotton washed with lN NaOH for 15
minutes followed by water wash to remove excess
¦ NaOH.
CHA-J Indicates cotton treated with chlorhexidine
acetate (no binding with cyanuric chloride)
followed by washing with*Joy.
In the Tables, calculations were made on the
. following molecular weights and nitrogen contents of the
con~pounds employed:
* TradeÇIark
~ 1 0 --
. . .
'7~'77
Antimicrobial Molecular Weight ~ Nitro~en
C-Cyanuric Chloride 184.5 22.7
CHA-Chlorhexidine 505.6 22.7
SPM-Streptomycin 581.6 16.86
AQR-Arquad 18-50 350 4.0
Cotton patches were prepared as above and the
. results of testing of the cyanuric-antimicrobial treated
cotton for inhibition of microbiological growth are set
forth in Tables l(a), (b), (c) and (d).
TABLE l(a?
.
~acteriological Results - Inhibition Against E. coli
Controls Dilution
- 1 ml0.1 ml . - .
NaOH TNTC1335
C-H20 TNTC699
C-J 55 . 7
C-A TNTC 207
C--T l'NTC 218
Unbound Antimicrobials
. _ _ . _ _
:: 20 CHA-H20
CHA-J TNTC 365
CHA-T g o
Bound Antimicrobials
.. __ . ... :
C-CHA-H20 2 O
C-CHA-J TNTC 365
C-CHA-A 289 25
C-CHA-T TNTC 307
C--SPM- H 2 0
C-SPM-J 3 o
C-SPM-A O O
:j C-SPM-T 2 0
C-AQR-H 2 18
C-AQR-J TNTC 382
C-AQR-A 3 l 7 3 0
C-AQR-T TNTC 220
;
, ` . .
` , ,
` ~879'77
. . .
TABLE l(b)
Bacteriological Results - Inhibition Against B. subtilis
Controls Dilution
1 ml 0.1 ml
NaOH 3
C-H20 278 31
C-J O O
C-A g o
C-T 1 0
Unbound Antimicrobials
CHA-H20 4 0
CHA-J O O
CHA-T . o o
Bound Antimicrobials
. .
, C-CHA-H20 7
.- C-CHA-J O O
:;
C-CHA-A 7 o
C-CHA-T O . O
C-SPM-H20 .6 0
C-SPM-J O O
. C-SPM-~ o o
C-SPM-T 11 0
.~;
C-AQR-H20 5 0
C-AQR-J 3 o
: . C-AQR-A 2 0
C-AQR-T 4 o
, ' . ,
., .
- 12 -
..
.. .
.. ..
~.. , , . . ; . ,........... . ~. ,
lC~137977
TABLE l~c)
Bacteriological Results - Inhibition Against Pseudomonas
aeruginosa
Controls Dilution
1 ml 0.1 ml
NaOH TNTC TNTC
C-H20 TNTC 1980
C-J TNTC 427
C-A TNTC TNTC
C-T TNTC 508
Unbound Antimicrobials
CHA-H20 206 26
CHA-J TNTC 2 94
CHA-T TNTC 763
Bound Antimicrobials
C-CHA-H20 11 O
C-CHA-J TNTC 2 9 4
C-CHA-A TNTC 508
C-CHA-T >300 68
C-SPM-H20 TNTC 381
C-SPM-J TNTC 572
C-SPM-A TNTC 345
C-SPM-T TNTC TNTC
C-AQR-H20 136 18
C-AQR-J >300 210
C-AQR-A >300 41
C-AQR-T 61 7
', ~'.
- 13 -
':
. , , ; . . ., ., ,. . - , ,
7~77
TABLE 1 ~d)
Bacteriological Results - Inhibition Against S. diastaticus
Controls Dilution
1 ml 0.1 ml
NaOH 42 5
C-H20 TWTC 50
C-J >300 31
C-A 64 7
C-T 66 8
Unbound Antimicrobials
CHA-H20 19 4
CHA-J 148 12
CHA-T >300 27
Bound Antimicrobials
i -.......... _ ._
C-CHA-H20 1 0
C-CHA-J 148 12
i~ . C-CHA-A 2 0
: C-CHA-T 16 . 2
C-SPM-H20 . 17 1 -
C-SPM-J 47 4
C-SPM-A 41 2
C-SPM-T 1 0
C-AQR-H20 0 0
C-AQR-J
. C-AQR-A O O
C-AQR-T O O
, ~ :
.: - 14 :
:
- . ~. :: .
t7~7
TABLE l(e ?
Mi~ rams of Nitrogen per Gram of Cotton Expressed as
I C~anuric Chloride and Antimicrobials
- Mg N per g . .
; Mg N per Cotton (Cor- Mg N per gMg Anti- ,
g of rected forCotton (Cor-microbial
SampleCotton NaOH Blank) rected for C) per g cotto~
.. ~
NaOH 0.25
C-H20 9.09 8.84 39.0
C-J 8.45 8.20 3601
C-A 8.35 8.10 35.6
C-T 7.42 7.17 31.6
C-AQR-J 8.87 8.62 0.40 10.0
C-AQR-A 10.33 10.08 1.98 49O6
C-AQR-T 9.92 9.77 1.60 40.0
C-AQR-H2O 9.32 9.07 0.23 5.8
C-SPM-J 11.65 11.40 3.20 19.0
C-SPM-A 10.06 9.81 1.71 10.1
,.,
C-SPM-T I5.36 15.11 7.94 47.1
C-SPM-H2O 11.57 11.32 2.48 14.7
C-CHA-J 16.65 16.40 8.20 29.6
C-CHA-A 18.05 17.80 9.70 35.0
C-CHA-T 14.73 14.48 7.31 26.4
C-CHA-H20 14.80 14.55 5.71 20.6
CHA-J. 0.85 0.60 2016
CHA-T 0.91 0.66 2.38
CHA-H20 0.97 0.72 2O60
, -:
~ I .
'
- ~,, . ':
~, . :-,
. : . : - : :
~` 1~)~3'7~ 7
The bac-teriological results for inhibitory activity
against the four ~icroorganisms in Tables l(a), (b), (c) and
(d) m~y be summarized generally as follows. The con~rols
in which cyanuric chloride alone was bound to cotton
(followed by washing wi~h water and detergents) showed little
orno inhibition of growth against E. coli, Pseudomonas aeruginosa
and S. diastaticus, but showed substantial effectiveness
against B. subtilis growth in the test results set forth
in Tables l(a), (b), (c) and (d). As will be noted,
B. subtilis growth was inhibited on all treated and untreated
cotton patches. It is believed that either the culture used
was weak or the number of organisms used was too small.
The experiment was repeated using a heavier inoculum of
B. subtilis and showed less inhibition of R. subtilis
by cyanuric chloride alone, as shown in Table 2(a) below. i
Two of the antimicrobials, namely chlorhexidine
(CHA) and streptomycin (SPM), after binding to cotton were
ineffective in preventing the growth of Pseudomonas aeruginosa,
` particularly after the cotton was washed with detergents.
In one instance chlorhexidine showed some inhibition against
Pseudomonas aexuginosa after only a water wash. Trimethyl
octyl decyl ammonium chloride (AQR) showed the best relative
results against this microbe after the cotton patches were
washed with water and detergents. A repetition of the
experiments as to Pseudomonas aeruginosa, as shown in
Table 2(b), generally confirms these results.
In Table l(a) cotton treated with chlorhexidine
alone followed by water washing and Tide detergent is shown
to have inhibitory activity against E. coli. Further, the
` 30 chlorhexidine could not be completely washed out of the cotton.
; - 16 -
,, '
~-- i
77
' Table l(e) shows the extent of antimicrobial bond-
ing by-analysis for nitrogen by the Kjeldahl method. As
shown in Table l~e), some of the chlorhexidine was directly
bound to the cotton (lines 25-27). The inhibiting effect of
chlorhexidine against E. coli was thus somewhat affected by
detergent washing. Trimethyl octyl decyl ammonium chloride
; also shows a reduction of inhibitory effect on E. coli after
detergent washing. Streptomycin showed excellent inhibition
of E. coli growth in all tests even after extensive washing
; 10 with water and detergents. Generally, streptomycin and
chlorhexidine showed about 70 to 90~ inhibition of
S. diastaticus growth. Trimethyl octyl dccyl ammonium
~ chloride was completely inhibitory in all the experiments
,~ against S. diastaticus.
, Thus, these above antimicrobials have been shown
~i, to be chemically bound to cotton via reaction with cyanuric
: ~1
, chloride and are useful to inhibit the growth of four
different classes of microorganisms even after extensive
washing of the treated cottons with water and three commonly
used commercially available detergents.
,'1 . .
,,tj, , The tests were repeated for further study of
,~
inhibitory activity against B. subtilis and Pseudomonas
aeruginosa and the results are shown in Tables 2(a) and (b).
~' 30
- 17 -
.
. '........ : .~ ' '. : ' , .
. .
TABLE 2(a)
Bacteriological Results - Inhibition Against B. subtilis
Controls Dilution
1 ml 0.1 ml
C-H20 31 5
C--H 2 O--J
C--H20--A
C-H20-T 78 7
Bound Antimicrobials
C--CHA--H 2
C-CHA-J 11 2
C-CHA-A 10 3
C-CHA-T - 4
C-SPM-H20 - 63 8
I C-SPM-J 18 3
3 C-SPM-A 121 18 :.
C-SPM-T 20 4
C-AQR-H20 o
C-AQR-J 13 2
C-AQR-A 0 0
C-AQR-T . 72 0
C-CM-H 2
C-CM-J 1 0
C-CM-A 25 2
C-CM-T 2 0
C-DM-H20
C-DM-J 72 12 ~ .
' C-DM-A 0 0
C-DM-T -2
'''"
- 18 - ~
: ` :
7~77
TABLE 2(b)
Bacteriological Resul~s - Inhibition Against Pseudomonas
aeruginose
Controls Dilution
1 ml 0.1 ml
C-H20 300 387
C-H20-J TNTC 636
C-H20-A TNTC TNTC
C-H20-T TNTC TNTC
Bound Antimicrobials
C-CHA-H2O -24
C-CHA-J >300 87
C-CHA-A 274 20
C-CHA-T >300 41
C-SPM-HzO 158
C-SPM-J >300 508
C-SPM-A >300 94
. . .
C-SPM-T >300 207
C-AQR-H2O 0 0
C-AQR-J . >300 84
C-AQR-A >300 179
C-AQR-T >300 182
C-CM-H 2 O O
C-CM-J >300 91
C-CM-A 2
C-CM-T >300 62
.,
C-DM-H20 >300 187
C-DM-J >300 224
C-DM-A >300 157
C-DM-T >300 178
., '
: -
: ,
~,, ' ' .
~ . .
7~77
.
The results shown in Tables 2(a) and (b) furtherillustrate the inhlbition of microorganism growth of
Bacillus subtilis and Pseudomonas aeruginosa by chlorhexidine
(CHA), streptomycin (SPM), trimethyl octyl decyl ammonium
chloride (AQR), and dilute mixtures !DM) and concentrated
mixtures (CM) of all three of these antimicrobials.
In the experiments shown in Table 2(a), a heavier
inoculum of B. subtilis was used and inhibition of B. subtilis
by chlorhexidine and trimethyl octyl decyl ammonium chloride
is clearly shown. Detergent washing as well as the concentra-
tion of the organism slightly affected the inhibitory ~;
properties of these chemically bound antimicrobials.
.
A dilute mixture of all three antimicrobials (DM)
shows good inhibition of growth of B. subtilis even after
extensive washing with water and detergentsO It has been
found that use of a more concentrated solution (DM) of anti-
~i microbial compounds shows even greater inhibition against
B. subtilis when bound to cotton regardless of the type of
washing which is used.
.~, . . .
~ 20 Chlorhexidine and trimethyl octyl decyl ammonium
.; . . ,
chloride show good inhibition toward Pseudomonas aeruginosa
when the cotton was washed only with water. There was less
. .
inhibitory effect when the cotton was washed with detergents,confirming the results shown in Table l(c). The dilute
mixture (DM) and streptomycln showed the least effectiveness
against Pseudomonas aeruginosa. The best resulks were
obtained by a concentrated mixture (CM) of all three anti-
microbials in the inhibition of Pseudomonas aeruginosa
growth.
~ -
:. , . '' - 20 -
.
~ .
. . ~ i; . , : .
~ 87~ 7 7
. .
Tables 2(a) and (b) show that good inhibition of
~rowth-of B. subtilis and Pseudomonas aeruginosa can be
obtained when these organisms are exposed to cotton trea~ed
With chlorhexidine or trLmethyl octyl decyl ammonium
chloride or mixtures of these two antimicrobials in the
proper concentrations. Cyanuric chloride alone had some
inhibitory effect against B. subtilis, but little effect
against Pseudomonas aeruginosa.
As shown in Table 2(c), the nitrogen values again
show substantial antimicrobial bonding to the cotton.
TABLE 2(c)
.
Milligrams of Nitrogen per Gram of Cotton Expressed as
Cyanuric Chloride and Antimicrobials
~ Mg N per g
¦ Mg N per Cotton (Cor- Mg N per g Mg Anti-
i g ofrected for Cotton (Cor- microbial per
Sample CottonN H Blank) rected for C) ~ Cotton
NaOH 0.25 -~
C-H2O 8.45 8.20 36.1
C-AQR-J 9.28 9.03 0.83 20.7
C-AQR-T 9.68 9.43 1.23 30.8
C~SPM-T 11.77 11.52 3.32 19.7
C-CHA-J 16.26 16.01 7.81 28.2
C-CHA-T 14.87 14.62 6.42 23.2
C-CM-J 15.13 14.98 6.78 38.6
C-CM-A 14.08 13.83 5.63 32.1
C-CM-T 16.40 16.15 7.95 45.3
C-CM-H2O 14.7 13.92 5.72 32.6
C-DM-J 12.77 12.52 4.32 24.6
; C-DM-A 13.33 13.08 4.88 27.8
C-DM-T 13.61 13.36 5.16 29.4
C-DM-H2O 12.20 11.95 3.75 21.4
.~ .
.,
., ~ .
- - 21 -
'
':
~i
.
.~ ' ' .
: ~
~` ~ ` ~087977
; In further work done, the antimicrobials were
bound'onto insoluble starch by cyanuric chloride. The
antimicrobials we~e applied in unbound and bound form on a
starch powder which a~ter exposure to the cyanuric chloride
and the antimicrobials, was well washed with water, followed
by an acetone rinse to remove excess reagent and to then
permlt air drying. It is indicated that chlorhexidine
acetate (CHA) and streptomycin (SPM) are adsorbed onto the
starch and are not completely removed by water washing since
they exhibit good antimicrobial activity without being
chemically bouncl with cyanuric chloride. Arquad 18-50
(AQR) also exhibited some adsorption onto the starch, but
is shown to be effective against E. coli only when bound via
cyanuric chlorid'e. '
The antimicrobials, streptomycin, chlorhexidine
acetate and*Arquad 18-50 have also been bound onto leather
.. . . .
(cowhide) via cyanuric chloride. In the case of chlorhexidine
acetate and Arquad 18-50, these antimicrobials appear to be
as effective unbound as when bound by cyanurlc chloride,
since there is substantial adsorption o~ these antimicrobials
on~to the leather. In the case of streptomycin, however,
there appears to be little adsorption of the SPM onto the
leather and when bound to leather by cyanuric chloride, it is
indicated that streptomycin' has substantial antimicrobial
activity against B. subti~is and S. diastaticus.
In later work, to examine the necessity for the
use of acetone or dioxane solvents for cyanuric chloridè,
an aqueous solution at a pH of about 9-10 was used and the
concentration of cyanuric chloride was reduced from 2.5%
`30 -to 1~. In two different sets of experiments the time of
reaction of the antimicrobials with the cyanuric chloride
* Trade Mark
22 -
was reduced from 18 hours to one hour, as shown in Tables
3~a), (b), (c) and ~d). From these Tables the results lead
to the conclusion that the cyanuric chloride can be ~ound
to a substrate (in this case, cotton squares, as for the
Tables l(a), (b), (c) and (d) above) in an aqueous alkaline
solution and that the concentration of cyanuric chloride
may be substantially reduced when i-t is to be used as a -
blnding agent only, in most cases.
The reduction of reaction time of the antimicrobials
from 18 hours to one hour indicates that a substantial
amount of the antimicrobial is bound to the cyanuric chloride
within the first hour of the reaction and that additional
reaction time does not add much more antimicrobial material.
Accordingly, it should be concluded that reaction time of the
antimicrobials need be no more than an hour, except in those
cases where an absolute maximum of antimicrobial bound onto
the substrate is required. The reaction time is indicated
by the number suffix 1 or 18 in the Tables.
.
TABLE 3(a)
Bacteriological Results - Inhibition Against E. coli
. .
Controls Dilution
1 ml 0.1 ml
NaOH TNTC TNTC
C TNTC TNTC
Unbound Antimicrobials
~'l
l AQR-l TNTC 250
i~ AQR-18 TNTC 500
CHA-l 500 64
CHA-18 TNTC 166
SPM-l 107 21
SPM-18 23 2
:
'` ~ ' ,, ' '- :
: . .
~C~8'79'~
Controls Dilution
1 ml 0.1 ml
Bound Antimicrobials :
C-AQR-l 18 4
C-AQR-18 TNTC 171
C-CHA-l 12
C-CHA-18 8 2
C-SPM-l 14 2
C-SPM-18 . 57 8 ~ ~:
.::
TABLE 3(b)
Bacteriological Results - Inhibition Against B. subtilis
Controls Dilution :
, 1 ml 0.1 ml
.1 - . . .
NaOH . 148 16 ~ :
C 264 24
Unbound Antimicrobials
AQR-l 0 0
AQR-18 0 0
CHA-l 1 0
CHA-18 0 0 : :
SPM-l 65 5
SPM-18 14 2
Bound Antimicrobials
C-AQR-l 0 0
C-AQR-18 0 O
C-CHA-l 0 0
C-CHA-18 0 0
:ii C-SPM-l 31 3
.~ C-SPM-18 7 o
- 24 - .
.
: ` '
. .
:
~ABLE 3(c)
Bacteriological Results - Inhibition Against Pseudomonas
aerugino sa
. ' :
; Controls Dilution
1 ml 0.1 ml
NaOH TNTC TNTC
I C TNTC TNTC
.~ Unbound Antimicrobials
; - AQR-l TNTC 127
- AQR-18 320 45 -^
CHA-l TNTC 380
CHA- 18 TNTC 79
,; SPM-l - . TNTC 320
SPM-18 TNTC 310
Bound Antimicrobials
~ . .
. C-AQR-l TNTC 167
` C-AQR-18 174 26 ;~
!l C-CHA-l TNTC 420
`1 C-CHA-18 TNTC 84 .
I C-SPM-l TNTC 624
,; C-SPM-18 TNTC 87
,
~i
.', ,
. ~ .
,~, .
:
. - 25 -
i
, .. ~
, :,
: ~ -
.~. . . .
'7~77
,
TABLE 3(d) .
Bacteri-ological Results - Inhibition Against S. diastaticus
Controls Dilution
1 ml 0.1 ml
NaOH TNTC 106
C 570 61
Unbound Antimicrobials
AQR-l 0 0 ~
AQR-18 2 0 ;:
CHA-l 54 6
CHA-18 37 4
SPM-l 810 95
SPM-18 112 13
Bound Antimicrobials ~.
,
C-AQR-l 0 0
C-AQR-18 . 3 1 ~ .
C-CHA-1 15
C-CHA-18 28 . 3
C-SPM-1 440 54
C-SP~-18 82 9
.~ .`. .:
.
- 26 -
:
9~7~7
TABLE 3(e)
Milligrams of Nitrogen per Gram of Cotton Expressed as
,
Cyanuric Chloride and Antimicrobials
. Corrected for Mg Anti-
Mg N per NaOH Blank Mg microbial per
, Sample g of Cotton N per g Cotton g Cotton
NaOH 0.44
AQR-l 0.66 0.22 5.5
CHA-l 1.41 0.97 3.5
SPM-l 0.54 ~ 0.10 0.53
AQR-18 0.87 0. 43 10.0
CHA-18 1.66 1.22 4.4
SPM-18 0.86 0.42 2.5
~ C 2.24 1.80 8.0 - .
.~l C-AQR-l 2.42 1.98 4.5
C-CHA-l 3.45 3.02 4.4
i, C-SPM-l 2.73 2. 29 2.9
.~ C-AQR-18 2.46 2.02 5.4
.l C-CHA-18 3.72 3.28 5.3
`~ C-SPM-18 2.99 2.55 4.4
"~ .
Notes: C = Cyanuric Chloride - 1~ aqueous solution pH 9 - 10.
: AQR-l = Exposure to AQR of one hour.
;~ AQR-18 = Exposure to AQR of eighteen hours.
~ .
.
.. ~ 27 ~
:` ~
'
~ -
'~ , ~ ., . : ' ,
'7~7
'.:
The results shown in Tables 3(a), (b), (c) and
(~) indicate generally that the inhibition ~f E. coli is
enhanced by the binding of the antimicrobials through -
cyanuric chloride. In the case of B. subtilis, the unbound
antimicrobials, as well as the bound antimicrobials,
inhibited yrowth of that organism, but it should be noted
that in all of the experiments shown in Tables 3(a), (b),
(c) and (d), a water wash only was used. Pseudomonas
aeruginosa was somewhat inhibited by the bound and unbound
. I .
10 . AQR and it was measurably affected by an 18 hour exposure of
antimicrobial rather than the one hour treatment. Accordingly,
for difficult to inhibit microorganisms such as Pseudomonas
aeru~inosa, it is recommended that a maximum of antimicrobial
::~ be employed by the use of a more concentrated solution of
~j
; cyanuric chloride as the binding agent and by a more
extensive exposure of antimicrobial to the cyanuric chloride
J treated substrate.
.. . . .
S. diastaticus was inhibited by both bound and
unbound AQR and CHA, while bound SPM somewhat inhibited the
growth of S. diastaticus. Again, the extended exposure of
the antimicrobial over 18 hours has a marked effect on the
inhibition of S. diastaticus.
From the Tables 3(a), (b), (c) and (d), it is
also indicated that the cyanuric chloride alone when used
in a 1% aqueous solution was not as antimicrobial as that
used for the Tables l, wherein a 2.5% solution in dioxane
.. . .
was used.
Table 3(e) shows the milligrams of antimicrobial
` per gram of cotton deposited or bound on the substrate as
determined by the Kjeldahl method. As shown in Table 3(e),
.
.,
. ' `
~; :
8~77
there was only abou-t 8 mg of cyanuric chloride per græ.m of
cotton bound to the substrate as compared to from 31.~ ~o 39
mg of cyanuric chloride per gram of cotton as shown in ~able
l~e). Accordingly, the binding of cyantlric chloride ~o the
- cellulosic substrate appears to be proportional to the
concentration of the cyanuric chloride solution used. As
the concentration o cyanuric chloride is increased, ~he
antimicrobial effect of the cyanuric chloride comes apparent.
In early work done the results showed that cyanuric
chloride when used in a concentration of 2.5~ substantially
inhibited growth of E coli When chlorhexidine acetate
O ~ ,
streptomycin and Arquad 18-50 were bound to the cotton
substrate via cyanuric chloride, the results showed variable
efficiency depending, for example, on the test organlsrn
used. In general, the chlorhexidine acetatej whether washed
with water or commercial detergents showed complete inhibi-
tion of E. coli and the streptomycin nearly comp]ete
inhibition. The*Arquad 18-50, however, only showed complete
inhibition when washed with All and very poor inhibition
when washed with Joy or*Tide.
.
Furthermore, it was also found during work
leading up to the present invention that not all antimicrobial
compounds retained their activity on being bound to a
substrate material. ~sing the procedures described herein-
~efore to determine the inhibitory properties of various
agents, it can be seen from the data presented in Table 4
that, using E. coli as test organism, neomycin effects
practically total inhibition of growth when bound to the
cotton substrate but, in contrast, cyanuric chloride
(CYC), pxoflanine (POF), tetracycline (TCC) and bacitracin
* Trade Mark
~ 29 -
,
. , ' . .
:~ .
:
797~7
-:
(BCT) proved ineffective from a practical viewpoint compared
with the value for the C-NaOH control. Since the type,
number and location of reactive moieties in sueh agents is
eritical to the aetivity the compound exhibits, it must
be eonsidered highly surprising that when such moieties
are involved in the bonding of the moleeule to the substrate,
and are therefore unavailable, activity is still found to
exist. These data teac~ then, that not all antimicrobial
agents on being so bound, successfully retain a desirable
level of activity.
TABLE 4
Baeteriological Results - Inhibition against E. coli
; Controls Dilution
1 ml 0~1 ml
C-~aOH TNTC 7,300
C TNTC 253
Bo_nd Antimicroblals
POF TNTC 376
~, TCC TNTC 1,399
NCM 4 0
BCT TNTC 2,226
:
Attempts to chemically bond antimicrobials with
eyanuric fluoride indicate that the amount of cyanuric
fluoride which would bond to the cotton was substantially
`30 less than the cyanuric chloride bound under the same
experimental conditions. Accordingly, the amount of
antimicrobial that could be bound to a cellulosic substrate
by cyanuric fluoride was also substantially less, i.e.
,
_ 30 _
'~
:`
~ 791~7
!~ in the order of about 20~ of that which could be bonded
via cyanuric chloride.
lt has also been foudn that a fresh solution
; o cyanuric chloride is more effective than aged solutions.
~.
Accoxdingly, it is recommended that the cyanuric chloride
be used within 24 hours after being prepared to assure
maximum bonding to the substrate.
The method and composition of the invention
-' thus provide antimicrobial surfaces for a variety of
uses wherein persistence, but non-migrating antimicrobial
action, is necessary or desirable. The chemical bonding of
,l the antimicrobial to the substrate provides resistance to
removal by washing and accordingly, is particularly suitable
for use on clothing, bedding, bandages, filters, packaging,
shoes, powders, and a number of other applications.
~ Chemical bonding of the antimicrobial to the substate also
3l prevents ingestion or other undesirable removal of the
antimicrobial from the substrate.
It will thus be seen that the objects set forth
above, among those made apparent from the preceding
.~ ,
description, are efficiently attained and, since certain
changes may be made in carrying out the above process and in
` the composition set forth without departing from the scope
of the invention, it is intended that all matter contained
in the above description shall be interpreted as illustrative
and not in a limiting sense.
.. . .
:~'
,, . .
~ - - 31 -
,
: .
.- . ,
