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Comparative Recovery of Serratia marcescens Using Bags versus
Gloves as Described in ASTM E1174-21 Health Care Personnel
Handwash Method
Elizabeth Moyer,aGregory Cole,aEleanor Harding,aMarilena Jamieson-Popp,aJanice L. Fulsa
a
Henkel Corporation, Stamford, Connecticut, USA
ABSTRACT The ASTM E1174-21 Health Care Personnel Handwash method is prescribed
by the U.S. Food and Drug Administration (FDA) to demonstrate the efficacy of antiseptic
handwashing products. The standardized method allows for marker bacteria to be collected
from the hands by using either a bag or a glove. Two recent studies utilizing the different
collection methods testing the same product showed substantial differences in results.
We sponsored two independent studies to compare the bag and glove collection methods
following contamination with Serratia marcescens. Overall, there was no difference between
collection methods for bacteria recovered (P=0.603). The distribution of recovery for the
bag method was slightly less variable than for the glove method. Statistical differences
were observed within each lab based on the collection day. The day-to-day variability
is critical to consider for future multiple-day studies. Additionally, hand size appears to
impact recovery, especially for the glove method, with both small and medium hand
sizes resulting in higher recovery than large and extralarge hand sizes (P=0.015), whereas
hand size did not impact recovery with the bag method (P=0.315). While it appears that
both the bag and glove methods can be used, our findings suggest that gloves may not
be the best option for subjects with large to extra-large hands. Additional work looking
at bacterial recovery following product treatment is warranted to understand the impact
of large hands in the bag versus glove recovery method.
IMPORTANCE Antiseptic hand wash products are evaluated using the standard ASTM
E1174-21 to demonstrate their antibacterial efficacy. Often products are tested at multiple
labs, and the need to understand variables that may play a role in the outcome of the
study is important. This work allows us to evaluate the impact that the two collection
methods, bags and gloves, have on bacteria recovery. If differences are observed, standard-
ization to one method may be critical to ensure similar test results when planning studies
at multiple labs.
KEYWORDS antibacterial, antimicrobial efficacy, antiseptic, clinical methods,
handwashing, health care personnel hand wash
In 2017, the U.S. Food and Drug Administration (FDA) issued a final monograph for anti-
septic ingredients used in a health care setting. In this monograph, the FDA prescribed the
use of the American Society for Testing and Materials (ASTM) E1174-13 Health Care Personnel
Handwash method to demonstrate the efficacy of antiseptic ingredients used in handwash-
ing agents (1, 2). This method was originally established as an ASTM standard test method in
1987 and was referenced in the 1994 Tentative Final Monograph for Health-Care Antiseptic
Drug Products (3). E1174 has gone through several revisions since 1987. The standard has
been revised an additional five times, with the latest revision being published in 2021 (4).
Some examples of the revisions made over the years include the addition of Escherichia coli
as an alternative test organism, changes to inoculum ranges, and removal of multiple washes
to reflect the current FDA requirement of only a single wash. The most recent revision to
Editor Eleanor A. Powell, University of
Cincinnati
Copyright © 2023 Moyer et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Elizabeth Moyer,
elizabeth.moyer@henkel.com.
The authors declare a conflict of interest.
Funding for this research was sponsored by
Henkel Corporation. All authors are either
current or were employees of Henkel
Corporation at the time of the studies. None of
the authors benefitfinancially or otherwise
from the outcome of the studies.
Received 21 April 2023
Accepted 8 May 2023
Published 18 May 2023
May/June 2023 Volume 11 Issue 3 10.1128/spectrum.01288-23 1
METHODS AND PROTOCOLS
E1174-21 added a precision and accuracy statement. In 2021, the ASTM subcommittee
E35-2001 completed an interlaboratory statistical analysis on studies run at multiple labs
using a single test product (5). The analysis determined the greatest introduction of variabili-
ty at baseline was attributed to subjects (44%), hands for the same subject (18%), laboratory
(22%), and studies run within a single lab (15%) (5).
All research methods have inherent variability that must be understood, i.e., inde-
pendent, dependent, and extraneous variables (6). For instance, in E1174, independ-
ent variables such as wash time and number of washes can be varied to measure the
effect on log
10
reduction. Factors contributing to the effectiveness of both soaps and
the methods to test them have been previously considered and summarized (7–9).
Variability has been mostly attributed to extraneous variables such as subjects, differ-
ent labs, and hands.
FDA’s new requirement to support the efficacy of the antiseptic ingredients is for two inde-
pendent laboratories to test the activity of the antiseptic ingredient (1). To meet this require-
ment, studies were performed on an antiseptic product at two independent labs. The results
were significantly different. As a result, we wanted to further examine E1174 and determine
what differences in execution may have led to the differing results. In reviewing the steps
outlined in the standard, it was determined that the two labs differed in how the bacteria
were recovered from the hands. E1174 allows for either the use of plastic bags or gloves for
recovery. Upon review of the historical versions of E1174, both bags and gloves were found
to be a part of the original 1987 version. While we cannot pinpoint historically why both
bags and gloves were allowed, we deduct, from the reference found in the ASTM standard,
that the use of gloves came from the evaluation of surgical scrubs (5). Published studies
demonstrating equivalency between bags and gloves cannot be found. The objective of
this study was to determine if bacterial sampling collection using bags was equivalent to
the bacterial sampling collection using gloves following contamination with Serratia marces-
cens as part of the Health Care Personnel Handwash method. Two independent laboratories,
Henkel Research@Elm (Henkel), and SGS Stephens, Inc. (SGS), executed these studies com-
paring the bag and glove methods.
RESULTS AND DISCUSSION
Bag versus glove variability. There was not a statistically significant difference in
the bacterial counts from samples collected using the bag method (average log
10
recovery,
8.81) and samples collected using the glove method (average log
10
recovery, 8.77) (P=0.603)
(Table 1). The distribution of data for the bag method was slightly less variable, having a stand-
ard deviation of 0.15 compared to a standard deviation of 0.21 for the glove method (Fig. 1).
Collection day impact on bacterial recovery. There was a statistically significant
difference in bacterial counts from the first bacterial collection (day 1) to the second bacterial
collection (day 8) at both independent laboratories. For the bag collection method, SGS had
observed a 0.21-log
10
recovery difference between days (P=0.005), while Henkel saw a
0.05-log
10
difference (P=0.314). With the glove collection method, both sites had a signifi-
cant difference between days, with Henkel having a 0.27-log
10
difference (P=0.001), while
SGS had a 0.20-log
10
difference (P=0.010) (Fig. 2). Day-to-day variability was observed at both
labs, which could be especially critical to consider for multiple-day studies, such as pivotal
studies where data can be collected over multiple days or weeks.
TABLE 1 Log
10
bacterial recovery means by collection method at two independent laboratories
b
Lab No. of samples
Log
10
bacterial recovery for:
Log
10
difference
a
Bags
a
Gloves
a
Henkel 24 8.81 60.11 8.78 60.22 0.04 60.25
SGS 22 8.80 60.19 8.76 60.20 0.04 60.26
All data 46 8.81 60.15 8.77 60.21 0.04 60.25
a
Values are means 6standard deviations.
b
While no significant differences were observed between methods by either lab, the bag method trended higher
log
10
recovery than the glove method.
Comparative Recovery of S. marcescens in ASTM E1174 Microbiology Spectrum
May/June 2023 Volume 11 Issue 3 10.1128/spectrum.01288-23 2
Hand size impact on bacterial recovery. Bacterial recovery was highest for subjects
with small hands and decreased with medium and large hands for both collection methods
(Fig.3).Whenusingtheglovecollectionmethod,therewasastatisticallysignificant effect of
hand size, with small hands recovering 8.88 log
10
, medium hand sizes recovering 8.82 log
10
,
and large/extra-large hand sizes recovering 8.54 log
10
(P=0.015). There was not a statistically
significant effect on bacterial recovery based on hand size when using the bag collection
method, with recoveries ranging between 8.84 log
10
,8.82log
10
, and 8.75 log
10
, respectively
(P=0.315). Due to the inherent variability within plate count methods, two counts are con-
sidered different if the difference is .0.3 log
10
(10).Onlywiththeglovemethodwastherea
difference greater than 0.3 log
10
between hand sizes.
Sex, race, and age impact on bacterial recovery. The data were evaluated by demo-
graphics, including sex, race, and age grouping. For sex, there was a statistically significant
difference in bacterial recovery, with the glove method observing a higher log
10
recovery
of 8.76 in female participants, while males had a log
10
recovery of 8.54 (P=0.001), which
correlates with males typically having larger hands. There was no statistical difference uti-
lizing the bag method for sex (P=0.604). Race and age categories did not impact bacterial
recovery for either method.
FIG 1 Log
10
bacterial recovery distribution by collection method. The bag method had a normal distribution and was slightly less
variable, having a standard deviation of 0.15. The glove method was nonnormally distributed with a standard deviation of 0.21.
FIG 2 Log
10
bacterial recovery means by study collection day. Differences were seen by day with both collection methods and with both labs. *,Pis statistically
significant at a 95% confidence level.
Comparative Recovery of S. marcescens in ASTM E1174 Microbiology Spectrum
May/June 2023 Volume 11 Issue 3 10.1128/spectrum.01288-23 3
Further implications. Understanding the potential sources of variation within a stand-
ardized method is critical in its successful execution and interpretation of results. Since the
FDA requires testing at multiple sites to confirm the antiseptic activity of a test article, there
is higher potential for variability in results.
It is important to note that these studies only examined baseline recovery. Additional
differences may be observed when a test product application is examined. With the full E1174
test method, the need for adequate neutralization and ensuring the hands are covered with
the stripping solution during massaging is critical to reliable results. In addition, bags are less
expensive and do not need to be tested for inhibitory properties as required for the gloves
to be used. Additional work looking at bacterial recovery following product treatment is war-
ranted to understand the impact of large hands in the bag versus glove recovery methods.
The surgical gloves used in these studies are extralarge for all subjects; however, it is often
observed that it is difficult to don large and extralarge subjects’hands. The hands are typi-
cally wet with either bacteria or product, and the insertion into the glove becomes difficult.
The data indicate that gloves may not be the best option for subjects with large to extralarge
hands. The bag recovery may be better, as there were no recovery differences between hand
size or sex.
MATERIALS AND METHODS
Subjects. An independent institutional review board approved both protocols involving human sub-
jects, and all research complied with all federal and institutional guidelines. Healthy adult subjects between 18
and 65 years of age with hands and wrists free of dermatoses, cuts, lesions, and other skin disorders were
FIG 3 Log
10
bacterial recovery by surgical glove size and collection method. With the bag method, there
was no difference in means between the three hand sizes (P= 0.316). With the glove method, there was
statistically less recovery for large/extralarge hand sizes than for small and medium hands (P= 0.015).
Comparative Recovery of S. marcescens in ASTM E1174 Microbiology Spectrum
May/June 2023 Volume 11 Issue 3 10.1128/spectrum.01288-23 4
recruited to participate in the studies. A total of 46 subjects were enrolled in and completed both studies (24
subjects at Henkel and 22 at SGS).
Study design. The studies were conducted in the spring of 2022 and utilized a randomized, single-
blinded, crossover design. Subjects were randomly assigned to one treatment group (bags or gloves) for
the first study visit and the opposite treatment group for the second visit. Each site followed the same
protocol. After informed consent was obtained, subjects participated in a 7-day washout during which
they were required to refrain from the use of antibacterial products. Subjects were provided a washout
kit containing nonantibacterial soaps, alcohol-based hand sanitizer, and gloves for home cleaning. The
alcohol-based hand sanitizer was included in this protocol due to the COVID-19 pandemic at the time of
study execution and is not known to be persistent.
After completion of the washout, subjects visited the study site to confirm any changes in health status,
and their hands were evaluated for cuts, scratches, or issues that would preclude participation. The hand size
of participants was measured according to a standard surgical glove sizing chart, which measured the distance
from the thumb bridge to the outer aspect of the hand and classifiedittosmall(7in.),medium,(8in.),large(9
in.), or extralarge (10 in.). Once enrolled, bacterial sampling was performed in two ways, collection in plastic
bags (bag method) and collection in sterile disposable surgical gloves (glove method). With both methods,
two laboratory technicians, known as hand massagers, were assigned to one subject. One massager was re-
sponsible for the right hand of the subject, and one massager was responsible for the left hand of the subject.
The same massagers were assigned to a subject and performed both sampling procedures. Subjects were
randomized so that half of the subjects had the bacterial sampling collected from the bag method, and half of
the subjects had the bacterial sampling collected from the glove method. All subjects then participated in a
second 7-day washout prior to returning to the study site for the final visit and bacterial sampling (day 8). At
the final visit, the subjects had the bacterial sampling collected by the method not performed during test
wash 1. Subjects remained in the study for 2 days as a follow-up period to monitor their hands for signs of skin
infection.
Microorganisms and growth conditions. The bacterial strain used in these studies was Ser ratia marces-
cens (ATCC 14756). It was obtained from American Type Culture Collection (ATCC) and was propagated accord-
ing to ATCC recommendations. Stock cultures were maintained using Microbank bacterial preservation system
(Pro-Lab, Diagnostics, Austin, TX) and stored at 80°C. The organism was grown in tryptic soy broth (TSB)
(Becton, Dickinson, and Company, Sparks, MD) at 20 to 25°C for 24 62h.A24-hbrothculturewasstreaked
onto Trypticase soy agar (TSA) (Becton, Dickinson, and Company, Sparks, MD) and incubated for 24 62hat20
to 25°C. A study challenge pool was made by transferring at least three isolated colonies from the TSA plate to
a sterile vessel of TSB. A series of at least two, but no more than five, 24-h broth transfers were made in 10 mL
of TSB. To achieve the appropriate volume of inoculum, TSB was inoculated with 0.1 mL of culture per 100 mL
of broth and incubated at 20 to 25°C for 24 64 h. This inoculum was used without dilution in the studies at a
titer between 5.0 10
8
CFU/mL and 4.0 10
9
CFU/mL.
Conditioning wash. A conditioning wash was performed prior to the start of each test to remove
any dirt and oil present on the hands. Subjects were asked to pass their hands under running tap water
tempered to 40 62°C. Two pumps of nonantimicrobial soap (Johnson & Johnson Head-to-Toe Wash &
Shampoo; Skillman, NJ) were dispensed into the cupped palm of one hand. The soap was spread over
the entire surface of the hands and the lower third of the forearms. Subjects washed for 15 62 s follow-
ing handwashing guidelines from the WHO, making sure all surfaces of the hands, fingers, and backs of
hands were washed (11). The hands were rinsed under running tap water tempered to 40 62°C for 30 s.
The subjects then dried their hands thoroughly using disposable paper towels. After drying, the hands
and wrists were soaked with 70% isopropyl alcohol for 30 s. The hands were then air-dried completely.
Bacterial contamination. Subjects’hands were contaminated with the marker organism following
the procedure outlined in ASTM E1174. Three separate 1.5-mL aliquots were placed into the hands and
rubbed by the subjects for 20 s. Between each aliquot, the hands dried for 30 s, with the final drying
being 90 s prior to recovery. The hands were immediately sampled using the prescribed bacteria recov-
ery methods. Following the sample recovery, the subjects’hands were decontaminated with isopropyl
alcohol, antibacterial soap, and topical antibiotic cream.
Bacterial recovery method. (i) Bag method. Plastic bags (nonsterile, low-density flat poly bag
[1.5 mil] or equivalent; 29.2 cm 31.8 cm) were placed on the subject’s right and left hands. Seventy-
five 62-mL aliquots of stripping solution with neutralizer (0.075 M phosphate buffer with lecithin and Tween
20) were added to each bag. Each bag was secured at the wrist, and the hands were massaged for 1 min in a
uniform manner. A 1-mL aliquot was obtained from the bagged hands within 1 min of completing the mas-
sageandplacedintoa9-mLtubeofButterfield’s phosphate-buffered water with lecithin and Tween 80.
(ii) Glove method. Synthetic polyisoprene sterile surgical gloves (unlined, powder-free gloves with
no antimicrobial properties in size extralarge) were pulled open by staff and placed on the subject’srightand
left hands. Seventy-five 62-mL aliquots of stripping solution with neutralizer (0.075 M phosphate buffer with
lecithin and Tween 20) were added to each glove. Each glove was secured at the wrist, and the hands were
massaged for 1 min in a uniform manner. A 1-mL aliquot was obtained from the gloved hands within 1 min of
completing the massage and placed into a 9-mL tube of Butterfield’s phosphate-buffered water with lecithin
and Tween 80.
Enumeration of bacteria from bags or gloves. Serial 10-fold dilutions were performed with
Butterfield’s phosphate-buffered water with lecithin and Tween 80 by using the initial 1-mL aliquot from
the bagged or gloved hands. Dilution aliquots were plated onto TSA using standard spread plate-counting
procedures. The plates were incubated at 20 to 25°C for 36 to 48 h to enhance pigmentation development.
Colonies displaying red pigmentation typical of S. marcescens were counted. Plates yielding 25 to 250 colo-
nies were counted using standard plate-counting procedures.
Comparative Recovery of S. marcescens in ASTM E1174 Microbiology Spectrum
May/June 2023 Volume 11 Issue 3 10.1128/spectrum.01288-23 5
Calculations and statistical analysis. The number of bacteria per hand was calculated by multiply-
ing the CFU per milliliter obtained in the plate count by 75, the volume in milliliters of stripping solution
used in the bag. The CFU per hand was then converted to log
10
counts and averaged, including the right
and left hands. Then, the average and standard deviation for each recovery method (glove or bag) were
calculated. The log
10
bacterial counts from each recovery method were tested for normality using the
Shapiro-Wilk and Anderson-Darling tests. Since the data were found to be not normally distributed, a
nonparametric Wilcoxon signed-rank test was used to compare the recovery methods (bag versus
glove). An analysis of variance (ANOVA) with
a
equal to 0.05 was used to assess other sources of data
variation, including the collection day within the study and demographic categories, including sex, race,
age, and hand size of subjects.
SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 0.1 MB.
ACKNOWLEDGMENTS
We thank SGS Stephens, Inc., Phoenix Arizona, where the second study was performed.
We thank the clinical staff of SGS, especially Andrea Waggoner and Tony Petrangeli.
Additionally, we thank James P. Bowman for advising on the statistical analysis design
and validating the results.
Funding for this research was sponsored by Henkel Corporation. All authors are either
current or former employees of Henkel Corporation at the time of the studies. None of the
authors benefitfinancially or otherwise from the outcome of the studies.
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