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Citation: Duffy, H.R.; Ashton, N.N.;
Blair, A.; Hooper, N.; Stulce, P.;
Williams, D.L. Regulatory Standard
for Determining Preoperative Skin
Preparation Efficacy Underreports
True Dermal Bioburden in a Porcine
Model. Microorganisms 2024,12, 2369.
https://doi.org/10.3390/
microorganisms12112369
Academic Editor: Suresh Joshi
Received: 18 October 2024
Revised: 1 November 2024
Accepted: 7 November 2024
Published: 20 November 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Article
Regulatory Standard for Determining Preoperative Skin
Preparation Efficacy Underreports True Dermal Bioburden in a
Porcine Model
Hannah R. Duffy 1,2 , Nicholas N. Ashton 1, Abbey Blair 1, Nathanael Hooper 1, Porter Stulce 1,2
and Dustin L. Williams 1,2,3,4,*
1Department of Orthopaedics, University of Utah, Salt Lake City, UT 84112, USA;
hannah.duffy@utah.edu (H.R.D.); n.ashton@utah.edu (N.N.A.); abbey.blair@hci.utah.edu (A.B.);
hooper.33@wright.edu (N.H.); u1270386@utah.edu (P.S.)
2Department of Biomedical Engineering, University of Utah, Salt Lake City, UT 84112, USA
3Department of Pathology, University of Utah, Salt Lake City, UT 84112, USA
4Department of Physical Medicine and Rehabilitation, Uniformed Services University of the Health Sciences,
Bethesda, MD 20814, USA
*Correspondence: dustin.williams@utah.edu
Abstract: Medical device companies and regulatory bodies rely on a nondestructive bacterial sampling
technique specified by the American Society for Testing and Materials (ASTM E1173-15) to test preopera-
tive skin preparations (PSPs). Despite the widespread use of PSPs, opportunistic skin-flora pathogens
remain the most significant contributor to surgical site infections, suggesting that the ASTM testing
standard may be underreporting true dermal bioburden. We hypothesized that ASTM E1173-15 may
fail to capture deep skin-dwelling flora. To test this hypothesis, we applied ASTM E1173-15 and a
full-thickness skin sampling technique, which we established previously through application to the backs
of seven pigs (Yorkshire/Landrace hybrid) following a clinically used PSP (4% chlorhexidine gluconate).
The results showed that samples quantified using the full-thickness skin method consistently cultured
more bacteria than the ASTM standard, which principally targeted surface-dwelling bacteria. Following
PSP, the ASTM standard yielded 1.05
±
0.24 log
10
CFU/cm
2
, while the full-thickness tissue method
resulted in 3.24
±
0.24 log
10
CFU/cm
2
, more than a 2 log
10
difference (p< 0.001). Immunofluorescence
images corroborated the data, showing that Staphylococcus epidermidis was present in deep skin regions
with or without PSP treatment. Outcomes suggested that a full-thickness sampling technique may better
evaluate PSP technologies as it resolves bioburdens dwelling in deeper skin regions.
Keywords: preoperative skin preparation; surgical site infection; skin microbiome; cup scrub method;
tissue blend method; porcine model
1. Introduction
Despite the clinical use of preoperative skin preparations (PSPs) through topical antisep-
sis, the patient’s endogenous flora is the primary source of surgical site contamination [
1
–
4
].
Culture data from surgical instruments, surgeons’ gloves, patients’ tissues, and hardware point
to the patient’s microflora as the principal source of microbial growth [
5
–
8
]. PSP-surviving
microbes account for 70–95% of surgical site infections (SSIs), greatly outnumbering contri-
butions from exogenous sources [
1
]. As such, SSI isolates mirror the natural geography of
the human skin microbiome. For example, surgeries involving the shoulders, breasts, and
spine incur much higher rates of infection with Cutibacterium acnes, a common resident of these
locations [
9
–
12
]. In all other anatomical regions, Gram-positive staphylococcal species such
as Staphylococcus epidermidis and Staphylococcus aureus are principal SSI contributors [
13
–
15
].
If this persistent surgical site contamination is to be addressed, the nature of PSP-surviving
microorganisms and how they evade prophylactic antiseptics must inform the development of
future testing methods, surgical practices, and next-generation antiseptic technologies.
Microorganisms 2024,12, 2369. https://doi.org/10.3390/microorganisms12112369 https://www.mdpi.com/journal/microorganisms
Microorganisms 2024,12, 2369 2 of 15
Thousands to millions of bacteria per cm
2
live throughout the skin’s surface and
deeper layers [
16
–
19
]. Both superficial and deep-dwelling bacteria have been identified
using various imaging techniques, including Gram stain and fluorescent imaging [
20
–
22
].
As early as 1985, Gram stains showed the presence of bacteria in hair follicle tracts and at
the edge of the pilosebaceous glands [
22
]. In recent years, researchers have used peptide
nucleic acid fluorescence to show bacterial aggregates on the stratum corneum and hair
follicle bulbs in healthy controls [
20
]. This investigation also quantified the bacterial skin
aggregates in biofilm: 92% of healthy patients had observable biofilm, with the majority
residing in the hair follicles (64%) or the stratum corneum (36%) [
20
]. Analyses of the
relative abundance of the 16S rRNA gene (common to all bacteria) also indicated that the
depth profiles of bacteria vary on skin characteristics and the presence of skin features [
23
].
On palm skin (hairless), bacteria are more plentiful in the first 0–1 mm of the skin than
depths greater than 1 mm [
23
]. In contrast, human facial skin (with hair) exhibits more
bacteria below 3 mm than between 1 and 2 mm [
23
]. Human hair follicles, which harbor
resident normal flora, extend 2–4.5 mm below the skin surface [
24
]. Likewise, sebaceous
glands and sweat glands extend into the dermis and provide environments for bacteria
growth [
22
,
23
]. Bacteria living in skin features across all dermal layers make up the human
skin microbiota and contribute to SSIs, a fact that researchers have been exploring for nearly
70 years [25–27].
The use of PSPs have dramatically reduced microbial bioburden and thus SSI rates
to 1–4% since their introduction as part of broader antiseptic techniques [
28
]. Current
skin disinfection with PSP is performed a few minutes before surgery in combination with
other SSI-preventing practices [
29
–
31
]. PSP products are effective on the skin’s surface,
often resulting in log
10
bacterial reductions in human subjects [
32
,
33
]. PSP products with
chlorhexidine gluconate (CHG) have become the clinical gold standard due to CHG’s long-
acting efficacy [
34
]. CHG and other antiseptics are highly potent upon contact with bacteria.
However, rapid antisepsis before surgery does not kill bacteria in the deeper regions of
the skin due to the depths of bacterial colonization [
26
,
35
]. It is these deeper-dwelling
microorganisms that are often overlooked by PSP testing practices.
Determining true PSP efficacy using appropriate testing methodologies to address
deeper-dwelling microbes is essential as bacteria that survive PSP pose a significant SSI risk.
The industry relies on the American Society for Testing and Materials (ASTM) standard
E1173-15, Standard Test Method for Evaluation of Preoperative, Precatheterization, or
Preinjection Skin Preparations, when testing PSP products [
36
]. Generally regarded as the
cup scrub method, this method has been the gold standard for PSP development since
1994 when 59 Federal Register 31402 was created [
37
]. Briefly, the cup scrub method
involves placing a sterile cup on the skin’s surface [
37
,
38
]. The cup is filled with solution.
A rubber spatula is used to rub the skin’s surface for approximately 1 min. Then, the
liquid is removed from the cup, serially diluted, and quantified to detect microorganisms
suspended in the liquid. This standard specifies levels of acceptable bioburden reduction
as 2 and 3 log
10
units for dry and moist sites, respectively. PSP products used clinically
are often approved by the Food and Drug Administration (FDA) based on cup scrub
method outcomes.
The cup scrub technique fits into a broader category of nondestructive methods
including swabs, scraping, and impressions. Generally, the cup scrub method is one of the
more thorough nondestructive methods for quantifying skin flora, as repeated scraping may
dislodge more bacteria than a swab. Of all nondestructive methods, the cup scrub method
has become especially consequential through sanctioning by the FDA. The widespread use
of the cup scrub method in antisepsis, skin conditions, and cosmetics demonstrates the
need for skin sampling methods [
39
–
44
]. Despite its prevalence, the cup scrub method fails
to consider bacteria living below the skin’s surface.
Unlike nondestructive techniques, destructive or biopsy-like methods better capture
microorganisms throughout various skin layers [
22
]. These methods are rarely performed,
however, as biopsies or other destructive techniques result in skin trauma for patients [
45
].
Microorganisms 2024,12, 2369 3 of 15
Moreover, studies that do use biopsies may produce confounding results conditional on
the anatomical location and size of the biopsy. Depending on the quantity of surface
area used, variation across the skin’s surface can yield a high bioburden in some samples,
while others may yield no growth [
46
]. Thus, manufacturers of antiseptic products opt to
demonstrate substantial equivalence through the 510(k) FDA pathway [
47
]. Generations of
510(k) predicates have perpetuated the use of the cup scrub method that fails to resolve
microbes of all skin layers. Destructive techniques are the only techniques that have
demonstrated sensitivity to deeper-dwelling microbes. Iterative destructive testing for PSP
products at scale is possible using a large animal model.
We previously established the tissue blend method using a pig model, wherein we
removed and processed 4
×
4 cm (16 cm
2
) full-thickness sections of dermal tissue (surface
to fascia) for testing PSPs [
48
]. This method addressed the major disadvantages of nonde-
structive methods. By homogenizing full-thickness tissue, we resolved bacteria living in all
skin layers and maximized the number of detectable microbes using optimal surface area
and depth profiles. The 4
×
4 cm excisions included a surface area roughly 14
×
larger than
a standard large biopsy punch. Surface area is a critical consideration of a PSP model as bac-
teria are dispersed in a nonhomogeneous pattern throughout the skin. Bacteria are found
in pockets throughout the skin’s varied anatomical features, including hair follicles, sweat
glands, and sebaceous glands, but are not organized systematically [
1
,
16
,
22
]. Samples with
larger surface areas incorporate more features housing bacteria, resulting in a more accurate
average. We employed this model to test two gold-standard PSPs with povidone-iodine
and CHG [
48
]. Neither antiseptic achieved a 2 log
10
reduction, highlighting the need for
improved PSP technology. The tissue blend method can be readily used to test current
and new PSP approaches in an animal model by using full-thickness tissue and large
surface areas.
In this study, we hypothesized that the ASTM standard E1173-15 would overlook
deep-dwelling bacterial communities compared to a previously established full-thickness
skin sampling method. If our hypothesis is supported, the tissue blend method is a good
model to test improved PSP technology with the long-term goal of targeting the problem
that underpins SSIs.
2. Materials and Methods
2.1. Supplies, Reagents, and Instruments
Alcohol-based Wet Skin Scrub Trays with CHG (CHG preoperative skin preparation kit, 4%
solution) were purchased from Medline (Northfield, IL, USA). Stainless steel cups were custom
fabricated from corrosion-resistant tubing purchased from McMaster-Carr (Elmhurst, IL, USA)
using 316 stainless steel (with a 1/4” wall thickness and a 1–1/2” outer diameter, 89495K49).
The cups were cut to approximately 1 in. in height, sanded, and polished. Glass rods with
rubber policemen scrapers (spatulas) were purchased from Cole-Parmer (Vernon Hills, IL, USA).
Columbia blood agar plates were acquired from Hardy Diagnostics (Santa Maria, CA, USA).
Petri dishes and agar were purchased from Fisher Scientific (Hampton, NH, USA). Dey-Engley
(D/E) neutralizing broth and tryptic soy broth (TSB) were purchased from MilliporeSigma
(Burlington, MA, USA). Brain heart infusion (BHI) broth was purchased from Research Products
International (Mt Prospect, IL, USA). Magic Bullet blenders, cups, and blades were purchased
from Amazon.com (Seattle, WA, USA).
AStaphylococcus epidermidis Monoclonal Antibody (primary, MA135788) was pur-
chased from ThermoScientific (Waltham, MA, USA). A Goat Anti-Mouse IgG Alexa Fluor
488 Polyclonal antibody was purchased from SouthernBiotech (Birmingham, AL, USA).
ProLong Gold Antifade Reagent Liquid was sourced from Cell Signaling Technology (Dan-
vers, MA, USA). The blocking buffer was created using 1% Bovine albumin, 0.1% Tween 20,
and 0.1% Triton-X 100 (Millipore Sigma/Sigma-Aldrich, Burlington, MA, USA) diluted in
phosphate-buffered saline (PBS). Visualization was conducted using a Leica DMi8 inverted
microscope purchased from Leica Microsystems (Wetzlar, Germany). Surgical tools, histo-
logical processing equipment/materials (water bath, slides, cover slides, and chemicals),
Microorganisms 2024,12, 2369 4 of 15
and other miscellaneous supplies were provided by the Bone and Biofilm Research Lab
(Salt Lake City, UT, USA).
2.2. Skin Sampling Methodology and Sample Collection
Seven female farm pigs (Yorkshire/Landrace hybrid) from Premier BioSource (San
Diego County, CA, USA) were euthanized as part of a separate study with approval and
oversight of the Institutional Animal Care and Use Committee (IACUC) at the University
of Utah (Protocol Number 21-09023, approved 29 September 2021). Necropsy began within
approximately 30 min of euthanasia administration. Hair was clipped along the back of
each pig. Each pig was marked according to the diagram in Figure 1with five locations
(zones) down the back. Wiping was performed on the skin’s surface at least three times
with sterile water and gauze to remove visible debris. One side of the pig received an
alternating alcohol-CHG surgical scrub, leaving the opposite side as a baseline control. PSP
was applied using the current clinical standard of care and according to the manufacturer’s
instructions; the CHG solution was applied on the specified sites in concentric circles
alternating with 70% isopropyl alcohol (each 3
×
) and allowed to dry for 5 min. The cup
scrub and tissue blend methods were then performed on each side of the pig.
Microorganisms 2024, 12, x FOR PEER REVIEW 5 of 16
Figure 1. Images of the sample collection methodology. (A) The pig’s back was bisected along the
sagial axis. The left side of the back was treated with a CHG PSP. The right side of the back was
cleansed with sterile water to serve as the control (native flora). Boxes (4 × 4 cm) were drawn with a
sterile skin marker. Medial boxes defined the location for the cup scrub method and lateral boxes
defined the area to be excised for the tissue blend method. (B) Pictured is a cylinder employed in
the cup scrub method and secured to the skin with pressure. (C) D/E broth (5 mL) was pipeed into
the cup. (D) The skin was agitated with a rubber policeman for 60 s. Endogenous flora was dislodged
and suspended in the D/E broth during agitation. Two mL of the D/E broth were removed via a
pipee for quantification. (E) Tissue in the lateral boxes was excised using a sterile scalpel and for-
ceps. Excised tissue was transferred to a sterile 50 mL conical tube. (F) Excised skin in D/E neutral-
izing broth ready for homogenization via blending.
2.3. Sample Processing and Bioburden Quantification
Personal blender cups and blades were cold sterilized using a similar process to pre-
vious work [48]. We exposed all internal parts of each blender cup and base to 200-proof
ethanol for >6 h. Blenders were run with ethanol for at least 15 s. Between use and before
the subsequent sterilization, the blender cups and blades were boiled for approximately 1
min. Before use, the ethanol was discarded, and each blender cup was filled with sterilized
deionized water. The water was blended for 15 s to remove/dilute residual ethanol.
Each full-thickness tissue sample and suspending D/E liquid was transferred to a
sterile blender cup and blended for approximately 5 min. Blending was performed in in-
tervals to avoid heating the sample solution. Blender cups were manually agitated up and
down to ensure thorough homogenization. Tissue blend samples were transferred back
into their respective conical tubes. Each tube was vortexed for 1 min and sonicated for 10
min.
Cup scrub samples were also vortexed for 1 min and sonicated for 10 min. Two hun-
dred µL of each sample mixture were plated and spread with a sterile loop on Columbia
blood agar to make a 0-dilution plate. Bioburden was quantified using a 10-fold serial
dilution series: 100 µL of the sample mixture was serially diluted in 900 µL of PBS carried
through to 10−4. Aliquots of each dilution (5 × 10 µL) were plated on Columbia blood agar.
The sample weight (if applicable), D/E volume, CFU, and surface area were used to quan-
tify the CFU/cm2 of each sample (except in the case of pig 1 where the sample weights
were estimated). The plates were incubated under aerobic conditions for 48 ± 4 h at 37 °C
in a jacketed incubator. Colony counts were used to represent the bioburden in units of
CFU/cm2.
Figure 1. Images of the sample collection methodology. (A) The pig’s back was bisected along the
sagittal axis. The left side of the back was treated with a CHG PSP. The right side of the back was
cleansed with sterile water to serve as the control (native flora). Boxes (4
×
4 cm) were drawn with a
sterile skin marker. Medial boxes defined the location for the cup scrub method and lateral boxes
defined the area to be excised for the tissue blend method. (B) Pictured is a cylinder employed in the
cup scrub method and secured to the skin with pressure. (C) D/E broth (5 mL) was pipetted into the
cup. (D) The skin was agitated with a rubber policeman for 60 s. Endogenous flora was dislodged
and suspended in the D/E broth during agitation. Two mL of the D/E broth were removed via a
pipette for quantification. (E) Tissue in the lateral boxes was excised using a sterile scalpel and forceps.
Excised tissue was transferred to a sterile 50 mL conical tube. (F) Excised skin in D/E neutralizing
broth ready for homogenization via blending.
The cup scrub method was performed using sterile, cylindrical cups (1” inner diameter)
placed on the skin. With the cup held securely in place, 5 mL of sterile D/E broth was
added to the cylinder with a pipette, 1 mL at a time to minimize splashing. A rubber
policeman was used to rub the skin within the cup for approximately 1 min. Strokes across
the skin were made randomly. The liquid was mixed, and 1–2 mL of the D/E broth was
removed with the pipet and placed in a sterile tube. The tube was capped and put in a
chilled cooler. Any remaining broth was discarded, and the cup was removed.
Microorganisms 2024,12, 2369 5 of 15
Full-thickness tissue samples were collected as previously described [
48
]. In short, the
skin samples were harvested using the aseptic technique: at least 1 cm apart and at least
5 cm away from the spine (Figure 1). The full-thickness samples were placed individually
into conical tubes (50 mL) filled with 35 mL of D/E broth to neutralize the CHG (Figure 1).
The samples were placed and transported in a chilled cooler to mitigate bacterial replication.
Half of the samples were treated with a CHG PSP, and the other half were treated with
sterile water (no antiseptic) to detect native pig flora (control). In total, 35 CHG-treated cup
scrub samples, 35 CHG-treated tissue blend samples, 35 control cup scrub samples, and
35 control tissue blend samples were collected (5 samples per treatment group for each
pig
×
7 total pigs). The samples were harvested using the aseptic technique. Instruments
were sterilized on-site using a glass bead sterilizer between locations within the same test
group and between groups.
2.3. Sample Processing and Bioburden Quantification
Personal blender cups and blades were cold sterilized using a similar process to
previous work [
48
]. We exposed all internal parts of each blender cup and base to 200-proof
ethanol for >6 h. Blenders were run with ethanol for at least 15 s. Between use and before
the subsequent sterilization, the blender cups and blades were boiled for approximately
1 min. Before use, the ethanol was discarded, and each blender cup was filled with sterilized
deionized water. The water was blended for 15 s to remove/dilute residual ethanol.
Each full-thickness tissue sample and suspending D/E liquid was transferred to a
sterile blender cup and blended for approximately 5 min. Blending was performed in
intervals to avoid heating the sample solution. Blender cups were manually agitated up
and down to ensure thorough homogenization. Tissue blend samples were transferred
back into their respective conical tubes. Each tube was vortexed for 1 min and sonicated
for 10 min.
Cup scrub samples were also vortexed for 1 min and sonicated for 10 min. Two hun-
dred
µ
L of each sample mixture were plated and spread with a sterile loop on Columbia blood
agar to make a 0-dilution plate. Bioburden was quantified using a 10-fold serial dilution series:
100
µ
L of the sample mixture was serially diluted in 900
µ
L of PBS carried through to 10
−4
.
Aliquots of each dilution (5
×
10
µ
L) were plated on Columbia blood agar. The sample weight
(if applicable), D/E volume, CFU, and surface area were used to quantify the CFU/cm
2
of
each sample (except in the case of pig 1 where the sample weights were estimated). The plates
were incubated under aerobic conditions for 48
±
4 h at 37
◦
C in a jacketed incubator. Colony
counts were used to represent the bioburden in units of CFU/cm2.
2.4. Process Control Samples
The tissue blend method was also performed without pig skin to verify that each
processing step was void of contamination. The blenders were sterilized according to the
same procedure outlined above. The D/E broth was blended, transferred to conical tubes,
vortexed, and sonicated, as previously described. CFU counts, if any, were determined in
the same way as the tissue samples.
2.5. Isolate Characterization
Bacterial colonies from each sample were classified according to morphology and
color. Representative colonies of each distinct morphology from each of the four study
groups (CHG tissue blend, CHG cup scrub, control tissue blend, and control cup scrub)
were isolated with sterile loops, streaked onto TSB agar, and incubated for approximately
48 h at 37 ◦C. Each bacterial isolate was cataloged and cryopreserved (−80 ◦C).
For further identification and Gram staining, bacterial isolates were cultured on TSB
agar and incubated at 37
◦
C for 24–48 h. Each selected isolate was characterized by Gram
staining and light microscopy. Additionally, we used the catalase test to categorize the
isolates further. All organisms were cross-compared using Gram staining, organism shape,
Microorganisms 2024,12, 2369 6 of 15
colony morphology, colony color, catalase classification, and previously identified isolates
using genotypic identification [48].
2.6. Location Analysis
We divided the pig back into zones from the neck to the rump. These zones were
drawn on the pig back (visible in Figure 1, vertical numbering in panel A). We reorganized
the bioburden (CFU/cm
2
) from each treatment group into 5 location zones: zone 1 (closest
to the neck), zone 2 (below zone 1), zone 3 (middle zone), zone 4 (below zone 3), and zone
5 (closest to the rump). We observed the data trend across zones 1–5.
2.7. Histology
Two skin samples from the same pig were fixed in 4% paraformaldehyde for approxi-
mately 20–36 h. The first sample was collected from an untreated control area below zone 5.
The second skin sample came from a CHG-treated area above zone 1. We dehydrated both
samples in increasing ethanol concentrations for a minimum of 2 h in each concentration
(70%, 95%, and 100%) and embedded each sample in paraffin wax using a Tissue-Tek
6 AI vacuum infiltration processor (Sakura Finetek USA, Inc., Torrance, CA, USA). The
samples were cut to approximately 5
µ
m sections on a Leica HistoCore Autocut R from
Leica Biosystems Nussloch GmbH (Nussloch, Germany). Once sectioned, each sample
was mounted to a glass slide. Each slide was washed in 100% xylene for 2
×
10 min to
deparaffinize and rehydrated in decreasing concentrations (95%, 70%) of ethanol. We rinsed
each sample with deionized water and let it sit in PBS for 10 min. In a similar way, positive
and negative control slides were also prepared using S. epidermidis ATCC 35989 grown on a
collagen plug.
We incubated the slides in a blocking solution consisting of 1% Bovine Serum albumin,
0.1% Tween-20, and 0.1% Triton-X 100 diluted in PBS for 1 h. We then incubated the slides
with a 1:100 dilution of an S. epidermidis Monoclonal Primary Antibody in PBS at room
temperature for 1 h. Previous work investigating microbial species in farm pigs showed
S. epidermidis to be a primary skin colonizer [
48
]. The S. epidermidis Monoclonal Primary
Antibody was chosen based on the relative distribution of S. epidermidis on pig skin [
48
].
Samples were washed in PBS. We performed a secondary incubation with a 1:500 dilution of
the Goat anti-Mouse Alexa Fluor Plus 488 Secondary Antibody in PBS at room temperature
for 1 h. This incubation was performed in the dark to prevent photobleaching. We washed
the incubated samples in PBS 3x and cover-slipped each slide using Prolong Gold Antifade
to preserve the immunofluorescent (IF) stain. Slides were visualized using light microscopy
We employed a green light filter and overlayed the fluorescent image with real light images.
2.8. Statistical Analysis
Multiple locations on each pig’s back were used for each sampling technique, which
introduced data clustering. Therefore, we analyzed the data using a mixed effects linear
regression (a multilevel model), accounting for locations nested along the pig’s back [
48
]. In
our model, the experimental condition was a fixed effect, and the pig was a random effect.
Given the small sample size, we specified the model to use a significance test based on a
t statistic, rather than the default z statistic, and fitted using Stata-17 statistical software
(StataCorp LLC, College Stata, TX, USA). The following comparisons were performed:
control tissue blend vs. CHG tissue blend, control cup scrub vs. CHG cup scrub, control
tissue blend vs. control cup scrub, and CHG tissue blend vs. CHG cup scrub.
3. Results
3.1. Microbial Quantification Outcomes
The tissue blend method captured more bioburden per cm
2
of porcine skin than the
cup scrub method (Figure 2). While CFU counts varied across the data, clear differences
were observed between the treatment groups. Both CHG-treated and control samples
showed statistically significant differences between the cup scrub and tissue blend methods
Microorganisms 2024,12, 2369 7 of 15
of sampling. (Figure 2). Process control data indicated that spurious contamination
was negligible.
Microorganisms 2024, 12, x FOR PEER REVIEW 8 of 16
Figure 2. The bioburden (Log10 CFU/cm2) determined in 7 pigs and differentiated by treatment
group and processing method. The blue bars represent CHG treatment (the left side of the pig’s
back). The gray bars represent the control samples (no antiseptic; right side of the pig’s back). The
process control bar represents all steps of the tissue blend method without skin present. The gray
circles represent individual data points from each sample. The error bars show the standard error
for the indicated treatment group (n = 5 per pig).
Figure 2. The bioburden (Log
10
CFU/cm
2
) determined in 7 pigs and differentiated by treatment
group and processing method. The blue bars represent CHG treatment (the left side of the pig’s back).
The gray bars represent the control samples (no antiseptic; right side of the pig’s back). The process
control bar represents all steps of the tissue blend method without skin present. The gray circles
represent individual data points from each sample. The error bars show the standard error for the
indicated treatment group (n = 5 per pig).
The difference between the cup scrub and tissue blend methods was especially pro-
nounced following CHG PSP from the left side of the pig’s back (see Figure 2, blue bars).
Specifically, the cup scrub method yielded 1.05
±
0.24 log
10
CFU/cm
2
, while the tissue blend
method resulted in 3.24
±
0.24 log
10
CFU/cm
2
, more than a 2 log
10
difference (p< 0.001).
A bioburden count of 0 signified that the total CFU/cm
2
was below detectable levels. This
phenomenon was observed following the application of a CHG PSP in 5/7 pigs using the cup
scrub method (Figure 3). In contrast, a result of 0 (below detectable levels) was observed in
only 1/7 pigs following CHG PSP using the tissue blend method (Figure 3). Without exception,
the tissue blend method detected more bacteria than the cup scrub method following a PSP in
every pig analyzed (Figure 3).
Microorganisms 2024,12, 2369 8 of 15
Microorganisms 2024, 12, x FOR PEER REVIEW 9 of 16
Figure 3. The outcomes of each individual pig indicating bioburden (Log
10
CFU/cm
2
) were ploed
as a function of treatment group, sampling methodology, and animal. The error bars represent the
standard deviation of the data set per treatment. Each gray circle indicates a single sample.
3.2. Representative Isolate Characterization
More microbial variety was observed in the control samples of both methods than
in the CHG PSP-treated samples (Figure 4). A greater quantity of unique isolates was
found in native pig skin cleansed with water (controls), with 30.1% and 27.9% of all iso-
lates present following the cup scrub and tissue blend methods, respectively. A total of
24.8% of the isolates came from the CHG PSP-treated samples from the tissue blend
method and 17.3% came from the CHG cup scrub group. Gram stain data showed that
most species cultured were Gram-positive cocci (82.7%). Some species were Gram-posi-
tive bacilli (12.8%). Other microorganisms did not grow after cryopreservation, did not
stain, or did not fit into any of the other categories (i.e., coccobacillus). One isolate was
identified as Gram-negative (0.4%). Most isolates were catalase-positive (94.7%).
Figure 4. A breakdown of representative bacterial isolates cultured from each of the various treat-
ment groups (n = 226). (A) The quantity of isolates cultured from each group (%). (B) A breakdown
of the isolates identified by Gram staining. (C) The distribution of isolates that were catalase positive
or negative.
Figure 3. The outcomes of each individual pig indicating bioburden (Log
10
CFU/cm
2
) were plotted
as a function of treatment group, sampling methodology, and animal. The error bars represent the
standard deviation of the data set per treatment. Each gray circle indicates a single sample.
Though not as pronounced, differences between the cup scrub and tissue blend methods
were also observed in the control groups. Using the cup scrub method, the average bioburden
per cm
2
of control skin samples was 2.62
±
0.21 log
10
CFU (Figure 2, gray bars). Using the tissue
blend method, the bioburden of control pig flora was 3.46
±
0.24 log
10
CFU/cm
2
(Figure 2,
gray bars). This difference was 0.84
±
0.45 log
10
CFU/cm
2
and this was statistically significant
(p< 0.001). As with the PSP-prepared side, the tissue blend method detected more bioburden
per cm
2
in every pig participating in this study, although this difference was nearly comparable
in pig 4 (Figure 3). Neither method in the control group produced a sample with a bioburden
below detectable limits (Figure 3).
Within methodology types, statistical equivalence was observed between the CHG-
prepared skin and the control skin processed using the tissue blend method, but not the
cup scrub method. Looking across each animal, there was no observable trend between
the CHG PSP samples and the control tissue blend samples (Figure 3). The comparison
between these groups was not statistically significant (Figure 2;p= 0.321). In contrast, the
difference in bioburden between the CHG and control cup scrub samples was statistically
significant (Figure 2;p< 0.001). Across the animals, the difference between these groups
ranged from about 1 log10 CFU/cm2to more than 2 log10 CFU/cm2.
3.2. Representative Isolate Characterization
More microbial variety was observed in the control samples of both methods than in
the CHG PSP-treated samples (Figure 4). A greater quantity of unique isolates was found in
native pig skin cleansed with water (controls), with 30.1% and 27.9% of all isolates present
following the cup scrub and tissue blend methods, respectively. A total of 24.8% of the
isolates came from the CHG PSP-treated samples from the tissue blend method and 17.3%
came from the CHG cup scrub group. Gram stain data showed that most species cultured
were Gram-positive cocci (82.7%). Some species were Gram-positive bacilli (12.8%). Other
microorganisms did not grow after cryopreservation, did not stain, or did not fit into any
of the other categories (i.e., coccobacillus). One isolate was identified as Gram-negative
(0.4%). Most isolates were catalase-positive (94.7%).
Microorganisms 2024,12, 2369 9 of 15
Microorganisms 2024, 12, x FOR PEER REVIEW 9 of 16
Figure 3. The outcomes of each individual pig indicating bioburden (Log
10
CFU/cm
2
) were ploed
as a function of treatment group, sampling methodology, and animal. The error bars represent the
standard deviation of the data set per treatment. Each gray circle indicates a single sample.
3.2. Representative Isolate Characterization
More microbial variety was observed in the control samples of both methods than
in the CHG PSP-treated samples (Figure 4). A greater quantity of unique isolates was
found in native pig skin cleansed with water (controls), with 30.1% and 27.9% of all iso-
lates present following the cup scrub and tissue blend methods, respectively. A total of
24.8% of the isolates came from the CHG PSP-treated samples from the tissue blend
method and 17.3% came from the CHG cup scrub group. Gram stain data showed that
most species cultured were Gram-positive cocci (82.7%). Some species were Gram-posi-
tive bacilli (12.8%). Other microorganisms did not grow after cryopreservation, did not
stain, or did not fit into any of the other categories (i.e., coccobacillus). One isolate was
identified as Gram-negative (0.4%). Most isolates were catalase-positive (94.7%).
Figure 4. A breakdown of representative bacterial isolates cultured from each of the various treat-
ment groups (n = 226). (A) The quantity of isolates cultured from each group (%). (B) A breakdown
of the isolates identified by Gram staining. (C) The distribution of isolates that were catalase positive
or negative.
Figure 4. A breakdown of representative bacterial isolates cultured from each of the various treatment
groups (n= 226). (A) The quantity of isolates cultured from each group (%). (B) A breakdown of
the isolates identified by Gram staining. (C) The distribution of isolates that were catalase positive
or negative. Data rounded to the nearest tenth.
3.3. Anatomical Analysis
We observed a mild correlation across the anatomical zones assigned vertically along the
pig’s back. While large differences in CFU counts were not observed between each anatomical
zone, there was a general “U” shape in the bar graphs (Figure 5). More specifically, there were
slightly more CFU counts per cm
2
of skin in zones 1 and 5 compared to zone 3 (Figure 5).
The values in zones 2 and 4 generally fell in between the end zones (zones 1 and 5) and the
middle zone (zone 3). This pattern was more obvious in the bioburden levels of the tissue
blend samples. The data within each zone mirrored the results shown in Figure 2.
Microorganisms 2024, 12, x FOR PEER REVIEW 10 of 16
3.3. Anatomical Analysis
We observed a mild correlation across the anatomical zones assigned vertically along
the pig’s back. While large differences in CFU counts were not observed between each
anatomical zone, there was a general “U” shape in the bar graphs (Figure 5). More specif-
ically, there were slightly more CFU counts per cm
2
of skin in zones 1 and 5 compared to
zone 3 (Figure 5). The values in zones 2 and 4 generally fell in between the end zones
(zones 1 and 5) and the middle zone (zone 3). This paern was more obvious in the bio-
burden levels of the tissue blend samples. The data within each zone mirrored the results
shown in Figure 2.
Figure 5. (A) The zones along the pig’s back were defined from superior to inferior in numerical
order 1–5. (B) Bioburden was categorized by the treatment and sampling methods as a function of
the anatomical location (zone).
3.4. Histological Analysis
Histological analyses corroborated the microbiological outcomes. For example, IF
images of CHG-treated and untreated pig skin showed the presence of bacteria (S. epider-
midis) in superficial and deep skin regions (Figure 6). Bacteria were observable with and
without the application of CHG PSP, especially along the hair follicle tract. More bacteria
were observed near the surface of the untreated pig skin than on the CHG-treated skin.
We observed pockets of bacteria in the dermis of both samples.
Positive and negative controls were also performed (see Supplementary Material).
An IF signal was observed on the positive control sample with S. epidermidis ATCC 35989
grown on a collagen plug. No signal was observed on the negative control incubated with-
out the primary antibody.
Figure 5. (A) The zones along the pig’s back were defined from superior to inferior in numerical
order 1–5. (B) Bioburden was categorized by the treatment and sampling methods as a function of
the anatomical location (zone).
3.4. Histological Analysis
Histological analyses corroborated the microbiological outcomes. For example, IF images
of CHG-treated and untreated pig skin showed the presence of bacteria (S. epidermidis) in
superficial and deep skin regions (Figure 6). Bacteria were observable with and without the
application of CHG PSP, especially along the hair follicle tract. More bacteria were observed
Microorganisms 2024,12, 2369 10 of 15
near the surface of the untreated pig skin than on the CHG-treated skin. We observed pockets
of bacteria in the dermis of both samples.
Microorganisms 2024, 12, x FOR PEER REVIEW 11 of 16
Figure 6. Cross-sections of porcine skin stained with IF for S. epidermidis. Fluorescent green indicates
the presence of bacteria. (A) The control, unwiped pig skin section collected from below zone 5
showed bacterial presence throughout the epidermal and dermal features. (B) CHG-treated pig skin
collected from above zone 1.
4. Discussion
The purpose of this study was to compare the bacterial bioburden in pig skin between
the cup scrub and tissue blend [48] methods following PSP. Our hypothesis that the cup
scrub method would not resolve bacteria in all skin layers was supported. Samples quan-
tified using the tissue blend method consistently cultured more bacteria from all skin re-
gions compared to the cup scrub method. IF images corroborated the tissue blend method
data, showing that S. epidermidis was present in deep skin regions with and without PSP
treatment. These outcomes underpinned the potential benefit of using a destructive skin
sampling method to test new PSP technologies.
The bacterial sampling methodology directly influenced bioburden levels after PSP
application. Using only the cup scrub method applied to pigs, CHG PSP approached the
FDA-required 2 log
10
reduction for PSPs [36] for dry areas with a log
10
reduction of 1.57 ±
0.45 log
10
CFU/cm
2
. Compared to the control cup scrub data, this bacterial reduction was
statistically significant (p < 0.001). In contrast, the CHG PSP kit did not meet the FDA-
required reduction when the tissue blend method was used with a log
10
reduction of 0.22
± 0.48 log
10
CFU/cm
2
. Using the tissue blend method, the PSP-treated skin was statistically
insignificant compared to the control skin wiped with sterile water (p = 0.231). This result
suggested that when bacteria from all layers of the skin were considered, CHG as a biocide
was statistically equivalent to a sterile water presurgical scrub without chemical disinfect-
ants. While CHG appears to exhibit promising outcomes as a PSP using the cup scrub
method, the tissue blend method leaves questions as to its true efficacy below the surface.
While considerable variation was observed between and within animals, overall
trends in the data between methods were reflected in the individual results of all seven
animals. Across the seven pigs included in this study, the CHG-treated cup scrub samples
consistently exhibited the lowest bioburden, even when the CHG-treated tissue blend
samples exhibited bioburdens of several log
10
CFU/cm
2
higher. Further highlighting the
limitations of the cup scrub method, 5/7 pigs had at least one sample where the bioburden
was below detectable limits when the cup scrub method was used following CHG appli-
cation. This was 5× the frequency of the CHG-treated tissue blend group. The bioburden
results from each animal individually highlighted the difference between the cup scrub
and tissue blend methods.
This discrepancy between the cup scrub and tissue blend methods following PSP was
due to the depth of skin each method considered. PSP chemicals were applied to the skin’s
surface where microbes were rapidly killed upon application. The cup scrub method prin-
cipally detected surface-dwelling bacteria impacted by PSP. In contrast, the tissue blend
Figure 6. Cross-sections of porcine skin stained with IF for S. epidermidis. Fluorescent green indicates
the presence of bacteria. (A) The control, unwiped pig skin section collected from below zone
5 showed bacterial presence throughout the epidermal and dermal features. (B) CHG-treated pig
skin collected from above zone 1.
Positive and negative controls were also performed (see Supplementary Material).
An IF signal was observed on the positive control sample with S. epidermidis ATCC 35989
grown on a collagen plug. No signal was observed on the negative control incubated
without the primary antibody.
4. Discussion
The purpose of this study was to compare the bacterial bioburden in pig skin be-
tween the cup scrub and tissue blend [
48
] methods following PSP. Our hypothesis that the
cup scrub method would not resolve bacteria in all skin layers was supported. Samples
quantified using the tissue blend method consistently cultured more bacteria from all skin
regions compared to the cup scrub method. IF images corroborated the tissue blend method
data, showing that S. epidermidis was present in deep skin regions with and without PSP
treatment. These outcomes underpinned the potential benefit of using a destructive skin
sampling method to test new PSP technologies.
The bacterial sampling methodology directly influenced bioburden levels after PSP applica-
tion. Using only the cup scrub method applied to pigs, CHG PSP approached the FDA-required
2 log
10
reduction for PSPs [
36
] for dry areas with a log
10
reduction of 1.57
±
0.45 log
10
CFU/cm
2
.
Compared to the control cup scrub data, this bacterial reduction was statistically significant
(p< 0.001). In contrast, the CHG PSP kit did not meet the FDA-required reduction when the
tissue blend method was used with a log
10
reduction of 0.22
±
0.48 log
10
CFU/cm
2
. Using the
tissue blend method, the PSP-treated skin was statistically insignificant compared to the control
skin wiped with sterile water (p= 0.231). This result suggested that when bacteria from all layers
of the skin were considered, CHG as a biocide was statistically equivalent to a sterile water
presurgical scrub without chemical disinfectants. While CHG appears to exhibit promising
outcomes as a PSP using the cup scrub method, the tissue blend method leaves questions as to
its true efficacy below the surface.
While considerable variation was observed between and within animals, overall
trends in the data between methods were reflected in the individual results of all seven
animals. Across the seven pigs included in this study, the CHG-treated cup scrub samples
consistently exhibited the lowest bioburden, even when the CHG-treated tissue blend
samples exhibited bioburdens of several log
10
CFU/cm
2
higher. Further highlighting
the limitations of the cup scrub method, 5/7 pigs had at least one sample where the
bioburden was below detectable limits when the cup scrub method was used following
Microorganisms 2024,12, 2369 11 of 15
CHG application. This was 5
×
the frequency of the CHG-treated tissue blend group. The
bioburden results from each animal individually highlighted the difference between the
cup scrub and tissue blend methods.
This discrepancy between the cup scrub and tissue blend methods following PSP
was due to the depth of skin each method considered. PSP chemicals were applied to the
skin’s surface where microbes were rapidly killed upon application. The cup scrub method
principally detected surface-dwelling bacteria impacted by PSP. In contrast, the tissue blend
samples captured up to 1–2 cm of skin and subcutaneous fat (full-thickness tissue). Thus,
the tissue blend method detected bacteria from all layers of the skin, including pockets
found along dermal features such as hair follicles and glands. Additionally, cylinders used
in the cup scrub method produced a sampling area of approximately 5 cm
2
, whereas each
tissue blend sample encompassed approximately 16 cm
2
. This larger surface area led to the
inclusion of a greater number of dermal features, thus resulting in a more accurate average.
Across greater depths and larger surface areas, the tissue blend processes accounted for
bacteria in more locations than the cup scrub method.
Based on these data, we estimated the effectiveness of the tissue blend method and
proposed a relative bacterial distribution. Operating under the assumption that most
bacteria on the surface of pig skin were eradicated using CHG PSP, the hundreds to
millions of CFUs detected after applying the tissue blend method came from below the
surface. These bacteria were not resolved using the cup scrub method. Specifically, there
was more than a 2 log
10
difference between the bioburden of the CHG-prepared cup scrub
and tissue blend samples. This result indicated that the tissue blend method applied in
domestic pigs may be over 100 times more sensitive to bacteria following the application of
CHG PSP than the cup scrub method. The data suggested that more than half of porcine
flora live under the surface of the skin. This conclusion in pigs was similar to previous work
investigating the scraping and swabbing methods and destructive methods in humans [
22
].
Histological analysis of porcine tissue supported the microbiological outcomes, in-
cluding bacterial distribution throughout skin layers. S. epidermidis was selected as the
representative species as it is a common skin commensal in both pigs and humans [
48
,
49
].
This decision was informed using previous genetic sequencing information from samples
collected using the tissue blend model in pigs [
48
]. Generally, the IF signal was localized at
the surface of the skin and along the hair follicle (base and shaft). While the signal intensity
varied from panel A to panel B, this was likely due to microbiota differences between the
sample collection sites more than any impact of PSP application. The sample from panel
A was from a posterior region below zone 5, with frequent proximity to fecal matter. The
sample from panel B was taken above zone 1 from a CHG-prepared region close to the neck
of the pig. In this study, we specifically tagged S. epidermidis even though it is just one of
many natural colonizers of pig skin. Previously, we found S. epidermidis to be a primary
colonizer of pig skin [
48
]. It is likely that there were various other primary colonizers of
the area close to the pig neck (panel B) that the S. epidermidis IF protocol did not detect.
Notwithstanding differences in location, an IF signal from S. epidermidis was observed
along hair follicle shafts and in the subcutaneous tissue on both samples, suggesting the
need for models such as the tissue blend method.
Anatomical location was a major consideration in this study. We designed the collec-
tion of each sample type along vertical columns down the pig’s back. When we analyzed
the bioburden quantity from zone 1 to zone 5, we detected a higher quantity of bacteria at
the peripheries of the dorsum, at the neck and rump, compared to the center of the back,
indicating some anatomical variability in pig-back bioburden. Notwithstanding differences
across zones, the same trends between the cup scrub method and the tissue blend method
were observed.
While data from this study showed that the cup scrub method has limitations, it
may be useful in some cases. The cup scrub method was previously used successfully in
many investigations spanning clinical dermatology and cosmetic research in addition to
antisepsis [
39
–
44
]. This non-destructive technique is important for researchers in a clinical
Microorganisms 2024,12, 2369 12 of 15
environment when biopsies and preclinical models are impossible to incorporate; 16 cm
2
wounds are not appropriate for patient volunteers. Thus, the path of this methodology
from research to an ASTM standard to a regulatory document outlining PSP approval is
understandable. However, by endorsing the standard and encouraging its use for PSPs, the
FDA has incorporated a significant oversight into its approval process. Unfortunately, this
has led the scientific community to believe that this standard accurately represents skin
flora. Data from this study showed that this is not true; our results showed that a diverse
and significant quantity of the natural flora was found out of reach of this skin sampling
method. While the cup scrub method may be an acceptable method, its limitations must be
recognized when developing new PSP methodologies.
The institutionalization of the cup scrub method may be hampering the development
and innovation of PSP products and techniques. We previously called out this issue as a
“blind spot” in PSP testing [
48
]. The lack of consideration given to deep-dwelling bacteria
is concerning because the bacteria that evade PSP application are the colonizers of surgical
wounds and thus the culprits of SSIs. We conclude that CHG as an antiseptic does not
diffuse adequately within the time allotted in the operating room (<5 min) and may need
future consideration. The early phase development of the next generation of PSPs might
be aided by pivoting away from clinical testing towards a variation of our porcine model
using full-thickness skin sampling.
This study was not without limitations. By necessity, we collected samples after
euthanasia. Cup scrub and tissue blend sample processing was initiated as soon as all
specimens were collected and transported to the lab for analysis (a 7 min walk). We
minimized bacterial replication by beginning analysis immediately after sample collection
and storing the samples in a chilled cooler to prevent log phase growth. If bacterial
replication had occurred, it likely would have been reflected equally across all samples
and would not affect the overall study outcomes. Additionally, the representative isolates
collected from the samples were not meant to be comprehensive, only representative of the
types of microorganisms seen. The genetic identification of pig skin species was performed
previously and not repeated for this investigation [48].
We acknowledge that this protocol was created in alignment with ASTM standard
E1173-15 but was modified in specific ways due to the nature of our animal investiga-
tion [
36
]. First, ASTM E1173-15 is a human standard that we adapted for a porcine model.
This standard specifies that threshold testing should be performed before the cup scrub
method is used. We did not perform this screening as we could not exclude any of our
animals based on the initial bioburden. Additionally, a D/E neutralizing broth was used as
the suspension solution to neutralize the CHG upon skin contact instead of the solution
proposed in the ASTM standard to avoid false negative cultures. Finally, the standard
recommends clipping 48 h before testing. Clipping was performed immediately before the
collection process to avoid unnecessary animal anesthesia. We removed all hair and debris
during the water step. This choice was made specifically to mirror how human skin is
prepared before surgery. We are currently performing a clinical study using discarded skin
from reconstruction surgery to test the clinical translatability of the data collected in pigs.
The tissue blend method applied in pigs was useful in detecting bacteria in all skin
regions following PSP. We showed that discrepancies in assumed PSP effectiveness were
due to variations in sampling techniques. The data uncovered limitations of the ASTM
standard E1173-15 for testing PSP products and emphasized the need for and benefit of
an animal model for future PSP development. The results of this study were consistent
with our previous work on the tissue blend method [
48
]; previous animal investigations
and the data from this study exhibited considerable overlap. Additionally, IF imaging
supported our microbial quantification data. As the tissue blend method applied in a
porcine model continues to be used and developed, we encourage researchers and PSP
manufacturers to incorporate full-thickness sampling as part of their testing strategy. The
tissue blend method is a robust methodology that may be used to create next-generation
PSP technologies.
Microorganisms 2024,12, 2369 13 of 15
5. Conclusions
This study suggested that the cup scrub method, a clinical standard for antiseptic
approval based on ASTM standard E1173-15, may be underreporting the quantity of
bacteria surviving PSP, leading surgical staff to believe PSP-treated skin is “sterile”, when,
in fact, it is not. When developing new PSP technologies, the tissue blend method applied
in pigs may better resolve true biocidal efficacy in deeper skin regions.
Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/microorganisms12112369/s1. Figure S1. Cross-sections of Staphylococcus epi-
dermidis grown on a collagen plug were stained as positive and negative process controls. (A) Underwent
the normal IF treatment protocol including incubation with a S. epidermidis monoclonal antibody (primary)
followed by a Goat Anti-Mouse IgG Alexa Fluor 488 polyclonal antibody (secondary). Fluorescence was
observed. (B) Underwent a modified IF treatment including incubation with the Goat Anti-Mouse IgG
Alexa Fluor 488 Polyclonal secondary antibody only. No fluorescence was observed.
Author Contributions: Conceptualization, D.L.W., N.N.A. and H.R.D.; methodology, H.R.D., N.N.A.
and D.L.W.; validation, H.R.D. and N.N.A.; formal analysis, H.R.D., N.N.A. and D.L.W.; investi-
gation, H.R.D., N.H., A.B., P.S. and N.N.A.; resources, D.L.W. and N.N.A.; data curation, H.R.D.;
writing—original draft preparation, H.R.D.; writing—review and editing, D.L.W., N.N.A., H.R.D.,
A.B., N.H. and P.S.; visualization, H.R.D.; supervision, N.N.A.; project administration, H.R.D.; fund-
ing acquisition, N.N.A. All authors have read and agreed to the published version of the manuscript.
Funding: This study was funded, in part, by the generosity of a departmental grant through the L.S.
Peery Foundation. Additional support was provided by the National Science Foundation Graduate
Research Fellowship Program Grant Number 2139322. Any opinions, findings, conclusions, or
recommendations expressed in this material are those of the authors and do not necessarily reflect
the views of the National Science Foundation.
Data Availability Statement: The original contributions presented in the study are included in the
article, further inquiries can be directed to the corresponding authors.
Acknowledgments: The authors thank Marissa Badham, Li Peniata, Brooke Kawaguchi, Jonelly Shoaf,
the University of Utah Comparative Medicine Center staff, and other Bone and Biofilm Research Lab
members for their technical support. The authors also thank Gregory J. Stoddard of the University of
Utah Study Design and Biostatistics Center for providing biostatistics assistance, the Center being
funded in part by the National Center for Advancing Translational Sciences of the National Institutes
of Health under Award Number UL1TR002538. The content is solely the responsibility of the authors
and does not necessarily represent the official views of the National Institutes of Health.
Conflicts of Interest: The authors declare no conflicts of interest.
References
1.
Wenzel, R.P. Surgical site infections and the microbiome: An updated perspective. Infect. Control Hosp. Epidemiol. 2019,40, 590–596.
[CrossRef] [PubMed]
2.
Seidelman, J.L.; Baker, A.W.; Lewis, S.S.; Advani, S.D.; Smith, B.; Anderson, D.; for the Duke Infection Control Outreach Network
Surveillance Team. Surgical site infection trends in community hospitals from 2013 to 2018. Infect. Control Hosp. Epidemiol. 2023,44,
610–615. [CrossRef] [PubMed]
3.
Kirkby, M.; Sabri, A.B.; Scurr, D.; Moss, G. Microneedle-Mediated Permeation Enhancement of Chlorhexidine Digluconate:
Mechanistic Insights Through Imaging Mass Spectrometry. Pharm. Res. 2022,39, 1945–1958. [CrossRef] [PubMed]
4.
Mangram, A.J.; Horan, T.C.; Pearson, M.L.; Silver, L.C.; Jarvis, W.R. Guideline for prevention of surgical site infection, 1999.
Hospital Infection Control Practices Advisory Committee. Infect. Control Hosp. Epidemiol. 1999,20, 97–132. [CrossRef]
5.
Nablo, B.J.; Rothrock, A.R.; Schoenfisch, M.H. Nitric oxide-releasing sol–gels as antibacterial coatings for orthopedic implants.
Biomaterials 2005,26, 917–924. [CrossRef]
6.
Hetrick, E.M.; Schoenfisch, M.H. Reducing implant-related infections: Active release strategies. Chem. Soc. Rev. 2006,35, 780.
[CrossRef]
7.
Chu, N.S.; Chan-Myers, H.; Ghazanfari, N.; Antonoplos, P. Levels of naturally occurring microorganisms on surgical instruments
after clinical use and after washing. Am. J. Infect. Control 1999,27, 315–319. [CrossRef]
Microorganisms 2024,12, 2369 14 of 15
8.
Agarwal, A.; Lin, B.; Agarwal, A.G.; Elgafy, H.; Schultz, C.; Agarwal, A.K.; Goel, V.K.; Sigler, V.; Karas, C.; Gidvani, S.; et al.
A Multicenter Trial Demonstrating Presence or Absence of Bacterial Contamination at the Screw-Bone Interface Owing to Absence
or Presence of Pedicle Screw Guard, Respectively, During Spinal Fusion. Clin. Spine Surg. A Spine Publ. 2020,33, E364–E368.
[CrossRef]
9.
Achermann, Y.; Goldstein, E.J.C.; Coenye, T.; Shirtliff, M.E. Propionibacterium acnes: From Commensal to Opportunistic
Biofilm-Associated Implant Pathogen. Clin. Microbiol. Rev. 2014,27, 419–440. [CrossRef]
10.
Grice, E.A.; Kong, H.H.; Conlan, S.; Deming, C.B.; Davis, J.; Young, A.C.; NISC Comparative Sequencing Program; Bouffard, G.G.;
Blakesley, R.W.; Murray, P.R.; et al. Topographical and temporal diversity of the human skin microbiome. Science 2009,324, 1190–1192.
[CrossRef]
11.
Levy, P.Y.; Fenollar, F.; Stein, A.; Borrione, F.; Cohen, E.; Lebail, B.; Raoult, D. Propionibacterium acnes Postoperative Shoulder
Arthritis: An Emerging Clinical Entity. Clin. Infect. Dis. 2008,46, 1884–1886. [CrossRef] [PubMed]
12.
Sampedro, M.F.; Huddleston, P.M.; Piper, K.E.; Karau, M.J.; Dekutoski, M.B.; Yaszemski, M.J.; Currier, B.L.; Mandrekar, J.N.;
Osmon, D.R.; McDowell, A.; et al. A Biofilm Approach to Detect Bacteria on Removed Spinal Implants. Spine 2010,35, 1218–1224.
[CrossRef] [PubMed]
13.
Otto, M. Staphylococcus colonization of the skin and antimicrobial peptides. Expert. Rev. Dermatol. 2010,5, 183–195. [CrossRef]
[PubMed]
14.
Takagi, S.; Tobara, K. Notes on the vertical distribution of the human sweat glands. Jpn. J. Physiol. 1954,4, 169–174. [CrossRef]
[PubMed]
15.
Moriarty, T.F.; Kuehl, R.; Coenye, T.; Metsemakers, W.-J.; Morgenstern, M.; Schwarz, E.M.; Riool, M.; Zaat, S.A.; Khana, N.; Kates,
S.L.; et al. Orthopaedic device-related infection: Current and future interventions for improved prevention and treatment. EFORT
Open Rev. 2016,1, 89–99. [CrossRef]
16.
Pistone, D.; Meroni, G.; Panelli, S.; D’auria, E.; Acunzo, M.; Pasala, A.R.; Zuccotti, G.V.; Bandi, C.; Drago, L. A Journey on the Skin
Microbiome: Pitfalls and Opportunities. Int. J. Mol. Sci. 2021,22, 9846. [CrossRef]
17. Grice, E.A.; Segre, J.A. The skin microbiome. Nat. Rev. Microbiol. 2011,9, 244–253. [CrossRef]
18.
Folléa, G.; Saint-Laurent, P.; Bigey, F.; Gayet, S.; Bientz, M.; Cazenave, J.P. Evaluation bacteriologique quantitative d’une methode
de desinfection cutanee chez les donneurs de sang. Transfus. Clin. Biol. 1997,4, 523–531. [CrossRef]
19. Selwyn, S.; Ellis, H. Skin Bacteria and Skin Disinfection Reconsidered. Br. Med. J. 1972,1, 136–140. [CrossRef]
20.
Ring, H.C.; Bay, L.; Kallenbach, K.; Miller, I.M.; Prens, E.; Saunte, D.M.; Bjarnsholt, T.; Jemec, G.B. Normal skin microbiota is
altered in pre-clinical hidradenitis suppurativa. Acta Derm. Venereol. 2017,97, 208–213. [CrossRef]
21.
Acosta, E.M.; Little, K.A.; Bratton, B.P.; Lopez, J.G.; Mao, X.; Payne, A.S.; Donia, M.; Devenport, D.; Gitai, Z. Bacterial DNA on the
skin surface overrepresents the viable skin microbiome. Elife 2023,12, RP87192. [CrossRef] [PubMed]
22.
Selwyn, S. Evaluating skin disinfectants
in vivo
by excision biopsy and other methods. J. Hosp. Infect. 1985,6, 37–43. [CrossRef]
[PubMed]
23.
Nakatsuji, T.; Chiang, H.-I.; Jiang, S.B.; Nagarajan, H.; Zengler, K.; Gallo, R.L. The microbiome extends to subepidermal
compartments of normal skin. Nat. Commun. 2013,4, 1431. [CrossRef]
24.
Kalb, R.L.; Fowler, G.C. Pfenninger and Fowler’s Procedures for Primary Care. In Pfenninger & Fowler’s: Procedures for Primary
Care; Elsevier: Amsterdam, The Netherlands, 2020; pp. 244–252.
25.
Swan, H.; Gonzalez, R.I.; Harris, A.; Couslon, C.; Hopwood, M.L. Use of a quaternary ammonium compound for the surgical
scrub. Am. J. Surg. 1949,77, 24–37. [CrossRef] [PubMed]
26. Lovell, D.L. Skin bacteria; their role in contamination and infection of wounds. Arch. Surg. 1945,51, 78–80. [CrossRef]
27. Montes, L.F.; Wilborn, W.H. Location of bacterial skin flora. Br. J. Dermatol. 1969,81, 23–26. [CrossRef]
28. Dobson, G.P. Trauma of major surgery: A global problem that is not going away. Int. J. Surg. 2020,81, 47–54. [CrossRef]
29. Enzler, M.J.; Berbari, E.; Osmon, D.R. Antimicrobial prophylaxis in adults. Mayo Clin. Proc. 2011,86, 686–701. [CrossRef]
30.
Saleem, Z.; Ahsan, U.; Haseeb, A.; Altaf, U.; Batool, N.; Rani, H.; Jaffer, J.; Shahid, F.; Hussain, M.; Amir, A.; et al. Antibiotic
Utilization Patterns for Different Wound Types among Surgical Patients: Findings and Implications. Antibiotics 2023,12, 678.
[CrossRef]
31. Salkind, A.R.; Rao, K.C. Antibiotic prophylaxis to prevent surgical site infections. Am. Fam. Physician 2011,83, 585–590.
32.
Nishihara, Y.; Kajiura, T.; Katsuhiro, Y.; Kobayashi, H.; Okubo, T. A Comparative Clinical Study Focusing on the Antimicrobial
Efficacies of Chlorhexidine Gluconate Alcohol for Patient Skin Preparations. J. Infus. Nurs. 2012,35, 44–50. [CrossRef] [PubMed]
33.
Bashir, M.H.; Hollingsworth, A.; Thompson, J.D.; Shortridge, D.; Lake, S.P.; Deeken, C.R.; Zimbler, D.; Mashiatulla, M.; Li, L.;
Singh, M.P.; et al. Antimicrobial performance of two preoperative skin preparation solutions containing iodine and isopropyl
alcohol. Am. J. Infect. Control 2022,50, 792–798. [CrossRef]
34.
Allegranzi, B.; Bischoff, P.; de Jonge, S.; Kubilay, N.Z.; Zayed, B.; Gomes, S.M.; Abbas, M.; Atema, J.J.; Gans, S.; van Rijen, M.;
et al. New WHO recommendations on preoperative measures for surgical site infection prevention: An evidence-based global
perspective. Lancet Infect. Dis. 2016,16, e276–e287. [CrossRef] [PubMed]
35.
Lovell, D.L. Preoperative skin preparation with reference to surface bacteria contaminants and resident flora. Surg. Clin. N. Am.
1946,26, 1053–1059. [PubMed]
36.
ASTM E1173-15; Standard Test Method for Evaluation of Preoperative, Precatheterization, or Preinjection Skin Preparations.
ASTM: West Conshohocken, PA, USA, 2022.
Microorganisms 2024,12, 2369 15 of 15
37.
Topical Antimicrobial Drug Products for Over-the-Counter Human Use; Tentative Final Monograph for Health-Care Antiseptic Drug
Products, 59 FR 31402. 59. 1994. Available online: https://www.federalregister.gov/citation/59-FR-31402 (accessed on 14 October 2024).
38.
Williamson, P.; And, B.A.; Kligman, A.M. A new method for the quantitative investigation of cutaneous bacteria. J. Investig.
Dermatol. 1965,45, 498–503. [CrossRef] [PubMed]
39.
Hosseini, P.; Mundis, G.M.; Eastlack, R.; Nourian, A.; Pawelek, J.; Nguyen, S.; Akbarnia, B.A. Do Longer Surgical Procedures
Result in Greater Contamination of Surgeons’ Hands? Clin. Orthop. Relat. Res. 2016,474, 1707–1713. [CrossRef]
40.
Petersen, R.L.W.; Scholz, C.F.P.; Jensen, A.; Brüggemann, H.; Lomholt, H.B. Propionibacterium acnes phylogenetic type III is
associated with progressive macular hypomelanosis. Eur. J. Microbiol. Immunol. 2017,7, 37–45. [CrossRef]
41.
Ryan-Kewley, A.E.; Williams, D.R.; Hepburn, N.; Dixon, R.A. Non-antibiotic isotretinoin treatment differentially controls
Propionibacterium acnes on skin of acne patients. Front. Microbiol. 2017,8, 1381. [CrossRef]
42.
Grimshaw, S.G.; Smith, A.M.; Arnold, D.S.; Xu, E.; Hoptroff, M.; Murphy, B. The diversity and abundance of fungi and bacteria
on the healthy and dandruff affected human scalp. PLoS ONE 2019,14, e0225796. [CrossRef]
43.
Kaveeshwar, S.; Duvall, G.; Jones, D.L.; O’Hara, N.N.; Klein, A.; Diedrich, A.M.; Kolakowski, L.; Lai, J.K.; Hasan, S.A.; Henn, R.F.;
et al. Risk factors for increased shoulder Cutibacterium acnes burden. JSES Int. 2020,4, 464–469. [CrossRef]
44.
Murphy, B.; Hoptroff, M.; Arnold, D.; Eccles, R.; Campbell-Lee, S. In-vivo impact of common cosmetic preservative systems in
full formulation on the skin microbiome. PLoS ONE 2021,16, e0254172. [CrossRef] [PubMed]
45.
Updegraff, D.M. A culteral method of quantitatively studying the microoorganisms in the skin. J. Investig. Dermatol. 1963,43, 129–137.
[CrossRef] [PubMed]
46.
Miller, B.S.; Olszewski, A.M.; Bedi, A. A Microcurrent Dressing Reduces Cutibacterium Acnes Colonization in Patients Undergoing
Shoulder Arthroplasty or Arthroscopy: A Prospective Case Series. HSS J. 2023,19, 92–96. [CrossRef]
47.
Food and Drug Administration. Premarket Notification 510(k). 2024. Available online: https://www.fda.gov/medical-devices/
premarket-submissions-selecting-and-preparing-correct-submission/premarket-notification-510k (accessed on 14 October 2024).
48.
Duffy, H.R.; Godfrey, R.W.; Williams, D.L.; Ashton, N.N. A Porcine Model for the Development and Testing of Preoperative Skin
Preparations. Microorganisms 2022,10, 837. [CrossRef] [PubMed]
49.
Joglekar, P.; Conlan, S.; Lee-Lin, S.-Q.; Deming, C.; Kashaf, S.S.; Program, N.C.S.; Kong, H.H.; Segre, J.A.; Barnabas, B.B.; Black,
S.; et al. Integrated genomic and functional analyses of human skin–associated Staphylococcus reveal extensive inter- and
intra-species diversity. Proc. Natl. Acad. Sci. USA 2023,120, e2310585120. [CrossRef]
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