Pesticide Formulation and Delivery Systems:
Innovating Legacy Products for New Uses
STP 1558, 2013
Available online at www.astm.org
DOI:10.1520/STP155820120184
Matthew Hardwick,1 Thomas Walsh,2 and Margaret Cotton1
Fabric Challenge Assays: New Standards for
the Evaluation of the Performance of Textiles
Treated with Antimicrobial Agents
REFERENCE: Hardwick, Matthew, Walsh, Thomas, and Cotton, Margaret,
“Fabric Challenge Assays: New Standards for the Evaluation of the Performance of Textiles Treated with Antimicrobial Agents,” Pesticide Formulation
and Delivery Systems: Innovating Legacy Products for New Uses on November 1–3, 2011 in Tampa FL; STP 1558, M. Bernards, Editor, pp. 1–14,
doi:10.1520/STP155820120184, ASTM International, West Conshohocken,
PA 2013.
ABSTRACT: Textiles treated with antimicrobial agents are emerging as new
strategies to reduce acquisition of healthcare-associated infections (HAIs).
Essential to development/validation of these textiles are standard methods for
the testing antimicrobial textile efficacy. Our laboratory has developed new testing methods, the fabric challenge assays, to recapitulate each transmission
method and test the efficacy of antimicrobial textiles in a more “real world” simulation. 5 105 colony-forming units/ml (CFU/ml) MRSA suspensions were
grown. 15 15 cm2 swatches of control, antimicrobial, hydrophobic barrier, and
VTT003 fabric were inoculated with MRSA either by aerosol, splatter, or direct
contact. Inoculated fabric was left at room temperature for 0, 30, or 60 min. Fabric was then transferred to buffer and shaken for 3 min at 400 rpm. A liquid suspension (0.1 ml) was then plated onto blood agar, grown overnight at 37 C, and
colonies were counted. In the aerosol test, at 0 min, VTT003 significantly
reduced MRSA by 78.52 6 10.26 % compared to control fabric. At 30 min, antimicrobial reduced MRSA levels by 91.48 6 8.52 %. In the splatter test, at 0 min,
antimicrobial, hydrophobic barrier, and VTT003 fabrics reduced MRSA levels
by 98.56 6 1.44, 83.91 6 13.16, and 100.00 6 0.03 %, respectively. At 30 min,
hydrophobic barrier and VTT003 reduced levels by 82.63 6 17.37 and
100.00 6 0.00 %, respectively. At 60 min, hydrophobic barrier and VTT003
Manuscript received November 26, 2012; accepted for publication January 23, 2013; published
online February 20, 2013.
1
MedStar Health Research Institute, Laboratory of Clinical Investigations, Washington, D.C.
20010, United States of America.
2
Weill Cornell Medical College, Department of Microbiology and Immunology, New York,
New York 10021, United States of America.
C 2013 by ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA
Copyright V
19428-2959.
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STP 1558 ON PESTICIDE FORMULATION AND DELIVERY SYSTEMS
abrogated MRSA levels. In the contact test, at 0 min, hydrophobic barrier and
VTT003 reduced MRSA levels by 99.06 6 0.94 and 97.08 6 2.92 %, respectively. At 30 min, hydrophobic barrier and VTT003 reduced MRSA levels by
100.00 6 0.03 and 19.38 6 19.38 %, respectively. At 60 min, hydrophobic barrier and VTT003 abrogated MRSA levels. The fabric challenge assays are a
novel method for evaluation of antimicrobial textile performance and should be
considered in the development of standards and testing methods for all antimicrobial textiles intended for use in healthcare infection control strategies.
KEYWORDS: antimicrobial, textiles, HAIs
Introduction
Healthcare-associated infections (HAIs) and increasing bacterial resistance have
emerged as a major challenge to the healthcare system. In the United States,
there are an estimated 1.7 106 HAIs yearly with nearly 100,000 associated
deaths [1]. HAI pathogens may be spread between patients via vectors such as
healthcare workers (HCWs) and fomites, including clothing. Work clothes may
become contaminated during patient care, and thus have the potential for transmission of medically important pathogens to both patients and staff [2–8].
The patient environment inside the hospital also has a great potential to
contribute to the transmission of HAIs [9]. Linens, such as privacy curtains and
staff clothing, which are not changed between patients, have the greatest potential to become fomites [10]. Hospital linens and HCW clothing are frequently
contaminated with the bacteria most commonly responsible for nosocomial
blood stream infections such as Staphylococcus aureus, methicillin-resistant
Staphylococcus aureus (MRSA), Enterococcus spp., and vancomycin-resistant
Enterococcus (VRE) [11]. Materials and technologies are being developed to
resolve this problem. One approach is the development of antimicrobial textiles. The goal of such textiles is to reduce microbial contamination of fabrics
by those organisms commonly associated with HAIs and, ultimately, to reduce
transmission of these organisms [1].
In this study, we utilize a 65 % polyester and 35 % cotton blend for both
control and experimental fabrics (antimicrobial, hydrophobic barrier, and
VTT003). VTT003 is treated with both an organo-silane-based quaternary ammonium compound antimicrobial agent (Semeltec; provided by Vestagen
Technical Textiles LLC, Orlando, Fl) and hydrophobic barrier chemistry. Antimicrobial fabric is control fabric treated exclusively with the organo-silane
antimicrobial agent. Hydrophobic barrier fabric is control fabric treated exclusively with the same hydrophobic barrier chemistry used in VTT003.
Several testing methods have been established for determining the
antimicrobial activity of textiles. Such methods as ASTM E2149 [12] and
AATCC100 [13] are excellent for effectively calculating efficacy of antimicrobial fabrics with increasing inoculum concentrations and exposure times. Both
methods, however, test textiles in a highly artificial setting. In the ASTM
E2149 protocol, textiles are inoculated through submersion in a liquid
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HARDWICK ET AL., doi:10.1520/STP155820120184
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inoculum. In the AATCC100 protocol, textiles are laid horizontally and inoculated and tested with a liquid inoculum applied to and maintained on the textile
surface. During the course of daily use, however, textiles such as HCW garments never encounter such conditions. Therefore, these and other established
standard testing protocols do not address the real-world transmission mechanisms encountered by fabrics during normal use in a hospital environment
[14–19].
Each potential transmission mechanism delivers inoculum in a different
way with dynamic possible surface interactions for each. Therefore, it becomes
important when developing protective garments for HCWs to recognize that
the quantifiable antimicrobial response to one method may not necessarily
reflect a similar response when subjected to another delivery method [20]. In
healthcare environments, HCW textiles, including lab coats and scrub suits,
may become exposed to microbes either through contact with bodily fluids or
direct physical contact with an inoculated patient, another HCW, or environmental surface. Testing methods should reflect these “real world” modes of
inoculation.
The purpose of this study was to develop test methods that build upon current testing protocols, use biologically relevant inocula and exposure times,
and use real-world types of exposures. The testing methods described in this
study simulated the three main mechanisms of inoculation of fabrics in healthcare environments: aerosol, splatter, and direct contact. Aerosol inoculation
mimics airborne microbial exposures such as sneezing or coughing. Splatter
inoculation mimics bulk fluid exposures such as blood, urine, or vomit. Finally,
direct contact inoculation mimics physical contact between the fabric and a
contaminated surface, patient, or other HCW.
Materials and Methods
MRSA Cultures
Frozen isolates of MRSA (strain #33591; American Type Culture Collection
(ATCC), Manassas, VA) were streaked onto sterile 100 mm BBL Trypticase
Soy Agar plates supplemented with 5 % sheep blood (Becton Dickinson,
Franklin Lakes, NJ) and grown overnight (O/N) at 37 C. MRSA suspensions
were made by transferring single colonies to sterile 25 ml Erlenmeyer flasks
containing 20 ml sterile tryptic soy broth (Sigma Aldrich, St. Louis, MO) and
incubated at 37 C O/N with constant shaking (200 rpm). All suspensions were
adjusted to contain a final bacterial concentration of 5 106 colony-forming
units (CFU)/mL. Final bacterial concentrations were confirmed by manual
counting on a hemacytometer. For all experiments, 0.1 ml O/N MRSA suspensions (final concentration of 5 105 CFU/ml) were mixed in sterile flasks with
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STP 1558 ON PESTICIDE FORMULATION AND DELIVERY SYSTEMS
sterile buffer solution composed of 0.03 mM KH2PO4 (Fischer Scientific, Pittsburgh, PA) in sterile filtered water supplemented with 0.1 % sterile calf bovine
serum (ATCC).
Preparation of Fabric and Glove Box
15 cm 15 cm (225 cm2) swatches of control, antimicrobial, hydrophobic barrier, and VTT003 (possessing both antimicrobial and hydrophobic barrier properties; Vestex; Vestagen Technical Textiles, Orlando, FL) fabrics were cut.
Fabric swatches were attached vertically to sterile clipboards inside a sterilized
Precise HEPA-Filter Glove Box (Labconco Corporation, Kansas City, MO).
All experiments were carried out in the glove box. After each experiment, the
inside of the glove box was disinfected with 70 % ethanol. Between experiments, sterile conditions were maintained inside the glove box using a UVG54 Handheld UV Lamp (UVP, Upland, CA).
Aerosol Fabric Challenge
Sterilized mini-spray bottles (VWR International, Bridgeport, NJ) were filled
with fresh MRSA suspensions and primed by spraying five times into an empty
50 ml conical tube. A single spray has a volume of 0.93 ml. Spray volumes
were confirmed for each experiment. With the base of the spray bottle positioned exactly 10 cm from surface of fabric, a single spray (0.93 ml) was
applied to each of four swatches of control, antimicrobial, hydrophobic barrier,
and VTT003 fabrics. Inocula were left on fabric swatches for 0, 30, or 60 min
at room temperature (RT). Fabric swatches were then transferred to 250 ml
reinforced-neck Erlenmeyer flasks (VWR International) containing 50-ml recovery buffer (KH2PO4 buffer solution containing 39 g of Dey/Engley (D/E)
neutralizing broth; Thermo Scientific, Lenexa, KS).
Splatter Fabric Challenge
Fresh MRSA suspension (0.93 ml) was directly applied to each of four
swatches of control, antimicrobial, hydrophobic barrier, and VTT003 fabrics
via sterile pipetting. Inocula were left on fabric swatches for 0, 30, or 60 min at
RT. Fabric swatches were then transferred to 250 ml reinforced-neck Erlenmeyer flasks (VWR International) containing 50 ml recovery buffer.
Direct Contact Fabric Challenge
Fresh MRSA suspension (0.93 ml) was saturated onto a single sterile cotton
swab. Swabs were then used to evenly spread inocula across the entire surface
of each of four swatches of control, antimicrobial, hydrophobic barrier, and
VTT003 fabrics. Inocula were left on fabric swatches for 0, 30, or 60 min at
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HARDWICK ET AL., doi:10.1520/STP155820120184
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RT. Fabric swatches were then transferred to 250 ml reinforced-neck Erlenmeyer flasks (VWR International) containing 50 ml recovery buffer.
Recovery of Bacteria
For all experiments, bacteria remaining on the fabric swatches were recovered
via wrist-action shaking using a Model 75 Wrist-Action Shaker (Burrell Scientific, Pittsburgh, PA) for 3 min at room temperature (RT) and 400 rpm. Recovered bacteria were serial diluted 1:10 three times (100 %, 10 %, 1 %, and
0.1 %) in filter-sterilized phosphate-buffered saline (MP Biomedicals, LLC,
Solon, OH) and 0.1 ml of each dilution was plated on sterile 100 mm BBL
Trypticase Soy Agar plates supplemented with 5 % sheep blood (Becton Dickinson, Franklin Lakes, NJ). Plates were then incubated at 37 C for 24 h, then at
RT for 24 h before colonies were manually counted. Results were reported as
mean CFU/100-mm plate 6 standard error of the mean (SEM). As suggested in
previous studies, only plate counts within the range of 25–250 CFU per plate
were used for data analysis except where counts were too low for all dilutions
[21]. All experiments were performed a minimum of three independent times.
Statistical Analysis
Paired student’s t test was used to compare % reduction of experimental fabric
values to control fabric for each time point. The specified p values are as indicated either in the text or figure legends.
Results
Aerosol Fabric Challenge
The aerosol fabric challenge was developed to mimic common airborne microbial exposures such as sneezing or coughing. To this end, we aerosolized MRSA
inocula and exposed them to control, antimicrobial, hydrophobic barrier, and
VTT003 fabric swatches. Upon aerosol inoculation, the control fabric immediately absorbed the liquid inoculum, whereas the inoculum formed small beads
on hydrophobic barrier and VTT003 fabric and was not absorbed (Fig. 1(a)).
Antimicrobial fabric partially absorbed the inoculum with some fluid remaining
as small beads adhering to the fabric (Fig. 1(a)). At 0 min, while both antimicrobial and hydrophobic barrier fabrics reduced MRSA levels relative to control
fabric, only VTT003 significantly reduced MRSA by 78.52 6 10.26 % compared
to control fabric (Fig. 1(a); p ¼ 0.0166). At 30 min, only antimicrobial alone significantly reduced MRSA levels by 91.48 6 8.52 % (Fig. 2; p ¼ 0.0086). Neither
hydrophobic barrier nor VTT003 reduced MRSA at 30 min. None of the fabrics
significantly reduced MRSA levels at 60 min. These data are relatively
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STP 1558 ON PESTICIDE FORMULATION AND DELIVERY SYSTEMS
FIG. 1—Visual representation of the fabric challenge assays. Each fabric was
inoculated with buffer containing 0.1 % (weight/volume) Ponceau S stain either
by aerosol (a), splatter (b), or direct contact (c) as previously described.
Images were then captured by digital photography.
inconclusive. However, the formation of inoculum droplets on all non-control
fabrics suggests that the recovery of organism from these fabrics may reflect recovery from the droplets and not the actual fabrics themselves. Moreover, organisms remaining in droplets sitting on top of the fabrics do not have access to the
antimicrobial agent further confounding the determination of antimicrobial
efficacy.
Splatter Fabric Challenge
The splatter fabric challenge was developed to mimic bulk fluid exposures
such as blood, urine, or vomit as might be seen in the healthcare setting. To
this end, we applied MRSA inocula via direct pipetting to both control and experimental fabric swatches. Upon splatter inoculation, control fabric immediately absorbed the liquid inoculum (Fig. 1(b)). While antimicrobial fabric
absorbed some of the inoculum, some liquid fell from the fabric (Fig. 1(b);
data not shown). Little to none of the inoculum remained on hydrophobic barrier and VTT003 fabrics with the bulk of the fluid running off of the fabric
(Fig. 1(b); data not shown). At 0 min, antimicrobial, hydrophobic barrier, and
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HARDWICK ET AL., doi:10.1520/STP155820120184
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FIG. 2—Experimental fabric reduction of MRSA bacterial levels following exposure to an aerosol inoculum. 5 105 CFU/ml MRSA was exposed via aerosol spray to control, antimicrobial, hydrophobic barrier, and VTT003 fabrics
for 0, 30, or 60 min. Data are presented as mean % control values 6 SEM.
Each data point represents three independent experiments. *p < 0.05;
**p < 0.01. Exact p values are given in the text.
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STP 1558 ON PESTICIDE FORMULATION AND DELIVERY SYSTEMS
VTT003 fabrics significantly reduced MRSA levels by 98.56 6 1.44,
83.91 6 13.16, and 100.00 6 0.03 %, respectively (Fig. 3; p ¼ 0.0002,
p ¼ 0.0236, and p < 0.0001, respectively). At 30 min, while antimicrobial fabric reduced MRSA levels, only hydrophobic barrier and VTT003 significantly
reduced levels by 82.63 6 17.37 and 100.00 6 0.00 %, respectively (Fig. 3;
p ¼ 0.0415 and p < 0.0001, respectively). At 60 min, hydrophobic barrier and
VTT003 completely abrogated MRSA levels (Fig. 3; p < 0.0001 for both),
whereas no reduction was seen with antimicrobial fabric. These data indicate
that all experimental fabrics have a significant reduction of organism relative
to control fabric immediately following inoculation. Further, the results indicate that only hydrophobic barrier and VTT003 maintain this reduction in a
significant manner at later time points.
Direct Contact Fabric Challenge
Finally, the direct contact fabric challenge was developed to mimic physical
contact between the fabric and a contaminated surface, patient, or other HCW.
To this end, we applied MRSA inocula directly to control and antimicrobial
fabric swatches via cotton swabs. As with the other inocula, the liquid from
swabs was immediately absorbed by control fabric (Fig. 1(c)). Swab inoculation of antimicrobial fabric leads to partial inoculum absorption, while leaving
some small droplets and cotton fibers on the surface (Fig. 1(c)). Interestingly, it
was extremely difficult to apply the swab inoculum to hydrophobic barrier and
VTT003 fabrics. While most of the inoculum did not transfer, some did remain
on fabric surfaces as small droplets (Fig. 1(c)). For all time points, both hydrophobic barrier and VTT003 fabrics significantly reduced MRSA levels relative
to control fabric, while antimicrobial fabric had no impact (Fig. 4). At 0 min,
hydrophobic barrier and VTT003 reduced MRSA levels by 99.06 6 0.94 and
97.08 6 2.92 %, respectively (p < 0.0001 and p ¼ 0.0003, respectively). At
30 min, while VTT003 reduced MRSA levels by 80.62 6 19.38 %, only hydrophobic barrier fabric significantly reduced MRSA levels by 100.00 6 0.03 %
(p < 0.0001). At 60 min, hydrophobic barrier and VTT003 completely abrogated MRSA levels (p < 0.0001 for both). These data suggest that the direct
contact fabric challenge captures significant reductions in bacterial burden on
both hydrophobic barrier and VTT003 fabrics.
Discussion
HAIs are a major challenge affecting every corner of the healthcare arena.
HCW textiles treated with antimicrobial agents are of increasing interest as
efforts to reduce the transmission and acquisition of HAIs becomes more
urgent. While there are several standard methods available for testing antimicrobial textile efficacy, each is limited because of the artificial nature of the
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FIG. 3—Experimental fabric reduction of MRSA bacterial levels following exposure to a splatter inoculum. 5 105 CFU/ml MRSA was exposed via pipetting to control, antimicrobial, hydrophobic barrier, and VTT003 fabrics for 0,
30, or 60 min. Data are presented as mean % control values 6 SEM. Each data
point represents three independent experiments. *p < 0.05; ***p < 0.001;
****p < 0.0001. Exact p values are given in the text.
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10 STP 1558 ON PESTICIDE FORMULATION AND DELIVERY SYSTEMS
FIG. 4—Experimental fabric reduction of MRSA bacterial levels following exposure to a direct contact inoculum. 5 105 CFU/ml MRSA was exposed via
cotton swab to control, antimicrobial, hydrophobic barrier, and VTT003 fabrics for 0, 30, or 60 min. Data are presented as mean % control values 6 SEM.
Each data point represents three independent experiments. ***p < 0.001;
****p < 0.0001. Exact p values are given in the text.
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testing method. In the clinical environment, there are three primary means of
transmission and acquisition of HAIs: aerosol such as a cough or sneeze, aqueous splatter such as urine or vomit, and direct contact with patients, environmental surfaces, or other HCWs. The fabric challenge assays were developed
to improve upon existing antimicrobial textile testing methods by introducing
modes of inoculation that mimic “real world” exposures that such textiles
might face in the healthcare environment.
In this study, we present evidence that each of the fabric challenge assays
(aerosol, splatter, and direct contact) captures the significant reductions in
MRSA bacterial levels made by VTT003 fabric, as well as antimicrobial and
hydrophobic barrier fabrics, relative to control fabric. A major finding of our
study is that MRSA reduction by VTT003, as well as antimicrobial and
hydrophobic barrier fabrics, is dependent on the type of inoculation: aerosol,
splatter, or contact. VTT003 consistently reduced MRSA levels to near 0 %
control in both splatter and contact challenges. Hydrophobic barrier fabric
regularly reduced MRSA levels to near 0 % control in both splatter and contact challenges. In the splatter challenge, antimicrobial fabric trended to a
reduction of MRSA levels without achieving statistical significance. The
results from the aerosol challenge were both interesting and surprising. In
this challenge, while VTT003 and antimicrobial fabrics significantly reduced
MRSA levels at 0 and 30 min, respectively, no single fabric consistently
reduced MRSA levels relative to control fabric. One possible reason for this
result may be the fluid dynamics of the spray inoculum upon encountering
the hydrophobic nature of the fabrics. While both the hydrophobic barrier
and VTT003 fabrics contain hydrophobic barrier chemistry, the antimicrobial
fabric is also slightly hydrophobic. This property is evidenced by the only
partial absorption of the liquid inoculum and likely the result of the organosilane conferring hydrophobicity when applied as a surface modification [22]
and may be the result of the long chain alkyl substituents of the quarternary
ammonium compound. It is important to note that the organo-silane used in
both the antimicrobial and VTT003 fabrics is covalently bound to the textile.
This covalent bonding prevents leaching of the antimicrobial away from the
fabric. Leaching is further prevented by homopolymerization of the compound at the fabric’s surface. By beading up on the surface of these fabrics,
the MRSA is not subject to the antimicrobial agent, which requires direct
contact with the organism for its killing effect.
Results from both the splatter and direct contact challenges provide clear
and statistically significant evidence that MRSA levels are reduced at all time
points by both the hydrophobic barrier and VTT003 fabrics. VTT003 performs
significantly better relative to control fabric than does the hydrophobic barrier
fabric. VTT003 combines both the organo-silane antimicrobial agent and
hydrophobic barrier chemistry. These results suggest that this combination confers a higher reduction of MRSA bioburden on fabric than does the
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12 STP 1558 ON PESTICIDE FORMULATION AND DELIVERY SYSTEMS
hydrophobic barrier alone. The evidence presented in the splatter challenge
data indicates that fabric containing only the organo-silane reduces MRSA levels, although to a lesser extent. This data suggests that the antimicrobial agent
provides an additive effect to the hydrophobic barrier when combined in
VTT003 fabric.
The development of an industrial assay for determining efficacy of fabrics
treated with antimicrobial agents requires standardization and validation.
Standardization of the novel assays presented in this study will require interinstitutional reproducibility, whereas validation necessitates correlation of laboratory findings with a clinical outcome. For the purposes of validation, the
results of these assays strongly correlate with the findings of a blinded clinical
trial conducted by Bearman and colleagues [23]. In their study, the authors performed a prospective, crossover trial testing the effectiveness of scrubs made
from Semeltec-treated antimicrobial fabric at reducing on apparel bacterial bioburden. The study demonstrates 4–7 mean log CFU reduction of MRSA on the
antimicrobial-treated scrub shirts of hospital personnel relative to control scrub
shirts. We demonstrate similar reductions in MRSA on antimicrobial-treated
fabric relative to control fabric for each method of inoculation. Although more
studies are needed, Bearman’s findings validate our laboratory findings in the
clinical setting of an intensive care unit.
One serious concern for use of antimicrobial agents is the potential formation of microbial resistance. Quarternary ammonium ion compounds have widely
been used as disinfectants in the hospital environment without medically important emergence of resistant pathogens. Moreover, there are no systemic antimicrobial agents that are currently used in treatment of human infections that share
structural homology with the silane-based quarternary ammonium ion compounds used in these studies. Therefore, widespread use of quaternary
ammonium-based antimicrobials such as that used in this study should be nonresistance forming and, further, should not artificially drive emergence of pathogenic resistance to antimicrobial agents currently in clinical use.
In conclusion, the results presented in this study show that the fabric challenge assays is a novel standard for the evaluation of the performance of antimicrobial textiles. Furthermore, the fabric challenge assays accurately simulate
real-world acquisition of biologically relevant inoculums and accurately determine how antimicrobial textiles will differentially perform under these conditions. Therefore, the fabric challenge assays should be considered as the next
standard of testing for all antimicrobial textiles intended for use in healthcare
infection control strategies.
Acknowledgments
This work is funded in part from an investigator-initiated research grant from
Vestagen Technical Textiles, LLC (Orlando, FL).
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