Surface-attached sulfonamide containing quaternary ammonium antimicrobials for textiles and plastics

Article abstract of DOI:10.1039/c8ra10173f

With the risks associated with healthcare-associated infections and the rise of antibiotic resistant microorganisms, there is an important need to control the proliferation of these factors in hospitals, retirement homes and other institutions. This work explores the development and application of a novel class of sulfonamide-based quaternary ammonium antimicrobial coatings, anchored to commercially and clinically relevant material surfaces. Synthesized in high yields (60-97%), benzophenone-anchored antimicrobials were spray-coated and UV grafted onto plastic surfaces, while silane-anchored variants were adhered to select textiles via dip-coating. Surface modified samples were characterised by advancing contact angle, anionic dye staining, X-ray photoelectron spectroscopy and atomic force microscopy. After verifying coating quality through the above characterization methods, microbiological testing was performed on batch samples in conditions that simulate the natural inoculation of surfaces and objects (solid/air) and water containers (solid/liquid). Using the previously established Large Drop Inoculum (LDI) protocol at solid/air interfaces, all treated samples showed a full reduction (10⁵-10⁷ CFU) of viable Arthrobacter sp., S. aureus, and E. coli after 3 h of contact time. Additional testing of the walls of plastic LDPE vials treated with a UV-cured sulfonamide antimicrobial at a solid/liquid interface using the newly developed Large Reservoir Inoculum (LRI) protocol under static conditions revealed a complete kill (>10⁶ reduction) of Gram-positive Arthrobacter sp., and a partial kill (>10⁴ reduction) of Gram-negative E. coli within 24-48 h of contact.

Full text of DOI:10.1039/c8ra10173f

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Surface-attached sulfonamide containing
quaternary ammonium antimicrobials for textiles
Cite this: RSC Adv., 2019, 9, 3140
and plastics†
a
a
a
a
Alexander Caschera,
Kamlesh B. Mistry,
Joseph Bedard, Evan Ronan,
a
a
b
ac
Moiz A. Syed, Aman U. Khan, Alan J. Lough, Gideon Wolfaardt
a
and Daniel A. Foucher
*
With the risks associated with healthcare-associated infections and the rise of antibiotic resistant microorganisms,
there is an important need to control the proliferation of these factors in hospitals, retirement homes and other
institutions. This work explores the development and application of a novel class of sulfonamide-based
quaternary ammonium antimicrobial coatings, anchored to commercially and clinically relevant material
surfaces. Synthesized in high yields (60–97%), benzophenone-anchored antimicrobials were spray-coated and
UV grafted onto plastic surfaces, while silane-anchored variants were adhered to select textiles via dip-
coating. Surface modified samples were characterised by advancing contact angle, anionic dye staining, X-ray
photoelectron spectroscopy and atomic force microscopy. After verifying coating quality through the above
characterization methods, microbiological testing was performed on batch samples in conditions that
simulate the natural inoculation of surfaces and objects (solid/air) and water containers (solid/liquid). Using the
previously established Large Drop Inoculum (LDI) protocol at solid/air interfaces, all treated samples showed
5
7
a full reduction (10 –10 CFU) of viable Arthrobacter sp., S. aureus, and E. coli after 3 h of contact time.
Additional testing of the walls of plastic LDPE vials treated with a UV-cured sulfonamide antimicrobial at
a solid/liquid interface using the newly developed Large Reservoir Inoculum (LRI) protocol under static
Received 11th December 2018
Accepted 13th January 2019
DOI: 10.1039/c8ra10173f
6
4
conditions revealed a complete kill (>10 reduction) of Gram-positive Arthrobacter sp., and a partial kill (>10
reduction) of Gram-negative E. coli within 24–48 h of contact.
rsc.li/rsc-advances
a healthcare associated infection (HCAI) during acute care is
possible. Infections acquired at medical centres across North
America are responsible for the loss of billions of dollars due to
Introduction
Microbial threat
5
The proliferation of antibiotic resistance genes within patho- additional hospitalization stays and litigation. According to
genic microorganisms has become a primary concern of human recent reports published by the Center for Disease Control
society in the 21st century. With the gradual emergence of (CDC), there are an estimated 722 000 nosocomial infections
1
,2
methicillin-resistant
vancomycin-resistant Enterococci (VRE),
Staphylococcus
aureus
(MRSA),
each year in the US, corresponding to 1 infection for every 25
1,3
and many other patients admitted. Of those infected, there were 75 000 related
multi-drug resistant (MDR) bacterial strains, there is an ever- deaths during hospitalization, representing a 1 in 10 chance of
increasing threat of contracting an untreatable infection. This death from a contracted infection. At the global level, the
4
6
threat is further magnied by the concentration of infected World Health Organization (WHO) estimated that 7 of every 100
patients at hospitals and clinics, where the risk of contracting patients in developed countries and 10 of every 100 patients in
developing countries will contract a HCAI. Healthcare costs
associated with HCAIs are also astonishing, with WHO esti-
a
Department of Chemistry and Biology, Ryerson University, 350 Victoria Street,
mates placing the nancial burden in US dollars at $8 billion in
Toronto, Ontario, Canada M5B-2K3. E-mail: daniel.foucher@ryerson.ca
b
5
Europe and $6.5 billion in the United States. HCAIs are
acquired from multiple routes of exposure including respira-
Department of Chemistry, University of Toronto, 80 St. George Street, Toronto,
Ontario, Canada M5S 3H6. E-mail: alough@chem.utoronto.ca
c
7
4,8
9,10
Stellenbosch University Water Institute Secretariat, Faculty of Natural Science, tory, gastrointestinal,
cardiovascular,
and renal path-
11
Stellenbosch University, South Africa. E-mail: gmw@sun.ac.za
ways.
Pathogenic microorganisms suspended in water
†
Electronic supplementary information (ESI) available: (a) Synthetic details (b)
droplets can cause infections when inhaled or ingested by an
individual. These contaminated droplets can transfer patho-
gens to surfaces such as catheters, gloves and other medical
1
13
29
NMR H, C, Si NMR data (c) XPS and ToF-SIMS data, (d) AFM and SP
proles (e) microbiological data. CCDC 1842528 1842536 1842645 1842659. For
ESI and crystallographic data in CIF or other electronic format see DOI:
10
equipment. To combat the emergence and spread of MDR
1
0.1039/c8ra10173f
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bacterial strains in institutional settings, several preventative antimicrobial-treated object into an aqueous body of contami-
28,33
efforts, such as antibiotic stewardship and infection control nated liquid.
Another method that is used to test for
protocols have been implemented with varying success. Anti- microbial activity at solid/liquid interfaces utilizes a ow cell
biotic stewardship, which relies heavily on compliance by system, where a nutrient medium is continuously passed
34
doctors and patients, represents the controlled use of these through an inoculated ow channel. This type of test repre-
prescriptions and reduces resistance development by lessening sents a more dynamic, high shear solid/liquid system where the
the chance of exposing bacterial cells to sub-lethal drug nutrient medium is replenished during the course of the test
12,13
concentrations.
This can also take the form of antibiotic and simulates environments similar to catheter tubing or
rotation regimens, which regularly swap classes of antibiotics general plumbing. However, limits of this test involve the
before specic resistance genes can develop within a given potential for bacterial species to migrate upstream and estab-
14
microbial population. Infection control programs at hospitals lish a biolm colony in an untreated section of channel, as well
and clinics are used to check the spread of disease between as the difficulty of completely treating a full ow system from
3
5
patients and also require strict compliance by patients, visitors inuent to effluent ports.
1,15
and hospital staff.
These programs focus on hygienic prac-
Although there is signicant concern with the proliferation
tices and sterilization techniques to reduce the transfer of of pathogenic microorganisms on dry solid/air surfaces, solid/
microorganisms on contaminated surfaces and individuals. liquid contact surfaces are also present in many high-risk
However, adherence to these protocols have yet to prove their environments and oen provide safe breeding grounds for
efficacy in preventing the proliferation of healthcare associated pathogenic microbes. These surfaces include sinks and drains
1,16
10
7
infectious disease.
in hospitals and air cooling towers, where there is an aqueous
To address the rising concerns regarding infection control medium in sustained contact with a solid container. These
and contamination from material surfaces, researchers have microbial niches are areas of concern and can lead to the
investigated the application of surface attached antimicrobials to continuous distribution of microorganisms into new environ-
1
7–19
20–22
20,21,23
20,24
3,10
glass,
plastic,
textiles
and metals.
While the ments, as well as a continuous release of cells as a prolifera-
36
attachment chemistry differs with each surface, the common tion mechanism described by Bester et al. In certain cases,
functional active site design relies on the presence of a long chain mature biolms can develop within 24 h and start to release 10
C –C ) alkyl quaternary ammonium salt which is proposed to cells per mL.
6
36,37
(
The Liquid Reservoir Inoculum (LRI) method
1
2
24
impart an adsorbent “phospholipid sponge” effect on cellular was devised to replicate microbial interactions between solid/
membranes through measurable surface charge, thereby liquid interfaces conducted under static, low shear conditions
25–27
damaging cell function and integrity.
Long chain QAC in a controlled environment, as well as provide accurate infor-
materials demonstrate promise as effective microbial resistant mation regarding antimicrobial activity of constantly wet
3
8,39
coatings when in contact with Gram-positive and Gram-negative surfaces.
In this work we describe our efforts to prepare and
18,22,28
bacteria at a solid/air interface,
platform for producing self-sterilizing surfaces.
and could provide a viable evaluate a series of new shorter chain sulfonamide quaternary
2
2
ammonium antimicrobials that operate at solid/air interfaces
To avoid biolm formation on solid materials under diverse and explore how well these antimicrobial treatments suppress
conditions, modications to QACs through the addition of microbial proliferation at solid/liquid surfaces.
potent functional moieties have been investigated herein.
Dating back to their discovery in 1935, sulfonamide-based
antimicrobial drugs represent the initial stages of antibiotic
Experiments and methods
treatment in modern medicine. These compounds evolved into Materials
the premier treatment method for infectious disease, such as
All reagents and solvents were obtained from commercial
blood-borne and gastrointestinal infections, but have limita-
sources and used as received unless indicated otherwise. Stock
29
tions based on potential toxicity and allergic reaction.
cotton fabric was sourced from Gildan (cat. G2000-W). Stock
plastic polystyrene (PS) (cat. 89106-754) and clear polyvinyl
chloride (CPVC) (cat. 82027-788) was sourced from VWR Inter-
national, Nalgene-brand 5 mL low-density polyethylene (LDPE)
vials (cat. 6250-0005) were sourced from ThermoFisher Scien-
tic Inc. and LEXAN-brand polycarbonate (PC) was sourced
from Sabic. The trimethoxysilane propyl halide precursor was
However, these potent antibiotics may serve effectively as
attached sulfa-containing QAC antimicrobials. Even today, new
sulfonamide compounds have been developed as sulfa drugs
and have shown promising antimicrobial properties against
30,31
various bacterial and fungal strains.
While there has been signicant research effort into
preparing non-leaching contact active antimicrobial coatings,
there are a limited number of techniques used to assess anti-
microbial activity via immersive inoculation. More oen, the
primary goal of biotesting surface-immobilized antimicrobials
is to demonstrate that coated materials can prevent microbial
40
prepared as previously described by Isquith et al. The benzo-
phenone propyl halide precursor was prepared as previously
41
described by Saettone et al. All other experimental details are
included in the supplementary section.
28,32
proliferation through direct inoculation methods.
When
Antimicrobial compound characterization
immersive solid/liquid interface testing is performed on treated
surfaces, it is usually accomplished using the ASTM E2149 Nuclear magnetic resonance (NMR) experiments were carried
shake ask method which simulates submerging an out with a 400 MHz Bruker Avance II Spectrometer (Ryerson
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3
University) using deuterated chloroform (CDCl ) unless other- Large droplet inoculum antimicrobial tests
1
13
wise noted. H and C spectra were referenced to the residual
Several clinically relevant bacterial stock cultures were used in
these tests. Bacterial test species were grown overnight in 10 mL
CHCl (7.26 and 77.0 ppm, respectively) solvent signals, while
3
1
9
F resonance was referenced against the internal standard
ꢁ1
ꢃ
of 3 g L tryptic soy broth (EMD Millipore) at 30 C within
a shaking incubator, and cultures were washed twice via
centrifugation at 9000 ꢄ g to replace the growth media with
sterile water. Arthrobacter sp. (IAI-3), a Gram-positive bacterium
originally isolated from indoor laboratory air was inoculated
onto all treated and control test surfaces as the model organism
2
9
CFCl
3
.
Si NMR was referenced against the internal standard
1
tetramethylsilane (TMS). Peak assignments in the H NMR
spectra are given in d (ppm) and were made with the assistance
1
3
of 2D COSY spectra, while assignments in the C NMR spectra
proton-decoupled) were made with the assistance of 2D HSQC
(
spectra. High resolution mass spectrometry (HRMS) was carried
out using electrospray ionization time of ight (ESI-ToF).
Melting points were measured in open air using a Fisher
Scientic melting point apparatus. A Bruker-Nonius Kappa-
CCD diffractometer was used to obtain the X-ray information
of the crystal structures of two precursor materials 5 (CCDC
39
for bacterial survival on solid surfaces. Lab strains of Gram-
negative Escherichia coli (DH5a) and Gram-positive Staphylo-
coccus aureus (Ryerson University) were also tested on treated
materials. These strains were chosen since they are well char-
acterized and are present in biolms found within high-risk
environments. The large drop inoculum (LDI) method was
used to assess the antimicrobial efficacy of the antimicrobial
1842645) and 9 (CCDC 1842536) and two active antimicrobials
6a (CCDC 1842528), and 10 (CCDC 1842659) have been depos-
treatment at a solid/air interface and is a modication of the
ited with the Cambridge Crystallographic Data Centre.
43,44
ISO 22196/JIS Z 2801 standard procedure.
Triplicate treated
samples were inoculated with 100 mL bacterial aliquots of
subsequently determined concentration, and survival on the
Characterization of antimicrobial treated surfaces
Contact angle images of treated and untreated surfaces were taken sample was determined by spot plating, described as following.
using a Teli CCD camera equipped with a macro lens attached The inoculated droplets were naturally air-dried within a class
perpendicularly to the sample surface. Contact angle measure- II, type A2 biosafety cabinet (Model 3440009, Labconco Corp.) to
ments were performed using OCA15 contact angle soware by avoid contamination, and surviving cells were enumerated
Data Physics Corporation. X-ray photoelectron spectroscopy (XPS) upon drying, typically 3 h aer inoculation. Note that the nal
was performed using a ThermoFisher Scientic K-Alpha, and time- drying time depends on the evaporation of the inoculum liquid
of-ight secondary ion mass spectrometry (ToF-SIMS) was per- and can be hard to determine on fabric samples due to droplet
formed using an IonTOF ToF-SIMS IV at the Ontario Centre for the wicking. Enumeration was performed by rehydrating and vor-
Characterisation of Advanced Materials (OCCAM), located at the texing samples in 5 mL of a 0.9% saline retrieval solution, which
ꢁ1
University of Toronto. Atomic force microscopy (AFM) using an was then serially diluted and spot-plated onto 3 g L tryptic soy
ꢃ
Anasys nanoIR2 equipped with Contact Mode NIR2 Probes (reso- agar. Plates were then incubated at 25 C for a period of 5–7
ꢁ
1
nance frequency 13 ꢀ 4 kHz, spring constant 0.07–0.4 N m ), and d which allowed for visualization of colony forming units (CFU).
surface prolometry using a KLA-Tencor P16+ Surface Prolometer At each time point, bacterial survival on the treated samples was
were also performed at OCCAM. AFM data was processed using compared to survival on triplicate untreated control surfaces of
42
Gwyddion 2.50 soware.
the same material.
Antimicrobial treatment method
Liquid reservoir inoculum antimicrobial tests
2
2
ꢁ1
Coating of plastic test samples (6.25 cm ꢀ 1 cm coupons) was Arthrobacter sp. and E. coli were grown overnight in 3 g L
ꢃ
performed via an ESS AD-LG electrospray apparatus (S/N tryptic soy broth at 30 C within a shaking incubator, and
2
0073037, Athens, Georgia) set to spray a 1% (w/v) antimicro- cultures were washed twice via centrifugation at 9000 ꢄ g to
bial coating solutions of 1a–8a at 150 kPa. Consistent coating replace the growth media with sterile water. The LRI method
uniformity was achieved by spraying test surfaces for ꢂ3 s at an (Fig. 1) was used to assess the efficacy of the antimicrobial
average distance of 45 cm from the spray nozzle. UV curing of treatment at a solid/liquid interface and is a modication of the
benzophenone-anchored QAC 1a–8a coated plastics was per- ASTM E2149 standard procedure. Triplicate treated 5 mL
formed using a Novacure spot curing system with a mercury-arc LDPE tubes containing 2.7 mL of 0.9% sterile saline were
discharge lamp at a peak intensity of 10 W, 7 cm from the light inoculated with 300 mL bacterial aliquots of subsequently
33
ꢁ2
guide source. A peak intensity of 0.075 W cm monitored over determined concentration. These tubes were capped and placed
ꢁ
2
a 60 s curing period, delivered ꢂ5 J cm total dose UVA, as onto a VWR orbital shaker (cat. 57018-754) set to 150 rpm for
measured using an EIT UV Power Puck 2. Coating solutions of 48 h to prevent sedimentation. At 24 h and 48 h, 100 mL aliquots
sulfonamide QACs 1b–4b were created by dissolving 1% (w/v) of were removed from the sample, serially diluted and spot-plated
ꢁ1
the desired antimicrobial in an EtOH : H
2
O solvent, with the onto 3 g L
tryptic soy agar to enumerate the survival of
ratio varying between 30 : 70 to 90 : 10 based on the solubility of planktonic cells suspended within the liquid reservoir. Aer
the test antimicrobial compound. Coating of fabric test 48 h, sample shaking was suspended, the remaining liquid was
2
2
samples, (6.25 cm ꢀ 1 cm swatches of cotton), was performed replaced with 5 mL sterile saline, and the samples were gently
by dip coating the samples into a coating solution before inverted 10 times to rinse any loosely-adhered cells from the
allowing them to dry at room temperature.
tube walls. With the rinse solution discarded, the LDPE tubes
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Compound 10 was additionally characterized by single crystal
X-ray diffraction and an ORTEP representation of the molecule
1
13
found in the unit cell is displayed in Fig. 2. NMR ( H and C)
spectroscopy of all sulfonamide containing compounds, along
with HRMS analysis, support their structural identity (ESI).
Synthesis and characterization of sulfaQACs
Further conversion of the sulfonamide precursors to quaternary
ammonium salts was achieved using conventional Menshutkin
quaternization procedures, which involves heating a mixture of
1–8 and the appropriate haloalkyl silane or benzophenone
attachment functionality to reux in MeCN for extended
periods (Scheme 3). Compounds 1a–8a were recovered as white-
coloured crystalline solids, while 1b–4b were isolated as clear or
golden brown-coloured oily gums. These compounds were
further puried with successive washes of the crude product
Fig. 1 Simplified schematic representation of the Liquid Reservoir
Inoculation (LRI) method.
45
with Et
2
O (10 mL ꢄ 3). Compound 6a was additionally char-
acterized by single crystal X-ray diffraction and an ORTEP
representation of the molecule found in the unit cell as dis-
played in Fig. 3.
were then lled with 1 mL of sterile saline and vortexed for
1
1
min to dislodge any biolm formed on the tube surface. One
00 mL aliquot was then removed from each tube, serially
For
all
antimicrobial
comparisons,
propyl-
diluted and spot-plated to enumerate the survival of biolm
dimethyl(benzoylphenoxy)octadecylammonium bromide (11)
was used to provide a baseline antimicrobial activity for alkyl-
QAC antimicrobials. This compound was initially detailed by
cells attached to the walls of the sample tube. Agar plates were
incubated at 25 C for a period of 5–7 d, which allowed for
visualization of CFUs. At each time point, bacterial survival on
the treated samples was compared to survival on triplicate
untreated control surfaces of the same material.
ꢃ
41
Saettone et al. in 1988 for inclusion in sunscreens and inves-
22,46
tigated by Foucher et al. in 2017
as antimicrobial coatings
on plastic surfaces (Fig. 4).
Results and discussion
Synthesis and characterization of sulfonamide precursors
The addition of 3-(dimethylamino)propylamine to the appro-
priate sulfonyl chloride in CH Cl with NEt for 3 h at room
2
2
3
temperature followed by a workup in distilled water produced
sulfonamide precursors 1–6 in high yields (Scheme 1). Alkyl
sulfonamide precursors 7 and 8 were successfully synthesized
in moderate to high yields in the absence of NEt in order to
3
avoid salt formation, while varying the order of addition, with
the sulfonyl chloride added to a solution of 3-(dimethylamino)
2 2
propylamine in CH Cl .
The non-quaternary ammonium Scheme 2 Preparation of a UV curable sulfonamide 10.
sulfonamide 10 was prepared from the reaction of sulfonamide
precursor 9 with 4-hydroxybenzophenone (Scheme 2) and iso-
lated as a yellow crystalline solid.
Fig. 2 ORTEP representation of 10 found in the unit cell and selected
ꢃ
bond lengths ( A˚ ) and bond angles ( ): S(1)–O(1) 1.4325(10), S(1)–O(2)
1.4348(9), S(1)–N(1) 1.6203(11), S(1)–C(4) 1.7861(13), N(1)–C(3)
1.4701(17), O(3)–C(1) 1.4337(16), O(3)–C(13) 1.3619(16), O(4)–C(19)
1.2235(18), O(1)–S(1)–O(2) 118.04(6), N(1)–S(1)–C(4) 106.79(6), C(3)–
Scheme 1 Preparation of sulfonamides.
N(1)–S(1) 119.53(9), C(13)–O(3)–C(1) 117.60(10).
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solution for ꢂ5 min, followed by a 24 h curing period at room
temperature. Aer treatment, samples were exhaustively rinsed
with 10 mL aliquots of distilled water and the rinse solution was
monitored for antimicrobial leachate with bromophenol blue
stain. Once the rinse solution was clear of unadhered antimi-
crobial, additional samples from each batch were sacriced for
bromophenol blue dye staining to visualize coating quality
across the material surface. Commonly, only one rinse step was
required to remove excess QAC deposited during the coating
process. Coating uniformity was also visualized by submerging
test samples in a 400 ppm bromophenol blue dye solution,
where treated samples stained blue while leaving uncoated
Scheme 3 Preparation of sulfaQACs.
22
samples unchanged. The remaining samples were then used
for microbiological evaluation using the LDI procedure with
triplicate treated samples compared against controls for anti-
microbial activity. Plastic coupons were twice treated with 1%
(w/v) of the active benzophenone-anchored antimicrobial to
ensure complete coverage. Aer treatment, samples were rinsed
with distilled water, and the rinse solution tested for residual
antimicrobial leachate with bromophenol blue stain or by UV-
Vis spectroscopy. Once the rinse solution was clear of any
unattached antimicrobial, the samples were tested in triplicate
under the same conditions as the fabric samples. The mesityl
Fig. 3 ORTEP representation of 6a found in the unit cell and selected
bond lengths ( A˚ ) and bond angles ( ): S(1)–O(3) 1.431(2), S(1)–O(4)
ꢃ
1
.433(2), N(1)–C(1) 1.469(4), N(2)–C(3) 1.514(3), N(2)–C(4) 1.514(3),
N(2)–C(20) 1.499(4), N(2)–C(21) 1.504(4), O(1)–S(1)–O(2) 119.91(13), sulfonamide, 3a, was chosen as the primary test candidate
N(1)–S(1)–C(22) 107.40(13), C(1)–N(1)–S(1) 118.8(2), C(3)–N(2)–C(4)
among the non-uorinated sulfaQAC coatings, based on phys-
ical characteristics, availability, ease of processing and relatively
low cost.
1
12.5 (2).
Sulfonamide QAC coating on fabric and polymer samples
Silane-anchored QAC coatings bearing long chain alkyl Characterization of antimicrobial coated test surfaces
substituents have been used extensively on porous surfaces for
the purpose of protecting against pathogenic infections and to
Several tests were performed to conrm the qualities and
characteristics of each antimicrobial coated sample, including
control against odors caused by bacterial metabolites. The
dye stain exposure, contact angle, and surface charge
rationale behind these efforts is to effectively kill bacterial
measurements. Antimicrobial coated surface quality on poly-
styrene samples was investigated by advancing contact angle
species before the establishment of a microbial biolm, thereby
preventing the possibility of exposure to contaminated mate-
measurements to determine surface wettability (Table 1). Sul-
rials. Some of the products treated using these compounds
faQAC treated materials 1a–4a displayed an advancing contact
include sporting goods, medical curtains and fabrics, common
clothing and protective gear for infection control and odor
47
prevention. Fabric samples were treated with 1% (w/v) of the
active silane-anchored antimicrobial by submerging and
agitating cotton swatches within a desired antimicrobial
Table 1 Advancing contact angle and surface charge measurements
a
of sample materials
Advancing contact
angle ( )
Charge density
ꢃ
+
ꢁ2
Sample
([N ] nm
)
2
2
Control PS
91.7 ꢀ 1.0
60.4 ꢀ 4.9
51.1 ꢀ 6.5
61.0 ꢀ 1.6
47.6 ꢀ 0.6
73.9 ꢀ 2.1
66.7 ꢀ 4.2
56.7 ꢀ 1.9
N/A
1
2
3
4
5
6
1
a on PS
a on PS
a on PS
a on PS
a on PS
a on PS
7.3 ꢀ 0.9
21.6 ꢀ 6.6
23.5 ꢀ 0.7
3.5 ꢀ 1.0
19.2 ꢀ 3.7
15.3 ꢀ 6.8
22
1 on PS (2 coats)
115.0 ꢀ 17.9
69
35.7
21
on glass
a
Results collected from this work and ref. 22 were performed in
Fig. 4 Schematic representation of surface grafted branching of sul- triplicate.
faQAC 3a (left) and alkylQAC 11(right).
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ꢃ
ꢃ
angle between 47.6 and 61.0 aer application of two coats, carbon, nitrogen and sulfur content (Fig. 5). With relation to the
ꢃ
which is signicantly lower than untreated polystyrene (91 ), carbon content, there is a signicant decrease (3.99%) in carbon
but similar to the long chain QAC 11 (56.7 ). For the partially- present on the coated sample surface compared to the control
uorinated sulfaQAC treated materials 5a–6a, the advancing material, which corresponds to a proportional increase of other
contact angle was notably higher (73.9 and 66.7 ) than non- elements located within the coating material. There is also an
uorine containing sulfonamide QACs, but in a similar range increase in the size of the C1s A (286.6 eV) peak, which indicates
to the value reported for the attached benzophenone antimi- the presence of carbon–oxygen ether bond of the benzophenone
crobial on glass developed by Locklin et al. The presence of the moiety of 3a. Comparisons of the nitrogen content between
quaternary ammonia moiety within the sulfaQACs increase the the two samples showed a notable increase of 1.49% on the
wettability of these surfaces, while being partially offset by the treated samples. This increase is seen through the rise of the
ꢃ
22

ꢃ
ꢃ

27
50
relative hydrophobicity of the tail moiety.
N1s (402.5 eV) and N1s A (399.9 eV) peaks, which correspond to
To compliment the contact angle data, the charge densities the presence of cationic nitrogen and carbon–nitrogen bonding
51
of the sulfaQAC antimicrobials were determined indirectly within the sample coating. There is also a 0.31% increase in
using UV-Vis spectroscopy (Table 1). As outlined by Kugler et sulfur content found upon the coated samples, similar in
48
al., this technique involves complexing an anionic dye with the signicance and scale to the increase of nitrogen content.
quaternary ammonium cation present on the surface of anti- ToF-SIMS was also performed on 3a treated and control
microbial coatings. Aer saturating the coating surface in CPVC samples (Fig. S7–S11†) to identify and correlate changes
a uorescein dye solution over a 24 h period and rinsing with in surface structure as related to specic fragments of surface
52
distilled water, the bound dye molecules were detached by material. From positive and negative ion fragmentation
washing the stained samples in a pH-balanced cationic solu- patterns analyzed from sample materials, the presence of ben-
tion. The solution can then be analysed using a spectrometer to zoylphenolate, tetracarbonyl ammonium and bromide ions
determine dye concentration, which correlates to the number of were determined to exist on the treated surfaces and are
exposed charges present on a coated sample. Comparing the consistent with the composition of 3a. Through the graphical
surface charge and contact angle data values, there appears to analysis of ionic fragments found exclusively upon the treated
be no correlation between the two factors. This could be due to sample, the coating appears to form a relatively irregular
the restricted accessibility of bulky anionic dye molecules to the patterned surface with 40–80 mm hemi-spherical structures.
cationic charges contained within the coated material, or to Interestingly, there also appears to be small 20 mm diameter
types of graed surface structures formed through the coating structures present when analysing the 3a coated surface for
and curing process. This could also explain the relatively low benzoylphenolate, indicative of the benzophenone moiety used
surface charges of sulfaQACs compared to those found in our to anchor the compound to the CPVC surface (Fig. S11†). To
22
21
previous studies, as well as those reported by Locklin and ascertain additional details regarding the coating thickness
49
Murata. Therefore, the signicance of meeting a certain
threshold of surface charge as established in previous studies
may not be as signicant with sulfaQACs, since microbiological
testing performed with these materials exhibit similar antimi-
crobial properties.
Physical properties of 3a and 6a treated samples
To further determine the properties and characteristics of sul-
faQACs, ToF-SIMS and XPS were performed on a 3a treated
CPVC sample and compared to an untreated control (Table 2). A
comparative XPS analysis between the 3a coated sample and the
untreated control showed that there were three signicant
variations in binding energy peak intensities relating to the
Table 2 Select XPS survey data for control and 3a treated CPVC
samples
Atomic
concentration
Sensitivity
factor
Sample
Element
Control CPVC
Control CPVC
Control CPVC
C1s
N1s
S2p
C1s
N1s
S2p
77.16
0.35
0.93
73.17
1.84
1.24
1.000
1.800
1.670
1.000
1.800
1.670
3
3
3
a treated CPVC
a treated CPVC
a treated CPVC
Fig. 5 XPS data corresponding to the nitrogen content (top) and sulfur
content (bottom) for control (left) and 3a treated (right) CPVC samples.
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(
(
Table 3) and structure of sulfaQAC treated materials, AFM
Fig. 6 and 7) and SP (Fig. S18–S19†) were performed on 3a and
6
a treated PC coupons. Samples were prepared by masking
a section of the sample surface with an adhesive tape (Scotch
M®) prior to treatment, followed by a water rinse aer tape
3
removal to remove any adhesive residue. The coating thickness
of sulfaQACs (3a, 6a) on PC samples (Table 3) is considerably
thinner than those found previously for the long-chain alkyl-
QAC 11, which could relate to differences in compound solu-
22
bility. When analyzing the images created from contact-mode
AFM scanning on 3a treated PC plastic, the most strikingly
apparent detail involved the roughness of the coated surface
when compared to the relatively smooth control material. From
2
the 10 mm image from Fig. 6, numerous peaks and valleys
which have a maximum height difference around ꢂ0.15 mm can
Table 3 Thickness measurements of surface attached benzophenone
a
antimicrobials
Material
EL (nm)
AFM (nm)
N/A
SP (nm)
N/A
42
21
on glass
Fig. 7 AFM images of PC control (top) and 6a treated PC (bottom).
2
2
1
3
6
1 treated PS (2 coats)
N/A
N/A
N/A
366 ꢀ 148
80 ꢀ 29
468 ꢀ 159
43 ꢀ 13
a treated PC (2 coats)
a treated PC (2 coats)
be found scattered across the surface. Collectively, these
195 ꢀ 51
125 ꢀ 6
features provide
a root mean square (RMS) roughness
a
Results collected from this work and ref. 22 were taken at three
separate sample points.
measurement of ꢂ20 nm for a 3a coated surface, compared to
ꢂ4 nm for the control.
For analysis of 6a treated PC material, tapping-mode AFM was
required to obtain a surface image since there was powerful
deection of the cantilever probe when trying to position the
disengaged probe head over the coated surface. The image ob-
tained (Fig. 7) shows a similar change in surface coarseness,
providing a RMS roughness value of ꢂ51 nm, but was found to
have smoother plateau regions followed by sharp pits and valleys.
Efficacy testing of antimicrobial treatment at solid/air
interfaces
The viability and effectiveness of treating non-porous polymer
surfaces with benzophenone-anchored sulfaQACs, 1a–4a, was
22,39
investigated using the LDI biotesting procedure.
Treated
samples were tested in triplicate against an uncoated set of
controls by inoculating each sample with a subsequently
determined microbial cell load and allowing the samples to dry
under sterile conditions. The results of these tests (Fig. 8) show
promising antimicrobial activity against the Gram-positive
representative species, Arthrobacter sp. This desiccant tolerant
Gram positive bacteria shows good survivability on control
samples in comparison to S. aureus and E. coli (Fig. 13).
A similar LDI experiment with Gram-positive Arthrobacter sp.
was also investigated for silane-anchored sulfonamide QACs,
1b–4b, on virgin cotton fabric to determine whether these
Fig. 6 AFM images of PC control (top) and 3a treated PC (bottom).
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antimicrobial treatments remain effective on porous fabric
materials (Fig. 9).
This test was also expanded to include uorinated sulfaQACs,
5a and 6a. In comparison to the non-uorinated 1a–4a, these
more hydrophobic coatings are still effective surface attached
antimicrobials as shown through microbiological testing
(Fig. 10). This would represent a step towards making tailored,
hydrophobic antimicrobial coatings. Interestingly, shorter alkyl-
sulfaQACs 7a and 8a do not exhibit any signicant antimicrobial
activity against Arthrobacter sp. at solid/air interfaces (Fig. 10).
Fig. 11 Survivability of Escherichia coli when inoculated onto plastic
coupons treated with compounds 1a–4a, compared against untreated
A complimentary set of Gram-negative trials were also per- controls (n ¼ 3).
formed on treated plastic and cotton samples using E. coli as the
test species (Fig. 11 & 12). It was determined that these treated
surfaces were similarly effective against the Gram-negative E.
coli species at solid/air interfaces, suggesting that the presence
Fig. 12 Survivability of Escherichia coli when inoculated onto cotton
fabric treated with compounds 1b–4b, compared against untreated
controls (n ¼ 3).
Fig. 8 Survivability of Arthrobacter sp. when inoculated onto plastic
coupons treated with compounds 1a–4a, compared against untreated
controls. The measurement at 0 h (>10 CFU for Arthrobacter sp.)
of a large exterior peptidoglycan wall does not appear to impact
the mechanism of kill during desiccation.
7
To evaluate the comparative antimicrobial activity of sulfa-
indicated the initial number of bacterial cells inoculated onto sample
material and was determined concurrently to inoculation, while the QAC treated UV-cured materials, representative Gram-positive
measurement at 3 h represents a standardized amount of time to allow and Gram-negative species were chosen for microbiological
the inoculum to dry onto a sample surface (n ¼ 3).
testing (Fig. 13). Primary tests were performed using an
airborne isolate of Arthrobacter species, as used previously for
22
LDI testing. This Gram-positive bacterial species acts as
a model organism for solid/air interface testing, due to its
heightened survivability against desiccation at room tempera-
ture. Additional tests were performed with Gram-positive S.
aureus and Gram-negative E. coli due to their importance in
nosocomial infections, as well as their relation to prevalent
antibiotic resistant strains.
To further evaluate how the sulfonamide functionality may
impact the antimicrobial activity of QAC coatings, an analogous
Fig. 9 Survivability of Arthrobacter sp. when inoculated onto cotton
samples treated with 1b–4b, compared against untreated cotton
controls (n ¼ 3).
Fig. 13 Survivability of various microorganisms when inoculated onto
plastic samples treated with compound 3a and 10, compared against
7
untreated plastic controls. The measurement at 0 h (>10 CFU for
5
Fig. 10 Survivability of Arthrobacter sp. when inoculated onto plastic Arthrobacter sp. and Escherichia. coli, >10 CFU for Staphylococcus.
coupons treated with compounds 5a–8a, compared against untreated aureus) indicated the initial number of bacterial cells inoculated onto
controls (n ¼ 3).
sample material and was determined concurrently to inoculation (n ¼ 3).
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non-quaternary UV-curable sulfonamide 10 (Scheme 3) was
tested using the LDI procedure. For this test, PS sample mate-
rials were treated with mesityl-based sulfaQAC 3a or mesityl-
based sulfonamide 10 and tested for antimicrobial activity
using Arthrobacter sp. at 3 h, and at 24 h to determine if there is
a weaker antimicrobial effect over time. As shown in Fig. 13, the
ammonium-free mesityl sulfonamide coating prepared from 10
exhibited no antimicrobial activity. This would suggest that the
sulfonamide functionality of sulfaQACs are independently
unable to act as antimicrobial agents without the inclusion of
quaternary ammonia.
Fig. 14 Survivability of Arthrobacter sp. and Escherichia coli when
inoculated into LDPE vials treated with compound 11, compared
against untreated LDPE controls (n ¼ 3).
Efficacy testing of antimicrobial treatment at solid/liquid
interfaces
The LRI was designed to complement the LDI method and takes
inspiration from industrial standard practices ASTM E2149 (ref.
43,44
33) and ISO 22196/JIS Z 2801.
The predominant feature of
this procedure that differentiates it from the LDI method is that
the inoculum remains wet instead of drying out. This prevents
desiccation stress from negatively impacting cell survival, but
also gives microbial species an avenue to avoid contact with the
coated material. To account for this avoidance, the test involved
enumerating planktonic cells during the inoculation period and
using a harsh rinse procedure to strip and enumerate wall-
coated biolm cells at the end of the test period. The LRI
method provides additional advantages in relation to estab-
lished standard practices (ASTM E2149 and ISO 22196/JIS Z
Fig. 15 Survivability of Arthrobacter sp. and Escherichia coli when
inoculated into LDPE vials treated with compound 3a (mesityl sulfa-
QAC), compared against untreated LDPE controls (n ¼ 3).
remaining) E. coli biolm samples. The drop itself could be
related to the lower initial inoculum cell count compared to the
other samples, but should not affect the result of comparison
between the respective treated and control sets. Compared to
the results obtained for 3a (Fig. 15), the slight drop in E. coli
biolm retrivals for 11 is conserved, indicating some slight
antimicrobial activity for QAC compounds against Gram-
negative species. In contrast, against Gram-positive Arthro-
bacter sp., 3a showed excellent antimicrobial activity aer 24 h
and 48 h regarding planktonic cells (Fig. 15). This represents
a signicant drop in cell survivability compared to control
2801) currently used to test material surfaces for antimicrobial
activity. With relation to the ASTM E2149 method, both tests
use a liquid inoculum reservoir to inoculate test materials and
use mechanical agitation to evenly distribute cells within the
test chamber. The advantage brought about by the LRI method
involves using the test chamber as the subject of study by
applying the microbial treatment directly to the walls of the
container, instead of the studied material being placed into the
uncoated test chamber. Regarding the ISO 22196/JIS Z 2801
protocol, the sample is kept hydrated by using a cover lm to
seal and press the inoculum into the test surface. Although this
alleviates desiccation stress from the inoculated cells, the cover
slip can assist cells in avoiding direct contact with an antimi-
crobial material by providing a non-antimicrobial surface and
a thin liquid gap between the coverslip and the test surface. In
contrast, the LRI method avoids untreated surface contact with
the liquid inoculum and allows for separate sampling of free-
6
4
samples at these timepoints (>10 CFU and >10 CFU). Against
Gram-positive biolm cells, 3a can completely reduce the
number of viable cells below measurable CFU quantities when
4
compared against untreated controls (>10 CFU). With Gram-
negative E. coli. cells, 3a shows reduced antimicrobial activity
6
when compared to the Gram-positive trials. At 24 h (<10 CFU
5
remaining) and 48 h (<10 CFU remaining), there is a modest
reduction in the number of viable planktonic cells when
6
5
compared against untreated controls (>10 CFU and >10 CFU).
This trend of reduced antimicrobial activity is also apparent
when comparing treated and control Gram-negative biolms
32

oating planktonic cells and surface-adherent biolm cells.
To determine whether long alkyl chain or sulfonamide QAC
antimicrobial coatings can be effective at solid/liquid inter-
faces, 11 and 3a were selected as representative candidates for
the LRI method. The treated and control LDPE samples con-
taining either 11 or 3a were tested against Gram-positive
Arthrobacter sp. and Gram-negative E. coli, bacterial species
respectively, and showed varying levels of antimicrobial activity.
As shown in Fig. 14, there was no evidence of kill from the
5
cells, which shows moderate biocidal activity (<10 CFU
6
remaining) when compared against untreated controls (>10
CFU).
Conclusion
long alkyl chain-QAC coating prepared from 11 when compared Quaternary ammonium antimicrobial treated plastic surfaces
against control samples except for a slight drop in survivability are effective at killing pathogenic microorganisms at solid/air
3
3
on both the treated (>10 CFU) and control (<10 CFU interfaces. It has further been demonstrated that signicantly
3148 | RSC Adv., 2019, 9, 3140–3150
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shorter quaternary ammonium compounds bearing sulfon-
amide functionalities are equally effective at killing bacteria at
solid/air interfaces. These sulfaQACs coatings also have unique
surface structure properties, ranging from lower surface
M. Senoh, T. Louie, S. Michell, E. Butt, S. J. Peacock,
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+
ꢁ2
charges for sulfaQACs (15.3–19.5 [N ] nm ) when compared to
+
ꢁ2
previously studied alkylQACs (35.7–115.0 [N ] nm ), to
changes in surface wettability between non-uorinated (47.6–
ꢃ
ꢃ
6
1.0 ) and uorinated (66.7–73.9 ) QAC coatings. There are also
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sulfaQAC coatings when examined using AFM and SP micros-
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Conflicts of interest
There are no conicts to declare.
2
0 L. M. Porosa, K. B. Mistry, A. Mocella, H. Deng, S. Hamzehi,
A. Caschera, A. J. Lough, G. Wolfaardt and D. A. Foucher, J.
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Acknowledgements
Funding was provided by Nanosafe Technologies Coating 21 J. Gao, N. E. Huddleston, E. M. White, J. Pant, H. Handa and
Incorporated, Jupiter Florida, USA , and Viaclean Technologies
Philadelphia, PA, USA.
J. Locklin, ACS Biomater. Sci. Eng., 2016, 2, 1169–1179.
22 L. Porosa, A. Caschera, J. Bedard, A. Mocella, E. Ronan,
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