Journal of Food Protection 86 (2023) 100035

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Journal of Food Protection

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Research Paper

Assessment of Microbial Source Tracking Marker and Fecal Indicator

Bacteria on Food-Contact Surfaces in School Cafeterias
Su Jin Nam, Dong Woo Kim, Seung Hun Lee, Ok Kyung Koo ⇑
Department of Food and Nutrition, Gyeongsang National University, Jinju, Republic of Korea

A R T I C L E I N F O A B S T R A C T

Keywords: Food poisoning outbreaks in schools can affect many students, causing physical and psychological damage and
CrAssphage time and economic loss. Fecal indicator bacteria (FIB) have been used to monitor the contamination; however,
Human feces the detection is time‐consuming and confirms the contamination from all warm‐blooded animals. Microbial
Microbial source tracking source tracking (MST) is a molecular‐based detection method that is host specific. This study aimed to evaluate
Real‐time PCR
MSTs and FIBs for tracing contamination in the school cafeteria. The average total aerobic count was 0.89 to
School cafeteria
3.63 log CFU/100 cm2, and the faucets in the cooking area showed a significantly high aerobic count. The stove
valve, faucet, and hand‐washer were the most contaminated area, with a concentration of 1.90 to 6.80 log
CFU/100 cm2 from the frequent hand contact. Escherichia coli was not detected on any surfaces, and coliform
was detected on five surfaces: the sink and faucet in the food preparation area, the faucet in the cooking area,
the hand‐washer, and the toilet seat in the restroom with 0.33 to 3.64 log CFU/100 cm2. Human‐specific
crAssphage appeared on a faucet in the food preparation area, while HF183 was not detected. The result indi-
cates that the continuous monitoring of frequent hand‐contact areas is recommended to maintain the hygiene
condition in the school cafeteria.

School meals are provided to encourage healthy eating habits by
providing safe and high‐quality meals for students, as indicated in Kor-
ea’s School Meals Act of the Ministry of Education (Ministry of
Education (MOE), 2021). Schools with 50 or more students must hire
a licensed nutritionist to prepare an appropriate diet under the nutri-
tional standards for improved meal quality. A total of 11,903 schools
provide school meals to 5.38 million students, or 99.9% of all students
nationwide in Korea, as of 2020 (Ministry of Education (MOE), 2021).
The participation rates in school meals are 84.2% in the United States,
43.9% in the United Kingdom, and 74.9% in Germany (Lee et al.,
2014).
In school cafeterias, many meals must be prepared at once, and
cooking and serving these meals is time‐intensive, including prepara-
tion time for washing and disinfecting food ingredients, a heating time
to reach the central temperature to follow the standards, and a serving
time to provide the students. During this time, meals are likely exposed
to various hazardous factors such as contaminated food ingredients,
poor personal hygiene, and the cooking environment. A single contam-
ination event can lead to large‐scale food poisoning (Lee and Greig,
2010). According to the Ministry of Food and Drug Safety (MFDS) in
Korea, foodborne outbreaks in schools accounted for 9.4% of 1,562
outbreaks between 2015 and 2020, while 9.9% were in other cafete-
rias and 27.9% in restaurants. However, the number of illnesses in
schools is the highest among facilities, with 7,370 illnesses, with
27% of total illnesses (Ministry of Food and Drug Safety (MFDS),
2021). School cafeterias also occur worldwide, with 5,222 illnesses
in Japan from 2015 to 2018 and 77,628 in the USA from 2014 to
2017 (Ministry of Food and Drug Safety (MFDS), 2021). Food poison-
ing accidents in schools can cause fatal sequelae in students with
weakened immunity. In addition, the disease can spread from student
to home and from home to community, causing economic losses by
hospitalization costs, productivity decline, and the burden of the dis-
ease itself (Lee and Greig, 2010).
Hygiene management is critical to ensure the safety of school
meals. From an epidemiological investigation of 121 gastrointestinal
outbreaks in schools worldwide, bacterial contamination caused about
51% of outbreaks and viruses about 40% (Lee and Greig, 2010). The
leading causes of microbial contamination are food ingredients, work-
ers, and the environment, including facilities and equipment. In a pre-
vious study, Enterobacteriaceae counts in fresh produce and raw meat
delivered to schools were 0.86–6.78 log CFU/g and 2.08–5.18 log
CFU/g, respectively (Shin et al., 2008). Microbial contamination of

⇑ Corresponding author. Address: Department of Food Science, Chungnam National University, Daejeon, Republic of Korea.
E-mail address: okoo@cnu.ac.kr (O.K. Koo).

https://doi.org/10.1016/j.jfp.2022.100035
Received 24 June 2022; Accepted 23 December 2022
Available online 30 December 2022
0362-028X/© 2022 Published by Elsevier Inc. on behalf of International Association for Food Protection.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
S.J. Nam et al.
food ingredients can be controlled through proper disinfection with
chlorine concentrations of 100 to 130 ppm for 5 min and heating at
75℃ or higher for general food or 85℃ for shellfish for more than
one min, based on 5th School Meal Hygiene Management Guidelines
(Ministry of Education (MOE), 2021). However, inappropriate envi-
ronmental conditions and personal hygiene by food handlers can cause
re‐contamination. According to an observational survey of catering
services, none of the services, including schools, hospitals, business
companies, and healthcare sectors, received an adequate score for food
safety management. Schools had the lowest score showing a high risk
of cross‐contamination (Garayoa et al., 2017).
A human feces of 1 gram can contain 104 to 1011 pathogens, includ-
ing Escherichia coli, Shigella spp., and Salmonella spp., with more than
1010 infectious viral particles, including norovirus and Hepatitis A
virus (Lee and Greig, 2010). Additionally, about 0.1 mg of fecal is pre-
sent on human skin, which may cause fecal cross‐contamination by
human contact (Lee and Greig, 2010). Workers’ hands transfer bacteria
or viruses to meals and environmental surfaces during food handling
or after toilet use (Pickering et al., 2010). A previous study found that
of 816 food worker‐related outbreaks, 40% were due to hand contact
(Todd et al., 2009). Viruses can readily cause transmissions and sur-
vive on surfaces over long periods. Previous studies using bacterio-
phages φX174 showed that at least 14 people could be contaminated
with horizontal transmission by touching the same contaminated han-
dle (Rheinbaben et al., 2000). Most foodborne pathogens survive on
hands and contact surfaces for a prolonged time. Therefore, hand‐
contact surfaces require comprehensive management, as they can be
a hidden source of contamination.
Fecal‐oral route refers to the transmission of fecal particles of an
infected person through various media such as hands, mouth, flies,
soil, and water. The fecal‐oral route monitoring is critical for under-
standing the transmission route of pathogens in schools. Fecal indica-
tor bacteria (FIB) and microbial source tracking (MST) have been
mainly used to trace this route. FIB are bacteria that live in the intesti-
nes of warm‐blooded animals to identify the presence of fecal contam-
ination (Gyawali and Hewitt, 2020; Nam et al., 2022). FIB, a culture‐
based method, takes more than 2–3 days to obtain results. MST, a
molecular‐based method, does not require an incubation process and
can detect low levels of contamination (Gyawali and Hewitt, 2020;
Nam et al., 2022). The host‐specific MSTs are abundant in the environ-
ment, and they can identify the fecal sources by detecting genetic
markers of each animal species, such as crAssphage and HF183 for
humans, Pig2Bac for pigs, LA35 for chickens, and others (Gyawali
and Hewitt, 2020; Schiaffino et al., 2020). Bacteriophages are suitable
for MSTs that can coexist with the bacterial host resulting in the high-
est density of microorganisms (Schippa and Conte, 2014). CrAssphage
was first discovered in 2014. It is a bacteriophage abundant and speci-
fic to Bacteroides, a human enterobacterium (Dutilh et al., 2014). The
bacteriophage has since been suggested as an MST marker for monitor-
ing human fecal contamination, and studies have been mainly con-
ducted on wastewater or other aquatic environments. Currently,
crAssphage has been demonstrated in several studies as a potential
indicator to identify and track human fecal contamination. This study
aimed to investigate the sanitation status in school cafeterias through
hygiene indicator bacteria and MST markers and investigate the poten-
tial use of crAssphage to evaluate cross‐contamination by workers and
help with food poisoning epidemiological investigations.
Materials and methods
School cafeteria sample collection
Environmental surface, hand, and fecal samples were collected
using a swab sampling method in 10 school cafeterias in the Gyeong-
2
Journal of Food Protection 86 (2023) 100035
nam province area from December 2020 to September 2021 (Son
et al., 2022). Thirteen environmental surfaces expected to have fre-
quent contact with hands during food preparation, cooking, and other
activities were selected. The sampling was done when the cleaning and
disinfection were completed after serving lunch. The researchers wore
sterile latex gloves to minimize cross‐contamination from hands dur-
ing sampling. Each environmental surface sample was collected in a
100 cm2 area on stainless steel frames (one 10 by 10 cm or four 5
by 5 cm) according to the surface conditions using a 3 M Pipette Swab
Plus in 10 mL Buffered Peptone Water (3M Korea). The samples were
immediately transferred to the laboratory under refrigerated condi-
tions and processed within 24 h after collection. Hand samples were
collected from 11 school cafeteria workers in three schools using a
glove juice method (Pickering et al., 2010). Each worker wore a sterile
latex glove with 50 mL of 0.1% peptone water, and their hands were
massaged for one min to elute the microbes from the hand. Hand sam-
ples were stored at 4°C or lower and processed within 24 h after col-
lection. Fecal samples were also collected from the school cafeteria
workers using a self‐made feces collection kit with cotton swabs
(Poongsung). Fecal samples of 40 workers were collected from 10
schools through approval by the Institutional Review Board of the
Korea National Institute for Bioethics Policy (IRB, P01‐201912‐31‐
001) and stored at −74°C until processed.
Each school nutritionist provided general information, such as the
number of meals served, the number of food workers, the surface size
of the kitchen, and the hygiene management methods for investigation
of the variables in each school that can affect microbial contamination
in the environment.
Hygiene indicator bacterial analysis
The collected surface and hand samples were vortexed for one min
using a Vortex‐Genie 2 (Scientific Industries). TAC, coliform, and
E. coli were quantified using Petrifilm aerobic count plates 3M and Pet-
rifilm E. coli/coliform count plates 3M, as indicated in the Food Code,
Korea (Ministry of Food and Drug Safety (MFDS), 2021). One milliliter
of samples was inoculated in each dry film. The number of colonies
was counted after incubation at 30 and 37°C for 24–48 h for the aero-
bic count and E. coli/coliform counts, respectively.
Electronegative membrane filtration for MST marker concentration
MST marker concentration was performed using an electronegative
membrane filtration method, according to the previous study (Ahmed
et al., 2020). In the sample solutions, MgCl2 was added to a final con-
centration of 25 mM and vortexed for 1 min. The solution was filtered
using a vacuum pump with a mixed cellulose ester membrane (MCE
membrane, 25 mm diameter, 0.45 μm pore size, Merck Millipore).
The filter was then transferred to a sterile 1.7 mL conical tube, and
300 mg glass beads (1.0 mm diameter, Sigma‐Aldrich) were added
with 800 µL SM buffer (Dynebio). After vortexing for 10 min, centrifu-
gation was performed at 13,000 rpm for 10 min, and 500 µL of the
supernatant was transferred to a new 1.7 conical tube for the subse-
quent DNA extraction process.
DNA extraction
The DNA extraction was performed using a phenol–chloroform
method with the filter‐concentrated surface and hand samples
(Deiner and Altermatt, 2014). To each 500 µL concentrated sample,
10 µL of 25% SDS and 10 µL of proteinase K (20 mg/mL) were added,
and the resultant solution was incubated at 56°C for 60 min. Subse-
quently, 500 µL of phenol:chloroform:isoamyl alcohol mixture (PCI
25:24:1, Merck) was added to the sample, vortexed, and centrifuged
S.J. Nam et al. Journal of Food Protection 86 (2023) 100035

at 13,000 rpm for 10 min. Next, the supernatant was transferred to a
new tube, mixed with the PCI mixture, and centrifuged again to sepa-
rate the supernatant. In the treated supernatant, 50 µL of 3 M sodium
acetate and 500 µL of 100% EtOH were added, settled at −70℃ for
10 min, and then centrifuged at 13,000 rpm for 30 min to remove
the supernatant. After adding 600 µL of 70% EtOH to the pellet, the
sample was mixed gently, centrifuged at 13,000 rpm for 10 min, and
then, the DNA was collected with a TE buffer of 50 µL. According to
the manufacturer’s instructions, the fecal DNA samples were extracted
using a QIAamp Power Fecal DNA Kit (Qiagen). Each sample was
diluted to a DNA concentration of 20 ng/µL per reaction, measured
with a spectrophotometer (NanoDrop One; Thermo Scientific), and
stored in a −20°C freezer until use.
CrAssphage and HF183 detection and quantification
The conventional PCR reaction was conducted in a T100 Thermal
Cycler (Bio‐Rad Laboratories). PCR Mastermix reagent (COSMO Gene-
tech) was prepared with 0.5 µM of forward and reverse primers, 20 ng
template DNA, and PCR‐grade water. Real‐time PCR amplification was
performed using a CFX Connect Real‐Time System (Bio‐Rad Laborato-
ries) with Taqman probe‐based RT‐PCR. The reaction mixture con-
sisted of Thunderbird Probe qPCR Mix reagents (TOYOBO), 0.5 µM
of forward and reverse primer, 0.8 µM of probe primer, 20 ng of tem-
plate DNA, and PCR‐grade water. The oligonucleotide sequences of
each primer and the reaction steps for the detection and quantitative
analysis of crAssphage and HF183 are shown in Table 1. The target
DNAs of crAssphage (NC_024711, 16030‐16177) and HF183
(AB242142, 180‐305) were synthesized and inserted into the plasmid
pUC57 and pTOP Blunt V2, respectively (Macrogen). The plasmid with
target DNA was obtained using an AccuPrep Nano‐Plus Plasmid Mini
Extraction Kit (Bioneer). The obtained target DNA was diluted to 104
to 109 gene copies (GC)/reaction concentrations and used as stan-
dards. The optimal reaction conditions for RT‐PCR were determined
based on the following criteria: a slope factor of −3.09 to −3.59, cor-
responding to a PCR efficiency of 90% to 110%, and a correlation coef-
ficient of >0.98.
Statistical analysis
All data entry and statistical analyses were performed using Predic-
tive Analytics Software (PASW) version 18.0 software. Analysis of vari-
ance one‐way (ANOVA) followed by Duncan's multiple range tests was
used for determining mean significant differences (p < 0.05) between
microbiological data of each school and surface. The Pearson correla-
tion coefficient (R) was calculated to assess the relationship between
school cafeterias and microbiological data variations. The closer the
jRj value is to 1.0, the higher the correlation is between the two data;
0.0 < jRj < 0.2, suggesting no correlation; 0.2 < jRj < 0.4, weak cor-
relation; 0.4 < jRj < 0.7, correlation; 0.7 < jRj < 0.9, high correla-
tion; and 0.9 < jRj < 1.0, very high correlation (Akoglu, 2018).
Results and discussion
Evaluation of microbial contamination in school cafeterias
Ten schools selected for this study have students between 84 and
1,570 for each school, and the sizes of the kitchen are 72–238 m2,
and the dining areas are 146–470 m2. All facilities were modernized
between 2009 and 2019. Modernized kitchens have separate areas
of prepreparation, cooking, and washing stations under the HACCP
program (Son et al., 2022). Thus, these schools were selected by con-
sidering model schools in Korea. Thirteen food/hand‐contact surfaces
in the ten school cafeterias were analyzed for TAC, coliform, and E. coli
(Tables 2 and 3). The average TAC ranged between 0.33 and 6.80 log
CFU/100 cm2. The cooking and other areas had values of 2.74 and
2.53 log CFU/100 cm2, and that of the preparation area was 1.81
log CFU/100 cm2. The stove valve in the cooking area had a surface
concentration of 6.80 log CFU/100 cm2 which was significantly high
than any other surface. In contrast, the apron had the lowest aerobic
count at 0.89 log CFU/100 cm2, and the detection rate was the lowest
at 30%. Coliform was only detected on the following five surfaces with
a range of 0.33 to 3.64 log CFU/100 cm2: the sink and faucet in the
food preparation area, the faucet in the cooking area, and the hand‐
washer and toilet seat in the other area. According to the School Meal
Hygiene Management Guidelines (Ministry of Education (MOE),
2021), there is a zero‐tolerance policy for E. coli on food and
non–food‐contact surfaces. Although there is no official standard for
TAC and coliforms, keeping a low microbial level is recommended.
In a previous study by Harrigan & McCance (Harrigan and McCance,
1976), the microbiological contamination levels were evaluated on
cooking utensils, equipment, containers, and work surfaces in food
manufacturing and cooking facilities. The suggested standards have
been continuously applied in many research labs since 1976. Harri-
gan's standard recommends that a TAC of less than 2.7 log
CFU/100 cm2 is considered safe, and it should not exceed 3.4 log
CFU/100 cm2, and coliform of less than 1.0 log CFU/100 cm2 on
kitchen instruments, equipment, and surfaces is considered safe
(Harrigan and McCance, 1976). The faucet in the cooking area was
the only surface where the average concentration exceeded 3.4 log
CFU/100 cm2, and eighty percent of the schools exceeded 2.7 log
CFU/100 cm2 on the faucet in the cooking area. All faucets tested in
this study are for food products. The surface exceeded 2.7 log
CFU/100 cm2 in 3 of the 13 surfaces (23.1%), including the faucet
in the food preparation area, the stove valve in the cooking area,
and the hand‐washer in the restroom. However, all schools were
within the limit on the apron, the glove, and the rice cooker. Coliform
was detected on the faucet in school G’s cooking area and a toilet seat
in school H with values of 3.64 and 2.42 log CFU/100 cm2, respec-
tively. E. coli was not observed on any surfaces. Overall, the hygienic
condition is comparatively acceptable, satisfying the MOE standards.
A significantly high TAC contamination was detected in the faucets,
the stove valves, and the hand‐washers. These surfaces are in relatively

Table 1
Information on primers, probes, and reaction steps used in conventional and real-time PCR analysis of crAssphage and HF183

CrAsspahge HF183
0 0
Primer Forward 5 -TGTATAGATGCTGCTGCAACTGTACTC-3 50 -ATCATGAGTTCACATGTCCG-30
Reverse 50 -CGTTGTTTTCATCTTTATCTTGTCCAT-30 50 -CTTCCTCTCAGAACCCCTATCC-30
Probe 50 -FAM-CTGAAATTGTTCATAAGCAA-MGB-30 50 -FAM-CTAATGGAACGCATCCC-BHQ-1–30
Reaction step Conventional PCR 5 min at 95°C, 40 cycles of 30 sec at 95°C, 20 sec at 60°C and 30 sec at 72°C,
and 5 min of final extension at 72°C
Taqman probe-based RT-PCR 10 min at 50°C, 10 min at 95°C, 10 min at 95°C,
40 cycles of 15 sec at 95°C and 60 sec at 50°C 40 cycles of 15 s at 95°C and 60 s at 60°C
Reference Stachler et al., 2017 Park et al., 2020

3
 S.J. Nam et al.
Table 2
Detection and concentration of total aerobic bacteria in 13 surface samples of 10 school cafeterias

Samples Total aerobic count (log CFU/100 cm2)

Average Detection School A School B School C School D School E School F School G School H School I School J
rate

Food preparation Sink 2.18bcde 50.00% ND† ND ND 1.77 ± 1.95 ± 2.72 ± 2.10 ± ND 2.34 ± 0.16Cd ND
area 0.10Ab‡ 0.24ABb 0.07Db 0.04BCb
Faucet 2.87defg 80.00% 3.14 ± 2.59 ± 0.04Bd 2.34 ± ND 2.29 ± 0.14Ad 2.54 ± 4.51 ± 2.28 ± 3.25 ± 0.01Cf ND
0.20Cbc 0.03Abcd 0.03Bb 0.14De 0.04Ad
abc
Refrigerator 1.41 90.00% 0.65 ± 0.92Aa 0.33 ± 0.58 Aa
2.18 ± 1.24 ± 2.04 ± 0.77 ± ND 3.14 ± 0.16De 2.04 ± 0.33 ±
0.17Cbcd 0.34ABab 0.12BCbc 0.68Aa 0.08BCc 0.58Aa
Apron 0.89a 30.00% ND 1.49 ± ND 0.50 ± 0.71Aa ND ND ND 0.67 ± 0.58Aa ND ND
0.20Ab
Gloves 1.09ab 70.00% 0.65 ± 0.92Aa 0.33 ± 0.58Aa 0.49 ± 0.85Aa 1.98 ± 0.03Bb 1.96 ± 0.17Bb 0.83 ± ND 1.42 ± ND ND
0.75ABa 0.10ABbc
abcd § Aa Ba
Vegetable 1.94 87.50% 3.42 ± NT 3.24 ± 0.50 ± 0.71 1.42 ± 0.10 0.43 ± ND 3.00 ± 0.04Ce 1.60 ± 0.00 Ba
NT
cutter 0.04Cbc 0.09Cef 0.75Aa
fg Ac
Cooking area Stove valve 3.38 90.00% ND 1.97 ± 0.16 3.70 ± 0.05Ef 6.80 ± 4.26 ± 0.10 Fg
2.26 ± 1.90 ± 3.25 ± 0.01 De
2.59 ± 0.02 Ce
3.70 ±
0.06Gd 0.06Bb 0.05Aa 0.04Ec
4

Rice cooker 1.39abc 90.00% 1.00 ± 1.46 ± 1.65 ± 0.50 ± 0.71Aa 1.91 ± 0.21Bb 0.53 ± 1.96 ± 1.73 ± 0.38Bc 1.74 ± ND
1.41Aba 0.15ABb 0.07ABb 0.92Aa 0.10Bab 0.13Bab
Sink 2.33cdef 80.00% ND ND 3.19 ± 3.43 ± 0.02Fc 1.88 ± 0.03Bb 2.64 ± 2.07 ± 1.10 ± 1.87 ± 2.44 ±
0.06Eef 0.12Db 0.11Bb 0.17Ab 0.15Bbc 0.02Cb
g Dc Ce
Faucet 3.7 100% 4.22 ± 0.17 3.82 ± 0.05 3.88 ± 0.00Cf 2.89 ± 0.09 Bc
2.91 ± 0.06 Bf
2.25 ± 5.14 ± 5.28 ± 0.22Eg 4.12 ± 0.08Dh 2.44 ±
0.02Ab 0.00Ef 0.07Ab
Other area Hand-washer 3.14efg 88.90% ND 2.75 ± 0.06Bd 2.66 ± 3.39 ± 0.15Dc 4.72 ± 0.04Fh NT 2.70 ± 3.21 ± 0.02Ce 3.56 ± 0.14Eg 2.14 ±
0.03Bcde 0.07Bc 0.12Ab
Toilet seat 2.69defg 88.90% ND 2.72 ± 2.90 ± 1.24 ± 2.61 ± 0.04Ce NT 3.08 ± 3.79 ± 0.08Ff 3.10 ± 0.09Ef 2.06 ±
0.01CDd 0.00DEde 0.34Aab 0.02Ed 0.06Bb
Freezer 1.8abcd 90.00% 2.10 ± 2.35 ± 1.96 ± 1.39 ± 2.20 ± 1.10 ± ND 3.11 ± 0.04Ee 1.65 ± 0.33 ±
0.28CDab 0.01DEcd 0.17CDbc 0.12BCb 0.08CDcd 0.95ABa 0.16BCDa 0.58ABa
‡
Significant differences (p < 0.05) for the surface samples of school cafeterias are indicated by alphabets. The capital letter indicated significant differences according to schools on the same surface, and the small
letter indicated significant differences according to surfaces on the same school.
†

Journal of Food Protection 86 (2023) 100035

ND: Not Detected.
§
NT: Not Tested.
S.J. Nam et al. Journal of Food Protection 86 (2023) 100035

Table 3
Detection and concentration of coliform in 13 surface samples of 10 school cafeterias

Samples Coliform (log CFU/100 cm2)

School School School School D School School F School G School H School School
A B C E I J

Food preparation Sink ND† ND ND ND ND 0.33 ± 0.58a‡ ND ND ND ND

area Faucet ND ND ND ND ND 0.33 ± 0.58a ND ND ND ND
Refrigerator ND ND ND ND ND ND ND ND ND ND
Apron ND ND ND ND ND ND ND ND ND ND
Gloves ND ND ND ND ND ND ND ND ND ND
Vegetable ND NT§ ND ND ND ND ND ND ND NT
cutter
Cooking area Stove valve ND ND ND ND ND ND ND ND ND ND
Rice cooker ND ND ND ND ND ND ND ND ND ND
Sink ND ND ND ND ND ND ND ND ND ND
Faucet ND ND ND 0.93 ± 0.81a ND ND 3.64 ± 0.06c ND ND ND
Other area Hand-washer ND ND ND ND ND NT 0.67 ± 0.58a ND ND ND
Toilet seat ND ND ND ND ND NT ND 2.42 ± 0.04b ND ND
Freezer ND ND ND ND ND ND ND ND ND ND
‡
Significant differences (p < 0.05) for the surface samples of school cafeterias are indicated by alphabets.
†
ND: Not Detected.
§
NT: Not Tested.

frequent contact with hands, suggesting the possibility of cross‐
contamination by hand contact. Hands contained 2.49 log CFU/hand
of E. coli and 4.23 log CFU/hand of fecal streptococci, which can be
transferred to the surfaces by instant hand contact (Pickering et al.,
2010). The curved surfaces can also affect high contamination since
microorganisms quickly deposit on nonflat and uneven surfaces with
many gaps. Microbial deposition on the surface is mainly affected by
surface roughness when exposed to the same environmental condi-
tions, including temperature, relative humidity, and air quality
(Tamburini et al., 2015). Even if these surfaces are cleaned, the disin-
fectant may not reach every corner of the surface crevices where
microorganisms are not easily removed and may form a biofilm. The
biofilm promotes microbial growth and accumulation and improves
the disinfectant resistance of microorganisms, making them more chal-
lenging to remove and causing high microbial contamination. Stainless
steel is resistant to corrosion and rust, has good durability owing to its
high strength, and is relatively safe against microorganisms due to its
relatively low surface roughness compared to other materials. There-
fore, according to the School Meal Hygiene Management Guidelines
(Ministry of Education (MOE), 2021), using stainless steel for cafeteria
equipment, including worktables, hoods, trenches, ducts, and others, is
recommended. However, various materials such as brass and plastic
are used with stainless materials used in faucets, stove valves, and
hand‐washers, thus increasing the chances of contamination. There-
fore, surfaces with frequent hand contact require periodic sterilization
and cleaning. Rough surfaces or curved structures must be washed
more carefully to inhibit biofilm formation.
The apron had the lowest TAC with 0.89 log CFU/100 cm2 and was
detected in only 30% of schools, while values of more than 6 log
CFU/100 cm2 were confirmed in previous studies (Lim et al., 2021,
2017). Aprons are stored in a UV disinfection cabinet, maintaining a
low microbial level by surface decontamination, while previous stud-
ies did not keep them in a UV cabinet. Likewise, the gloves were kept
in the UV disinfection cabinet resulting in the second‐lowest count
with 1.09 log CFU/100 cm2. UV disinfection showed effective perfor-
mance in controlling microbiological contamination in school
cafeterias.
Genitourinary and gastrointestinal diseases can be transmitted
through human secretions in restrooms, including human feces, urine,
and vomiting. The hand‐washer and toilet seat in the restroom were
expected to have high concentrations of coliform and E. coli, the FIBs,
due to contamination from worker fecal particles. In previous studies,
detection rates of E. coli were 46.4% in toilet seats (Ogba and Obio,
2018) and 30.6% in hand‐washers (Matini et al., 2020). In this study,
E. coli was not detected in the restroom. The coliform was detected at
0.67 log CFU/100 cm2 on the hand‐washer of school G and 2.42 log
CFU/100 cm2 on the toilet seat of school H. Overall, restrooms had
a TAC detection rate of 88.9%, with average values of 3.14 and 2.69
log CFU/100 cm2 on hand‐washers and toilet seats, respectively.
Although this study showed relatively low detection rates of coliform
and E. coli compared to previous studies, personal hygiene is still
emphasized because microbiological contamination by fecal particles
in toilets can be critical. Contamination of hand‐washers can cause
the hands to be re‐contaminated after washing them. Covering the fau-
cet handle with a paper towel or installing a noncontact faucet can
reduce the spread of microorganisms.
Evaluation of microbial contamination in hand samples
Total aerobic bacteria of 3.82 to 5.81 log CFU/hand were detected
in 11 hand samples of school cafeteria workers. Coliforms were
detected in two of 11 samples with 1.22 and 1.92 log CFU/hand,
respectively. E. coli was not detected in any of the hand samples. This
result was similar to a previous study that analyzed employees’ hands
in childcare centers and kindergartens; TAC was up to 5.42 log CFU/
hand, and coliform was up to 3.78 log CFU/hand, with a detection rate
of 23.5%. According to the School Meal Hygiene Management Guide-
lines (Ministry of Education (MOE), 2021), the workers' hands in
school cafeterias should be E. coli negative. All hand samples met
the criteria. Transient microorganisms exist temporarily on human
skin due to external contact, and resident microorganisms are dis-
tributed in the stratum corneum, hair roots, and sweat glands. Patho-
genic bacteria are generally transient microorganisms readily removed
by hand washing (Todd et al., 2010). Thus, hand hygiene management
through hand washing is essential to prevent infectious diseases in
school cafeterias.
According to previous studies, 52.8% of microorganisms were
reduced after hand washing (Jeong et al., 2003). The detection rate
of fecal bacteria was 44% before hand washing, 23% after hand wash-
ing only with water, and 8% after regular soap washing (Burton et al.,
2011). However, resident microorganisms are not easily removed by
hand washing. Although most resident microorganisms are not patho-
genic, Staphylococcus aureus can cause food poisoning (Snyder, 2001).
When hand washing is not performed sufficiently well, pathogens may

5
S.J. Nam et al. Journal of Food Protection 86 (2023) 100035

Table 4 always be worn when handling food (Ministry of Education (MOE),

General information of each school cafeteria; the number of workers, number of 2021).
meals (lunch) per day, kitchen size, and disinfectant used for cleaning

School Workers Meals per Size of kitchen Disinfectant use Correlation between variables in school cafeterias
day (m2)

A 3 194 93 Sodium hydroxide

In order to analyze the factors affecting the microbiological con-
B 3 84 72 Sodium tamination on hand‐contact surfaces in the school cafeterias, general
hypochlorite†, information about each school cafeteria was collected (Table 4), and
70% ethanol‡ the correlation of these factors with the TAC was compared (Fig. 1).
C 13 1,570 94 Sodium hypochlorite
Positive correlations were observed between microbial contamination
D 4 229 79 Sodium hypochlorite
E 10 1,237 238 Sodium level and the number of meals, workers, kitchen size, and meals/work-
hypochlorite, ers (workload). The level of microbial contamination was 'correlated'
70% ethanol in meals and workers (respectively, R = 0.62), 'weakly correlated' in
F 4 115 83 Sodium hypochlorite kitchen size (R = 0.31), and 'highly correlated' in workload values
G 7 454 280 Sodium
hypochlorite,
(R = 0.74).
70% ethanol With more meals provided by school cafeterias, the working time is
H 6 470 91.8 Sodium hypochlorite extended due to increased pretreatment and cooking time. The
I 4 320 179 Sodium increased working time can cause more cross‐contamination between
hypochlorite,
ingredients, workers, and the environment (Kwak et al., 2012). In this
70% ethanol
J 3 112 148 Sodium hypochlorite study, increased microbial contamination with more food workers was
likely due to increased cross‐contamination, hand contact, and impro-
†
Sodium hypochlorite is used at a concentration of 200 ppm for disinfection per food handling (Eo et al., 2001). This result supports the positive
of equipment and utensil.
‡
correlation between microbial contamination and the number of meals
70% ethanol is used by spraying and drying for 5 min.
and workers. The imposed workload per worker was also compared.
According to a previous study, with fewer meals/workers, the perfor-
not be removed entirely. Workers’ hands inevitably contact food mate- mance of hygiene management for food handling, cleaning and disin-
rials, environmental surfaces, cooked food, and the workers them- fection, and equipment and utensils is accordingly higher (Kim and
selves throughout the food manufacturing process and are a Oh, 2005). In another study with a nutritionist survey, major factors
significant cause of cross‐contamination. Therefore, proper training responsible for poor hygiene management were inadequate personal
in handwashing is required at least once a month, and gloves should work habits and lack of time due to excessive workload (Kim and

Figure 1. Correlation between the total aerobic count of hand-contact surfaces in the school cafeterias and the variables of each cafeteria. A) Correlation with the
number of meals served at one time: R = 0.62. B) Correlation with the number of cook workers; R = 0.62. C) Correlation with the size of the entire kitchen area:
R = 0.31. D) Correlation with the number of meals/workers: R = 0.74.

6
S.J. Nam et al.
Table 5
The detection rate and concentration of crAssphage and HF183 in fecal samples of workers in each school cafeteria
School No. of fecal samples crAssphage
No. of positive (%)
A 3 1 (33.3)
B 3 1 (33.3)
D 4 ND
E 9 3 (33.3)
F 3 ND
G 6 ND
H 5 1 (20.0)
I 4 2 (50.0)
J 3 3 (100)
†
ND: Not Detected.
Oh, 2005; Yang et al., 2006). The excessive workload affects hygienic
practices, training, and education, resulting in a high correlation
between microbial contamination and workload (Yang et al., 2006).
Therefore, an appropriate workload for the cooks and hygiene educa-
tion to prevent cross‐contamination are critical to improving the
hygiene management performance and quality of school meals.
MST marker detection in school cafeterias
As MST markers, crAssphage and HF183 were used to detect
human fecal contamination in school cafeterias. HF183, a Bacteroides
16S rRNA genetic marker, was analyzed to compare the performance
of crAssphage (Gyawali and Hewitt, 2020). MSTs were first analyzed
in their feces to confirm this. Table 5 shows the detection rates and
concentrations of crAssphage and HF183 in the fecal samples of each
school worker. CrAssphage was detected in 11 out of 40 fecal samples
(27.5%), and the concentration ranged from 3.45 to 8.06 log GC/ng.
HF183 was detected in 16 out of 40 fecal samples (40.0%), and the
concentration range was from 3.26 to 7.28 log GC/ng.
In a previous study, we confirmed the distribution of crAssphage in
Korea, showing a detection rate of 39.4% in human feces with a con-
centration of 4.26–8.25 log GC/ng DNA, and HF183 was 29.8% and
4.64–8.48 log GC/ng DNA (Nam et al., 2022). The donor's age range
was 19 to 45 years, with an average age of 25.4 in the previous study
(Nam et al., 2022). However, the age range of the workers was higher,
from 33 to 60 years, with an average of 49.1 in this study. The age
range can cause a difference in the positive rate by the change in
gut microflora. The abundance of the phylum Bacteroidetes was higher
by 10.25% in 65 years and older than 6.14% in the age group of 30–40
(La‐Ongkham et al., 2020). In particular, Bacteroides uniformis, B. dorei,
and B. massiliensis were predominant only in the elderly subjects (La‐
Ongkham et al., 2020). Since HF183 is a genetic marker targeting
the B. dorei 16S rRNA gene, the detection rate of HF183 was increased
in this study, where the average age of fecal donors was relatively
high. The gut microbiota may vary depending on dietary habits, life-
style, and other environmental factors, and MST markers’ sensitivity
and specificity are influenced by geographic location (Schippa and
Conte, 2014). Thus, it is necessary to consider the variations and com-
bine MST markers for each host to increase the detection rate.
Distribution of MST marker in surface samples
Since MST can detect at low concentrations with high specificity,
the MST was applied to evaluate the food‐contact surfaces in school
cafeterias. CrAssphage was detected in the faucet in the pretreatment
area of school D with a concentration of 5.59 log GC/ng. However,
HF183 was not detected in any of the samples. Although it was not
detected in school D workers, we cannot exclude the potential contam-
ination from the workers or visitors. Thus, MST detection in the food‐
7
Journal of Food Protection 86 (2023) 100035
HF183
Concentration No. of positive (%) Concentration
(log GC/ng) (log GC/ng)
7.23 ND† -
5.1 ND -
- ND -
5.18–6.25 4 (44.4) 5.46–6.96
- 3 (100) 5.38–7.28
- 2 (33.3) 3.40–6.53
6.6 2 (40.0) 3.95–6.23
3.45–8.06 3 (75.0) 3.26–3.45
4.54–6.24 2 (66.7) 5.32–6.69
contact surfaces and the cooks’ feces can help trace the source of food
poisoning accidents caused by fecal microorganisms.
The detection of FIB showed limited correspondence to the MST‐
positive surfaces. According to previous research, the FIB contamina-
tion showed a weak negative correlation with the concentration of
HF183 (tau = − 0.19) and could not confirm the correlation with
the detection rate of HF183 (Nguyen et al., 2018). CrAssphage and
HF183 are MST markers specifically detected in human feces. In addi-
tion to the first identified prototypical crAssphage, a crass‐like phage
has been identified, which may increase the utility of crAssphage as
an MST marker (Nam et al., 2022). Therefore, various MST markers
make it possible to increase the detection rate of human fecal contam-
ination, and more accurate identification of contamination is possible
using both FIB and MST (Gyawali and Hewitt, 2020; Nam et al., 2022).
Limits of detection of FIB and MST markers by sanitizer
The low or limited detection rate of FIB and MST markers is likely
due to the sodium hypochlorite used in the cleaning process of
restrooms and cafeterias. Sodium hypochlorite is the most widely used
disinfectant in the food industry and effectively removes bacteria and
viruses. It dissociates to the hypochlorite ion (‐OCI) and proton (H+),
depending on the optimal pH, leading to conformational changes in
proteins, and destroying the native structure of the microorganism
because of a direct reaction or the formation of stable N‐Cl bonds with
the microorganisms (Ersoy et al., 2019). Culture‐based methods can-
not detect dead or damaged microorganisms on food‐contact surfaces.
Also, sodium hypochlorite has a robust oxidizing capacity, destroying
the viral DNA to detect MST markers through damage by base modifi-
cations and chlorinated base products (Kemp and Smith, 2005). In this
study, the presence of crAssphage suggests that the MST markers have
sufficient potential despite DNA loss due to the use of sodium
hypochlorite.
In summary, this study monitored hygiene status to investigate the
contamination in school cafeterias using MST markers such as HF183
and crAssphage. Overall, the hand‐contact surfaces in the school cafe-
teria were kept relatively clean. However, the stove valves and faucets
showed a high level of contamination by relatively less flat and curved
surfaces. Also, the low contamination level on aprons and rubber
gloves showed an adequate performance of the UV disinfection cabi-
net. It was found that the amount of workload had the most significant
influence on each school’s total microbial contamination level, and
there was a weak correlation with the number of meals, the number
of workers, and the kitchen area. Human fecal contamination was con-
firmed by detecting HF183 and crAssphage in the school cafeterias and
workers, indicating whether the cause of human fecal contamination is
internal or external. It is expected that the hygiene level in the school
cafeteria can be improved by controlling the workers’ workload and
microbial contamination through continuous hygiene monitoring.
S.J. Nam et al. Journal of Food Protection 86 (2023) 100035

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The authors declare the following financial interests/personal rela- Metagenomic analysis of microbial composition revealed cross-contamination
tionships which may be considered as potential competing interests: pathway of bacteria at a foodservice facility. Frontiers in Microbiology, 12, 1–12.
Lim, E. S., Lee, J. E., Kim, J. S., & Koo, O. K. (2017). Isolation of indigenous bacteria
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